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Development of a high-performance liquid chromatographic assay for human chorionic gonadotropin as an… Embree, Leanne 1985

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DEVELOPMENT OF A HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC ASSAY FOR HUMAN CHORIONIC GONADOTROPIN AS AN ALTERNATIVE TO THE OFFICIAL UNITED STATES PHARMACOPEIAL ANIMAL ASSAY By LEANNE EMBREE B.Sc.(Pharm), F a c u l t y of Pharmaceutical Sciences U n i v e r s i t y of B r i t i s h Columbia Vancouver, B. C , Canada, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES F a c u l t y of Pharmaceutical Sciences D i v i s i o n of Pharmaceutical Chemistry We accept t h i s t h e s i s as conforming to the r e g u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA August 1985 (c) Leanne Embree, 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date £<3 /$->S - i i -ABSTRACT Human c h o r i o n i c g o n a d o t r o p i n (HCG), a g l y c o p r o t e i n hormone w i t h two n o n i d e n t i c a l s u b u n i t s , i s produced by c h o r i o n i c t i s s u e i n pregnant women and by n e o p l a s t i c t i s s u e c o n t a i n i n g c h o r i o n i c e l e m e n t s . I t i s used i n the t r e a t m e n t of male hypogonadism and female s u b - f e r t i l i t y . Q u a n t i t a t i o n of HCG i s used t o monitor t h e r a p y , d i a g n o s e v a r i o u s d i s e a s e s t a t e s and diag n o s e and monitor pregnancy. Low l e v e l s of HCG i n the e a r l y and l a t e s t a g e s of pregnancy and i n v a r i o u s d i s e a s e s t a t e s has prompted the development of e x t r e m e l y s e n s i t i v e assay p r o c e d u r e s . C l i n i c a l l y , radioimmunoassay methods are most f r e q u e n t l y used due t o t h e i r p r e c i s i o n , s e n s i t i v i t y and c o s t . However, problems w i t h s p e c i f i c i t y have been no t e d . Commercial p r e p a r a t i o n s of HCG must meet the s t a n d a r d s o u t l i n e d i n the U n i t e d S t a t e s Pharmacopeia (USP). The assay p r o c e d u r e i n v o l v e s a r a t u t e r i n e w e i g h t b i o a s s a y . T h i s p r o t o c o l i s l e n g t h y t o pe r f o r m (5 d a y s ) , r e q u i r e s the s a c r i f i c e of a l a r g e number of a n i m a l s (minimum of 60 female r a t s per assay) and may need t o be r e p e a t e d i f the r e s u l t s do not meet the s t a t i s t i c a l r e q u i r e m e n t s of the as s a y . Due t o the use of a n i m a l s and the an i m a l c a r e f a c i l i t i e s r e q u i r e d , t h i s i s an e x p e n s i v e a s s a y . I n a d d i t i o n , the b i o a s s a y i s not s p e c i f i c f o r HCG. T h e r e f o r e , t h i s t h e s i s - i i i -r e p o r t s the a n a l y s i s of two commercial p r e p a r a t i o n s of HCG, as w e l l as USP Refe r e n c e S t a n d a r d HCG and c o m m e r c i a l l y a v a i l a b l e p u r i f i e d i n t a c t HCG and p u r i f i e d i n d i v i d u a l s u b u n i t s . V a r i o u s HPLC assay p r o c e d u r e s were e v a l u a t e d t o determine i f HPLC would be a v i a b l e a l t e r n a t i v e t o the o f f i c i a l USP b i o a s s a y . S i z e e x c l u s i o n HPLC, u s i n g one P r o t e i n Pak 125 sw column and two P r o t e i n Pak 300 sw columns i n d i v i d u a l l y and i n v a r i o u s c o m b i n a t i o n s , was used t o a s s e s s a l l the samples of HCG. Attempts t o i n c r e a s e r e s o l u t i o n of HCG from i n t e r f e r i n g components found i n the s e p r e p a r a t i o n s i n c l u d e d u s i n g b o t h 208 nm and 278 nm f o r u l t r a v i o l e t d e t e c t i o n , e v a l u a t i o n of 32 b u f f e r s as mobile phases w i t h the P r o t e i n Pak 300 sw column, f l u o r e s c a m i n e d e r i v a t i z a t i o n of HCG f o l l o w e d by f l u o r e s c e n c e d e t e c t i o n , c o n n e c t i o n of two s i z e e x c l u s i o n columns i n s e r i e s , and r e c y c l i n g on a P r o t e i n Pak 300 sw column. F u r t h e r a t t e m p t s t o i s o l a t e HCG from i t s p r o t e i n c o n t a m i n a n t s i n v o l v e d u s i n g i o n exchange HPLC w i t h a P r o t e i n Pak DEAE 5 pw column w i t h 20 d i f f e r e n t b u f f e r s as mobile phases as w e l l as re v e r s e d - p h a s e HPLC w i t h an U l t r a s p h e r e ODS column. The g r e a t e s t r e s o l u t i o n was o b t a i n e d w i t h one P r o t e i n Pak 300 sw column w i t h a phosphate b u f f e r (0.15 M, pH 7.0) f o r t h e m o b i l e phase and u l t r a v i o l e t d e t e c t i o n . L a t e x a g g l u t i n a t i o n i n h i b i t i o n s l i d e t e s t s and - i v -e l e c t r o p h o r e s i s t e c h n i q u e s were used t o e v a l u a t e commercial samples of HCG and c h r o m a t o g r a p h i c peak e l u a t e s . Commercial HCG samples appear t o c o n t a i n the i n d i v i d u a l s u b u n i t s of HCG and i n t a c t HCG a l o n g w i t h i m p u r i t i e s . The USP R e f e r e n c e S t a n d a r d HCG c o n t a i n s i n t a c t HCG but a l s o c o n t a i n s o t h e r u l t r a v i o l e t a b s o r b i n g components t h a t were p a r t i a l l y s e p a r a t e d by HPLC. E l e c t r o p h o r e s i s a l s o i n d i c a t e d t h a t t h i s HCG sample c o n t a i n e d i m p u r i t i e s . I n a d d i t i o n , the p u r i f i e d i n t a c t HCG and p u r i f i e d s u b u n i t samples c o n t a i n e d i m p u r i t i e s , as shown by HPLC. The s i z e e x c l u s i o n HPLC assay d e v e l o p e d u s i n g one P r o t e i n Pak 300 sw column was unable t o r e s o l v e i n t a c t HCG from the b e t a - s u b u n i t . T h i s assay would be u s e f u l f o r a q u a l i t a t i v e assay f o r p u r i t y of HCG p r e p a r a t i o n s . However, a t p r e s e n t , HPLC i s not a v i a b l e a l t e r n a t i v e t o the USP b i o a s s a y . -v-TABLE OF CONTENTS Chapter Page ABSTRACT i i LIST OF TABLES x i LIST OF FIGURES x i i SYMBOLS AND ABBREVIATIONS X V ACKNOWLEDGEMENT x v i i i INTRODUCTION 1 1. LITERATURE SURVEY 4 1.1 G o n a d o t r o p i c Hormones 4 1.2 Human C h o r i o n i c G o n a d o t r o p i n 4 1.2.1 P h y s i o l o g i c a l F u n c t i o n s o f HCG 6 1.2.2 Sources of HCG ' 8 1.2.3 Uses of HCG 9 1.3 C h e m i s t r y of HCG 11 1.3.1 Alp h a and Be t a S u b u n i t s o f HCG 11 1.3.2 Ca r b o h y d r a t e Content 17 1.3.3 S t r u c t u r e A c t i v i t y R e l a t i o n s h i p s For HCG 20 1.3.4 D i s u l f i d e Bonds of HCG 26 1.3.5 Immunochemistry 28 1.3.6 B i o s y n t h e s i s of HCG 33 1.4 P h a r m a c o k i n e t i c s of HCG and i t s S u b u n i t s 34 1.5 P h y s i c o c h e m i c a l P r o p e r t i e s of HCG 37 1.6 Methods of S e p a r a t i o n And A n a l y s i s of HCG 39 1.6.1 Immunoassays 41 - v i -C hapter Page 1.6.2 E l e c t r o p h o r e s i s 46 1.6.3 B i o l o g i c a l Assays 50 1.6.4 Chromatographic Methods 52 1.6.4.1 Column Chromatography o f HCG 53 1.6.4.2 HPLC of HCG 55 2. EXPERIMENTAL 62 2.1 S u p p l i e s 62 2.1.1 E l e c t r o p h o r e s i s 62 2.1.2 High Performance L i q u i d Chromatography 6 2 2.1.3 Human C h o r i o n i c G o n a d o t r o p i n 63 2.1.4 M i s c e l l a n e o u s 63 2.2 Equipment 64 2.2.1 E l e c t r o p h o r e s i s 64 2.2.2 High Performance L i q u i d Chromatography 65 2.3 F l u o r e s c a m i n e D e r i v a t i z a t i o n of HCG 65 2.4 Recombination of HCG S u b u n i t s 66 2.5 D i s s o c i a t i o n of I n t a c t HCG 66 2.6 P r e p a r a t i o n of Reagents and Stock S o l u t i o n s 66 2.6.1 H y d r o c h l o r i c A c i d (1.0 M) 66 2.6.2 Sodium Hydroxide (0.1 M) 67 2.6.3 G l a c i a l A c e t i c A c i d (0.1 M) 67 2.6.4 Urea S o l u t i o n (10 M) 67 2.6.5 Sodium Phosphate B u f f e r (0.02 M) With Urea (8 M) 67 2.6.6 Sodium Phosphate B u f f e r (0.02 M) ' 68 2.6.7 Sodium Dodecyl S u l f a t e -P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s 68 - v i i -C h apter Page 2.6.7.1 A c r y l a m i d e Stock S o l u t i o n 68 2.6.7.2 Ammonium P e r s u l f a t e S o l u t i o n 68 2.6.7.3 G e l B u f f e r S o l u t i o n 69 2.6.7.4 R e s e r v o i r B u f f e r 69 2.6.7.5 SDS-PAGE Working S o l u t i o n (10% A c r y l a m i d e ) 69 2.6.8 D i s c o n t i n u o u s P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s 69 2.6.8.1 S e p a r a t i n g G e l B u f f e r S o l u t i o n 69 2.6.8.2 S e p a r a t i n g G e l A c r y l a m i d e S o l u t i o n 70 2.6.8.3 S e p a r a t i n g G e l Ammonium P e r s u l f a t e S o l u t i o n 70 2.6.8.4 S e p a r a t i n g Gel Working S o l u t i o n (7.5% and 10% Ac r y l a m i d e ) 70 2.6.8.5 S e p a r a t i n g Gel P r e l i m i n a r y E l e c t r o p h o r e s i s B u f f e r 71 2.6.8.6 S t a c k i n g G e l B u f f e r 71 2.6.8.7 S t a c k i n g G e l A c r y l a m i d e S o l u t i o n 71 2.6.8.8 S t a c k i n g G e l R i b o f l a v i n S o l u t i o n 72 2.6.8.9 S t a c k i n g G e l Sucrose S o l u t i o n 72 2.6.8.10 S t a c k i n g G e l Working S o l u t i o n (2.5% A c r y l a m i d e ) 72 2.6.8.11 DISC-PAGE R e s e r v o i r B u f f e r 72 2.6.9 S t a i n For E l e c t r o p h o r e s i s 73 2.6.10 D e s t a i n For E l e c t r o p h o r e s i s 73 2.7 HPLC M o b i l e Phase P r e p a r a t i o n 73 2.7.1 Sodium S u l f a t e Sodium Phosphate Monobasic (0.1 M/0.02M) 73 2.7.2 Sodium Phosphate B u f f e r (0.01 M) 74 2.7.3 Sodium Phosphate B u f f e r (0.075 M) 74 2.7.4 Sodium Phosphate B u f f e r (0.10 M) 74 2.7.5 Sodium Phosphate B u f f e r (0.15 M) 75 - v i i i -C h apter Page 2.7.6 Sodium Phosphate B u f f e r (0.15 M) With Sodium C h l o r i d e 75 2.7.7 Sodium Phosphate B u f f e r (0.15 M) With EDTA (0.001 M) 75 2.7.8 Sodium Phosphate B u f f e r (0.15 M) Wit h Sodium C h l o r i d e (0.1 M) And EDTA (0.001 M) 76 2.7.9 Sodium Phosphate B u f f e r (0.15 M) With Ammonium C h l o r i d e 76 2.7.10 Sodium Phosphate B u f f e r (0.15 M) With P o t a s s i u m C h l o r i d e 76 2.7.11 Sodium Phosphate B u f f e r (0.15 M) With T r i e t h y l a m i n e (0.05 M) 77 2.7.12 Sodium Phosphate B u f f e r (0.15 M) Wit h A c e t o n i t r i l e 77 2.7.13 Sodium Phosphate B u f f e r (0.15 M) With I s o p r o p a n o l 78 2.7.14 Sodium Phosphate B u f f e r (0.15 M) With Methanol 78 2.7.15 Sodium Phosphate B u f f e r (0.20 M) 78 2.7.16 Sodium Phosphate B u f f e r (0.30 M) 79 2.7.17 Sodium Phosphate B u f f e r (0.40 M) 79 2.7.18 Sodium A c e t a t e B u f f e r (0.1 M) 79 2.7.19 T r i s A c e t a t e B u f f e r (0.1 M) 79 2.7.20 T r i s C h l o r i d e B u f f e r (0.02 M) 80 2.7.21 Ammonium B i c a r b o n a t e B u f f e r (0.05 M) 80 2.7.22 Ammonium B i c a r b o n a t e B u f f e r (0.1 M) 80 2.7.23 Ammonium Phosphate Monobasic B u f f e r 80 (0.05 M) 2.7.24 T r i f l u o r o a c e t i c A c i d : A c e t o n i t r i l e : Water 81 2.8 Sodium Dodecyl S u l f a t e - P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s 81 2.8.1 Gel P r e p a r a t i o n 82 2.8.2 Sample P r e p a r a t i o n 82 2.8.3 E l e c t r o p h o r e s i s C o n d i t i o n s 83 2.8.4 S t a i n i n g Procedure 83 2.8.5 P r e p a r a t i o n of Chromatographic Peak E l u a t e f o r SDS-PAGE 83 2.9 D i s c o n t i n u o u s P o l y a c r y l a m i d e Gel E l e c t r o p h o r e s i s 84 - i x -C hapter Page 2.9.1 G e l P r e p a r a t i o n 84 2.9.2 Sample P r e p a r a t i o n 85 2.9.3 E l e c t r o p h o r e s i s C o n d i t i o n s 85 2.10 L a t e x A g g l u t i n a t i o n I n h i b i t i o n S l i d e T e s t s f o r HCG 86 2.10.1 P r e g n o s i s S l i d e T e s t 86 2.10.2 Pregnancy L a t e x S l i d e T e s t (Beta S u bunit) 86 2.11 High Performance L i q u i d Chromatography 87 3. R e s u l t s And D i s c u s s i o n 89 3.1 E l e c t r o p h o r e s i s Of Commercial HCG P r e p a r a t i o n s 89 3.1.1 Sodium Dodecyl S u l f a t e -P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s 89 3.1.2 D i s c o n t i n u o u s - P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s 90 3.2 USP R e f e r e n c e S t a n d a r d HCG 91 3.2.1 Sodium Dodecyl S u l f a t e -G e l E l e c t r o p h o r e s i s 91 3.2.2 D i s c o n t i n u o u s - P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s 92 3.3 P r e l i m i n a r y S t u d i e s W ith S i z e E x c l u s i o n Chromatography U s i n g The P r o t e i n Pak 300 sw HPLC Column 92 3.3.1 E v a l u a t i o n of P r o t e i n Pak 300 sw Column Performance 92 3.3.2 Commercial HCG P r e p a r a t i o n s 94 3.3.3 O p t i m i z a t i o n of M o b i l e Phase B u f f e r 97 3.3.4 Two P r o t e i n Pak 300 sw Columns Connected i n S e r i e s 103 3.4 S i z e E x c l u s i o n chromatography U s i n g A P r o t e i n Pak 125 sw Column 105 -x-Chapter Page 3.4.1 P r o t e i n Pak 125 sw Column 105 3.4.2 P r o t e i n Pak 300 sw And 125 sw Columns Connected i n S e r i e s 107 3.5 Ion Exchange Chromatography 107 3.6 Reversed-Phase Chromatography 110 3.7 F l u o r e s c e n c e D e t e c t i o n Of The F l u o r e s c a m i n e D e r i v a t i v e Of HCG 113 3.8 L o c a t i o n Of I n t a c t HCG And I t s S u b u n i t s 113 . 3.8.1 USP Refer e n c e S t a n d a r d HCG 119 3.8.2 P u r i f i e d S u b u n i t s 119 3.8.3 P u r i f i e d I n t a c t HCG 123 3.9 Comparison Of P u r i f i e d And Commercial P r e p a r a t i o n s 126 3.10 R e c y c l e S i z e E x c l u s i o n HPLC 126 3.11 Recombination Of S u b u n i t s And D i s s o c i a t i o n Of I n t a c t HCG 130 4. Summary And C o n c l u s i o n s 133 5. R e f e r e n c e s 137 - x i -L I S T OF TABLES Table Page V a r i o u s B u f f e r s Used as M o b i l e Phase W i t h the P r o t e i n Pak 300 sw Column 100 I I V a r i o u s B u f f e r s Used as M o b i l e Phase With the P r o t e i n Pak DEAE 5 pw Column 109 - x i i -LIST OF FIGURES F i g u r e Page 1 The Amino-Acid Sequence of the A l p h a -S u b u n i t of HCG 13 2 The Amino-Acid Sequence of the B e t a -S u b u n i t of HCG 15 3 The S t r u c t u r e s of the N - G l y c o s i d i c And O - G l y c o s i d i c C a r b o h y d r a t e U n i t s of HCG 19 4 Chromatogram of G e l F i l t r a t i o n S t a n d a r d 93 5 Chromatogram of HCG ( A y e r s t L a b o r a t o r i e s ) On P r o t e i n Pak 300 sw Column W i t h D e t e c t i o n a t 280 nm 95 6 Chromatogram of HCG (K-Line P h a r m a c e u t i c a l s ) On P r o t e i n Pak 300 sw Column With D e t e c t i o n a t 280 nm 96 7 Chromatogram of HCG ( A y e r s t L a b o r a t o r i e s ) On A P r o t e i n Pak 300 sw Column W i t h D e t e c t i o n a t 208 nm 98 8 Chromatogram of HCG ( A y e r s t L a b o r a t o r i e s ) On A P r o t e i n Pak 300 sw Column W i t h M o b i l e Phase of Optimum pH 101 9 Chromatogram of HCG ( A y e r s t L a b o r a t o r i e s ) On A P r o t e i n Pak 300 sw Column W i t h Optimum M o b i l e Phase 102 10 Chromatogram of HCG ( A y e r s t L a b o r a t o r i e s ) On Two P r o t e i n Pak 300 sw Columns Connected In S e r i e s With 0.01 M Sodium Phosphate B u f f e r As M o b i l e Phase 104 11 Chromatogram of HCG ( A y e r s t L a b o r a t o r i e s ) On Two P r o t e i n Pak 300 sw Columns Connected In S e r i e s W i t h 0.15 M Phosphate B u f f e r as M o b i l e Phase 106 - x i i i -F i g u r e Page 12 Chromatogram of HCG ( A y e r s t L a b o r a t o r i e s ) On A P r o t e i n Pak 125 sw Column With D e t e c t i o n a t 208 nm 108 13 Chromatogram of HCG ( A y e r s t L a b o r a t o r i e s ) On A P r o t e i n Pak DEAE 5 pw Column 111 14 Chromatogram of USP R e f e r e n c e S t a n d a r d HCG On An U l t r a s p h e r e ODS Column 112 15 Chromatogram of P u r i f i e d I n t a c t HCG On An U l t r a s p h e r e ODS Column 114 16 Chromatogram of F l u o r e s c a m i n e D e r i v a t i v e Of HCG ( A y e r s t L a b o r a t o r i e s ) On A P r o t e i n Pak 300 sw Column W i t h F l u o r e s c e n c e D e t e c t i o n 115 17 Chromatogram of F l u o r e s c a m i n e D e r i v a t i v e Of HCG ( A y e r s t L a b o r a t o r i e s ) On Two P r o t e i n Pak 300 sw Columns Connected In S e r i e s With F l u o r e s c e n c e D e t e c t i o n 116 18 Chromatogram of F l u o r e s c a m i n e D e r i v a t i v e Of HCG ( A y e r s t L a b o r a t o r i e s ) On P r o t e i n Pak 125 sw And 300 sw Columns Connected In S e r i e s 117 19 Chromatogram of F l u o r e s c a m i n e D e r i v a t i v e Of HCG ( A y e r s t L a b o r a t o r i e s ) On A P r o t e i n Pak 125 sw Column 118 20 Chromatogram of USP Refe r e n c e S t a n d a r d HCG On A P r o t e i n Pak 300 sw Column W i t h D e t e c t i o n At 280 nm 120 21 Chromatogram of USP Refe r e n c e S t a n d a r d HCG On A P r o t e i n Pak 300 sw Column W i t h D e t e c t i o n At 208 nm 121 22 Chromatogram of P u r i f i e d HCG-Beta On A P r o t e i n Pak 300 sw Column W i t h D e t e c t i o n a t 280 nm 122 - x i v -F i g u r e Page 23 Chromatogram of P u r i f i e d HCG-Alpha On A P r o t e i n Pak 300 sw Column W i t h D e t e c t i o n a t 280 nm 124 24 Chromatogram of P u r i f i e d HCG On A P r o t e i n Pak 300 sw Column W i t h D e t e c t i o n a t 280 nm 125 25 Chromatogram of P u r i f i e d A l p h a -And B e t a - S u b u n i t s of HCG 127 26 Chromatogram of USP Refe r e n c e S t a n d a r d HCG And The P u r i f i e d A l p h a - And B e t a -S u b u n i t s 128 27 Chromatogram of A y e r s t L a b o r a t o r i e s And USP R e f e r e n c e S t a n d a r d HCG On Two P r o t e i n Pak 300 sw Columns Connected In S e r i e s 129 28 Chromatogram o f HCG ( A y e r s t L a b o r a t o r i e s ) A f t e r R e c y c l i n g S i x Times On A P r o t e i n Pak 300 sw Column 131 - X V -S Y M B O L S A N D A B B R E V I A T I O N S A l a A l a n i n e AMP Adenosine 5'-Monophosphate Arg A r g i n i n e Asn A s p a r a g i n e Asp A s p a r t i c A c i d B i s -a c r y l a m i d e N , N ' - m e t h y l e n e - b i s - a e r y l a m i d e CD C i r c u l a r D i c h r o i s m CFSH C h o r i o n i c F o l l i c l e - S t i m u l a t i n g Hormone CG C h o r i o n i c G o n a d o t r o p i n CHO Carb o h y d r a t e CM Carboxymethyl CPS Compendium Of P h a r m a c e u t i c a l s And S p e c i a l t i e s C - t e r m i n a l C a r b o x y - t e r m i n a l Cys C y s t e i n e Da D a l t o n DEAE D i e t h y l a m i n o e t h y 1 DISC D i s c o n t i n u o u s DNP 2 , 4 - D i n i t r o b e n z e n e s u l f o n i c A c i d DTT D i t h i o t h r e i t o l EDTA E t h y l e n e d i a m i n e t e t r a a c e t i c A c i d FITC F l u o r e s c e i n I s o t h i o c y a n a t e FSH F o l l i c l e - S t i m u l a t i n g Hormone - x v i -Fuc Fucose Gal G a l a c t o s e GalNAc N - A c e t y l g a l a c t o s a m i n e G i n Glutamine G l u G l u t a m i c A c i d GluNAc N - A c e t y l g l u c o s a m i n e G l y G l y c i n e GMP Guanosine 5'-Monophosphate HCG Human C h o r i o n i c G o n a d o t r o p i n HCG-alpha A l p h a - s u b u n i t o f HCG HCG-beta B e t a - s u b u n i t of HCG H i s H i s t i d i n e HPLC High-Performance L i q u i d Chromatography ICSH I n t e r s t i t i a l C e l l - S t i m u l a t i n g Hormone, L u t e i n i z i n g Hormone IEC Ion Exchange Chromatography IEF I s o e l e c t r i c F o c u s i n g l i e I s o l e u c i n e IU I n t e r n a t i o n a l U n i t Leu L e u c i n e LH L u t e i n i z i n g Hormone, I n t e r s t i t i a l C e l l - S t i m u l a t i n g Hormone Lys L y s i n e Man Mannose Met M e t h i o n i n e M Molar - x v i i -NANA N - A c e t y l n e u r a m i n i c A c i d OAAD O v a r i a n A s c o r b i c A c i d D e p l e t i o n ODS O c t a d e c y l s i l y l PAGE P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s Phe P h e n y l a l a n i n e p i I s o e l e c t r i c P o i n t Pro P r o l i n e PTFE P o l y t e t r a f l u o r o e t h y l e n e RIA Radioimmunoassay RRA R a d i o r e c e p t o r Assay SDS Sodium Dodecyl S u l f a t e SEC S i z e E x c l u s i o n Chromatography SE S u l p h o e t h y l Ser S e r i n e SP S u l p h o p r o p y l SPIA S o l P a r t i c l e Immunoassay TEMED N,N,N',N'-Tetramethylethylenediamine Thr Threonine T r i s T ris(hydroxymethy1)aminomethane TSH T h y r o i d - S t i m u l a t i n g Hormone Tyr T y r o s i n e USP U n i t e d S t a t e s Pharmacopeia V a l V a l i n e VPW V e n t r a l P r o s t a t e Weight - x v i i i -ACKNOWLEDGEMENT I w i s h t o thank Dr. K e i t h McErlane f o r h i s v a l u a b l e guidance t h r o u g h o u t the c o u r s e of t h i s s t u d y . H i s c o n s t a n t encouragement and k i n d n e s s i s g r e a t l y a p p r e c i a t e d . I am g r a t e f u l t o the members of my graduate committee, Dr s . Frank A b b o t t , John Brown, He l e n B u r t , and James O r r f o r t h e i r h e l p f u l a d v i c e . F i n a n c i a l s u p p o r t from the Canadian H e a r t F o u n d a t i o n , U n i v e r s i t y of B r i t i s h Columbia SPCA Animal A l t e r n a t i v e s Committee, and the U n i v e r s i t y Summer Graduate F e l l o w s h i p Committee i s g r a t e f u l l y acknowledged. Above a l l , I would l i k e t o thank my husband f o r h i s d e v o t i o n and encouragement. - 1 -INTRODUCTION Human c h o r i o n i c g o n a d o t r o p i n (HCG) i s a g l y c o p r o t e i n hormone w i t h two n o n i d e n t i c a l s u b u n i t s t h a t i s produced by p l a c e n t a l t i s s u e i n pregnant women and p a t i e n t s w i t h some t r o p h o b l a s t i c and n o n t r o p h o b l a s t i c d i s e a s e s . An immunosuppressive mechanism of a c t i o n has been s u g g e s t e d f o r HCG i n bo t h pregnancy and d i s e a s e . C o m m e r c i a l l y , HCG i s a v a i l a b l e as an i n t r a m u s c u l a r i n j e c t i o n f o r t h e t r e a t m e n t of male hypogonadism and female s u b - f e r t i l i t y . Methods f o r i s o l a t i o n of HCG r e p o r t e d i n the l i t e r a t u r e i n c l u d e e l e c t r o p h o r e t i c and c h r o m a t o g r a p h i c t e c h n i q u e s . Immunoassays and b i o a s s a y s f o r q u a n t i t a t i o n of HCG are used t o mon i t o r t h e r a p y , d i a g n o s e v a r i o u s d i s e a s e s t a t e s and diag n o s e and mon i t o r pregnancy. The o f f i c i a l a s say r e q u i r e d f o r p r e p a r a t i o n s of HCG i s t h a t c o n t a i n e d i n the U n i t e d S t a t e s Pharmacopeia (USP). T h i s i s a r a t u t e r i n e w e i g h t b i o a s s a y which r e q u i r e s the s a c r i f i c e of a l a r g e number of a n i m a l s (minimum of 60 female r a t s per a s s a y ) , t a k e s a l o n g time (5 days) t o p e r f o r m and may need t o be r e p e a t e d i f the r e s u l t s do n o t meet the s t a t i s t i c a l r e q u i r e m e n t s of t h e a s s a y . A l s o , the use of a n i m a l s and the n e c e s s a r y a n i m a l c a r e f a c i l i t i e s make t h i s an e x p e n s i v e a s s a y . S o p h i s t i c a t e d c h r o m a t o g r a p h i c assay p r o c e d u r e s have been d e v e l o p e d t o r e p l a c e l e s s r e l i a b l e b i o l o g i c a l a s s a y s -2-p r e v i o u s l y used by the USP. For d e t e r m i n i n g p o t e n c y , h i g h -performance l i q u i d chromatography (HPLC) was a c t u a l l y found to be more p r e c i s e than the b i o l o g i c a l a s s a y s f o r i n s u l i n and a number of a n t i b i o t i c s (Dennis 1984, G i l p i n 1983, Johnson 1983). Moreover, many o f f i c i a l a n t i b i o t i c a ssay p r o c e d u r e s have been changed from b a c t e r i o l o g i c a l p r o c e d u r e s t o gas- or h i g h p e r f o r m a n c e - l i q u i d c h r o m a t o g r a p h i c assay methods (USP 1980) . C o n t i n u i n g t h i s t r e n d i n d e v e l o p i n g s p e c i f i c , s e n s i t i v e , r a p i d and r e l a t i v e l y i n e x p e n s i v e c h r o m a t o g r a p h i c assay p r o c e d u r e s f o r compounds p r e s e n t l y b e i n g d e t e r m i n e d by b i o l o g i c a l a s s a y s , many r e p o r t s on HPLC a n a l y s i s of g l y c o p r o t e i n s (Borrebaeck 1984, C l a r k 1984, Dua 1984, F l a s h n e r 1983, Hearn 1982, Lambotte 1984, M e l l i s 1983) i n c l u d i n g g l y c o p r o t e i n hormones such as HCG ( F o r a s t i e r i 1982, Moudgal 1982, Parsons 1984, Putterman 1982, S h i m o h i g a s h i 1982; 1983) have r e c e n t l y appeared i n the l i t e r a t u r e . The s p e c i f i c aims of t h i s p r o j e c t were as f o l l o w s : 1. t o d e v e l o p an HPLC a n a l y t i c a l p r o c e d u r e s u i t a b l e f o r r e p l a c i n g the o f f i c i a l USP b i o a s s a y f o r a n a l y s i s of commercial HCG p r e p a r a t i o n s . 2. t o e v a l u a t e commercial p r e p a r a t i o n s of HCG and the USP R e f e r e n c e S t a n d a r d HCG by b o t h sodium d o d e c y l s u l f a t e and d i s c o n t i n u o u s p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s t e c h n i q u e s . - 3 -3 . t o c o n f i r m the l o c a t i o n of HCG and the i n d i v i d u a l s u b u n i t s of HCG by e v a l u a t i o n of p u r i f i e d i n t a c t HCG, HCG-alpha and HCG-beta and by a n a l y s i s of c h r o m a t o g r a p h i c peak e l u a t e w i t h the e l e c t r o p h o r e s i s t e c h n i q u e s and l a t e x a g g l u t i n a t i o n i n h i b i t i o n s l i d e t e s t s . - 4 -1. LITERATURE SURVEY 1.1 G o n a d o t r o p i c Hormones G o n a d o t r o p i c hormones are g l y c o p r o t e i n s w i t h n o n i d e n t i c a l s u b u n i t s t h a t are produced by the a n t e r i o r p i t u i t a r y and p l a c e n t a . They i n c l u d e f o l l i c l e - s t i m u l a t i n g hormone (FSH) and l u t e i n i z i n g hormone (LH or i n t e r s i t i t i a l c e l l - s t i m u l a t i n g hormone, ICSH) from the p i t u i t a r y , and c h o r i o n i c g o n a d o t r o p i n (CG) and c h o r i o n i c f o l l i c l e - s t i m u l a t i n g hormone (CFSH) from the p l a c e n t a (Murad 1980, Turner 1971). These g o n a d o t r o p i n s have a s t i m u l a t o r y e f f e c t on bo t h male and female gonads. In the male, LH f u n c t i o n s t o s t i m u l a t e the i n t e r s t i t i a l c e l l s of the t e s t i s ( L e y d i g c e l l s ) t o produce androgen (Turner 1971) and FSH .promotes spe r m a t o g e n e s i s by a c t i n g d i r e c t l y upon the s e m i n i f e r o u s t u b u l e s (Turner 1971). F o l l i c l e - s t i m u l a t i n g hormone and LH, i n the f e m a l e , a c t s y n e r g i s t i c a l l y t o promote e s t r o g e n s e c r e t i o n by f o l l i c l e s u n d e r g o i n g m a t u r a t i o n and t o cause o v u l a t i o n (Turner 1971). 1.2 Human Chorionic G o n a d o t r o p i n - 5 -A l t h o u g h human c h o r i o n i c g o n a d o t r o p i n (HCG) i s found n o r m a l l y o n l y i n pregnant women, i t produces a g r e a t v a r i e t y of gonadal a c t i o n s (Turner 1971) . M i n i m a l gonadal s t i m u l a t i o n of the male by HCG o c c u r s i n u t e r o . A f t e r b i r t h , HCG i s o n l y found i n males w i t h a t e r a t o m a t o u s tumor c o n t a i n i n g c h o r i o n i c elements (Murad 1980). I n j e c t i o n of HCG i n men causes d i f f e r e n t i a t i o n of L e y d i g c e l l s and s t i m u l a t e s and m a i n t a i n s s e c r e t i o n of t e s t i c u l a r androgens (Murad 1980, Turner 1971). Females w i t h t r o p h o b l a s t i c tumors may a l s o produce HCG. In t h e absence of t h e s e tumors, HCG has a s t r o n g l u t e o t r o p i c f u n c t i o n and i s i m p o r t a n t f o r m a i n t a i n i n g the f u n c t i o n of the corpus luteum d u r i n g the e a r l y s tage of pregnancy (Tojo 1982). The mechanism f o r the l u t e o t r o p i c e f f e c t of HCG can be d e s c r i b e d as f o l l o w s (Tojo 1982) : HCG b i n d s membrane r e c e p t o r s of g r a n u l o s a l u t e i n c e l l s and s t i m u l a t e s a d e n y l a t e c y c l a s e a c t i v i t y , promotes c y c l i c AMP-dependent p r o t e i n k i n a s e , and i n d u c e s o v a r i a n m e t a b o l i c changes ( e s p e c i a l l y g l y c o l y s i s ) and s t e r o i d o g e n e s i s t o produce and s e c r e t e p r o g e s t e r o n e from g r a n u l o s a l u t e i n c e l l s i n t o the m a t e r n a l c i r c u l a t i o n . As mentioned above, t r o p h o b l a s t i c tumors a l s o produce HCG and i t s s u b u n i t s (Murad 1980, Tojo 1982). Thus, i t i s t h o u g h t t h a t t h e p r i m a r y r o l e of HCG i s t o s t o p r e j e c t i o n of the f e t u s i n the i n i t i a l s t age of pregnancy (Mangano 1983) - 6 -a f t e r w h i c h i t i s s u b s t i t u t e d by o t h e r f a c t o r s w i t h the same f u n c t i o n . The HCG produced by tumors may p l a y a s i m i l a r immunosuppressive r o l e . In s t u d y i n g the i n v i v o a c t i o n of anti-HCG a n t i b o d i e s on n e o p l a s t i c t i s s u e , Mango (1983) found a s l o w i n g of tumor growth and a l o n g e r s u r v i v a l time f o r the a n i m a l s . The mechanism of a c t i o n of the anti-HCG a n t i b o d i e s may be t o s t o p the immunosuppressive a c t i o n of HCG s e c r e t e d by the n e o p l a s t i c c e l l s or t o c o n t r o l the tumor's growth by i n f l u e n c e on the e n d o c r i n e o r immunocompetent systems (Mangano 1983). B a r t o c c i (1983) showed t h a t the HCG m o l e c u l e i t s e l f e x h i b i t s immunosuppressive a c t i v i t y _in v i v o i n b o t h male and female mice. Removal of the gonads ( B a r t o c c i 1983) c o m p l e t e l y d e p l e t e d t h i s immunosuppressive a c t i v i t y . T h i s s u g gests the immunosuppressive a c t i o n of HCG i s mediated by a f a c t o r r e l e a s e d from t h e gonads ( B a r t o c c i 1983) . R e c e n t l y i t was r e p o r t e d t h a t HCG a l s o has an i n v i v o and i n v i t r o growth promoting e f f e c t on murine tumor c e l l s (Mukherjee 1984). T h i s g i v e s f u r t h e r s u p p o r t t o and e x p l a i n s p r e v i o u s o b s e r v a t i o n s t h a t the presence of HCG i s r e l a t e d t o poor p r o g n o s i s i n ca n c e r (Mukherjee 1984). 1.2.1 P h y s i o l o g i c a l F u n c t i o n s of HCG D u r i n g e a r l y pregnancy, s y n t h e s i z e d HCG i s s e c r e t e d by -7-the c h o r i o n i c v i l l i of the p l a c e n t a and appears i n the mat e r n a l b l o o d and u r i n e (Turner 1971). Serum HCG l e v e l s s t a r t t o i n c r e a s e a t the end o f the f i r s t month of pregnancy and r e a c h a maximum c o n c e n t r a t i o n of 40 t o 45 I n t e r n a t i o n a l U n i t s per mL d u r i n g the t h i r d month ( B r a u n s t e i n 1976). Human c h o r i o n i c g o n a d o t r o p i n appears i n the u r i n e and reaches maximum c o n c e n t r a t i o n i n the f i r s t t r i m e s t e r . Hyperemesis g r a v i d a r u m has been a s s o c i a t e d w i t h i n c r e a s e d c o n c e n t r a t i o n s of HCG i n bo t h b l o o d and u r i n e , s u g g e s t i n g t h a t i n c r e a s e d t r o p h o b l a s t i c a c t i v i t y might p l a y a r o l e i n t h i s c o n d i t i o n ( K a u p p i l a 1979). More r e c e n t f i n d i n g s ( K a u p i l a 1984) o f i n c r e a s e d c o n c e n t r a t i o n s of HCG i n p a t i e n t s w i t h v o m i t i n g i n e a r l y pregnancy s u p p o r t s t h i s h y p o t h e s i s . By t h e end of the f i r s t t r i m e s t e r , u r i n a r y HCG drops t o low l e v e l s and remains f a i r l y c o n s t a n t u n t i l a few days a f t e r p a r t u r i t i o n . The serum l e v e l s a l s o d e c r e a s e a f t e r the end o f the f i r s t t r i m e s t e r and remain a t around 10 I n t e r n a t i o n a l U n i t s per mL u n t i l p a r t u r i t i o n ( B r a u n s t e i n 1976). Human c h o r i o n i c g o n a d o t r o p i n resembles LH as i t c o n v e r t s the corpus luteum of the m e n s t r u a l c y c l e i n t o the corpus luteum of pregnancy. T h i s p r o l o n g s the l u t e a l p r o d u c t i o n of e s t r o g e n and p r e g n a n e d i o l r e q u i r e d f o r the c o n t i n u a t i o n of pregnancy (Tojo 1982, Turner 1971). The p l a c e n t a l s e c r e t i o n of t h e s e s t e r o i d hormones i n c r e a s e s by -8-the end of the f i r s t t r i m e s t e r so t h a t t h e c o r p u s luteum and o v a r i e s a r e not r e q u i r e d f o r maintenance of g e s t a t i o n . A l o n g w i t h g o n a d o t r o p i c a c t i v i t y , HCG a l s o e x h i b i t s t h y r o i d - s t i m u l a t i n g a c t i v i t y . T h i s i n t r i n s i c a c t i v i t y of the HCG m o l e c u l e was found t o be q u a l i t a t i v e l y d i f f e r e n t from o t h e r t h y r o i d - s t i m u l a t i n g hormones ( N i s u l a 1980). For example, u s i n g a mouse t h y r o i d b i o a s s a y , the maximum response t o HCG was obser v e d a t 9 hours w h i l e t h a t f o r t h y r o i d - s t i m u l a t i n g hormone (TSH) and a l o n g a c t i n g t h y r o i d s t i m u l a t o r o c c u r e d a t 2 hours and 22 hours r e s p e c t i v e l y ( N i s u l a 1980). 1.2.2 Sources o f HCG N o r m a l l y HCG i s found o n l y i n pregnant women but some t r o p h o b l a s t i c d i s e a s e s and n o n t r o p h o b l a s t i c tumors a l s o cause i t s p r o d u c t i o n . With t r o p h o b l a s t i c d i s e a s e s such as c h o r i o c a r c i n o m a , the l e v e l s of HCG remain e l e v a t e d or r i s e a g a i n a f t e r t e r m i n a t i o n of pregnancy (Tojo 1982). U r i n e and/or serum c o n c e n t r a t i o n s of HCG are used t o d i a g n o s e and m o n i t o r t r e a t m e n t o f p a t i e n t s w i t h t h i s d i s e a s e . Das (1984) demonstrated t h a t the HCG and human c h o r i o n i c s o m a t o t r o p i n d e t e c t e d i n the s e r a of p a t i e n t s w i t h c e r v i c a l c a n c e r i s e c t o p i c a l l y produced by the tumor. A l s o , squamous c e l l c arcinoma cases were found t o g i v e the h i g h e s t i n c i d e n c e of - 9 -H C G - p o s i t i v e s e r a . T r o p h o b l a s t i c tumors t h a t produce l a r g e amounts of HCG can l e a d t o h y p e r t h y r o i d i s m due t o the t h y r o t r o p i c a c t i v i t y of HCG (Murad 1980). Measurement of HCG i s a l s o v a l u a b l e i n p r e d i c t i n g the p r o g n o s i s of d i s e a s e i n p a t i e n t s w i t h gonadal and non-gonadal tumors w h i c h produce HCG. These i n c l u d e tumors found i n the stomach, l i v e r , l u n g , k i d n e y , p a n c r e a s , ovary and t e s t i s ( K l o p p e l 1983, Tojo 1982). Thus HCG and/or i t s i n d i v i d u a l s u b u n i t s have been proposed as markers f o r v a r i o u s tumors. 1.2.3 Uses of HCG I n t r a m u s c u l a r i n j e c t i o n of HCG i s used i n the t r e a t m e n t of male hypogonadism and female s u b - f e r t i l i t y . An i n  v i v o s t u d y of t e s t o s t e r o n e p r o d u c t i o n w i t h b o t h r a t and human L e y d i g c e l l s demonstrated a r e f r a c t o r y p e r i o d between 24 and 72 hours a f t e r a s i n g l e i n j e c t i o n of HCG ( C a l v o 1984, H u h t a n i e m i 1983). T h i s has l e d t o the a d m i n i s t r a t i o n o f HCG over l o n g p e r i o d s t o s t i m u l a t e t e s t o s t e r o n e p r o d u c t i o n i n c l i n i c a l s i t u a t i o n s (Calvo 1984). Massive doses of HCG produces s t e r o i d o g e n i c d e s e n s i t i z a t i o n of L e y d i g c e l l s (Smals 1984) which l a s t s a t l e a s t 48 h o u r s , so an a p p r o p r i a t e c l i n i c a l dosage f o r HCG i n the male must be used. In man, r e t e n t i o n of the t e s t e s i n the abdominal c a v i t y ( c r y p t o r c h i s m ) may be due t o anatomic o b s t r u c t i o n or -10-c e r t a i n h y p o p h y s i a l d y s f u n c t i o n s (Turner 1971). Human c h o r i o n i c g o n a d o t r o p i n has been used i n the t r e a t m e n t of c r y p t o r c h i s m not r e q u i r i n g s u r g e r y a t a dosage of 4000 USP u n i t s t h r e e times weekly f o r two t o t h r e e weeks or 1000 USP u n i t s t h r e e times weekly f o r s i x t o e i g h t weeks (CPS 1983). The dosage s c h e d u l e may v a r y depending on the age of the p a t i e n t . Delayed a d o l e s c e n c e i n the male may a l s o be t r e a t e d by HCG, a t a dose of 4000 t o 5000 USP u n i t s t h r e e times weekly f o r s i x t o e i g h t weeks, t o s t i m u l a t e the i n t e r s t i t i a l c e l l s e c r e t i o n of androgen. T h i s dosage regimen has a l s o been used i n the t r e a t m e n t of h y p o g o n a d o t r o p i c e u n u c h o i d i s m and hypogonadism ( a f t e r s e x u a l m a t u r i t y ) i n the male. B e f o r e e p i p h y s e a l c l o s u r e , s t i m u l a t i o n of the i n t e r s t i t i a l c e l l s by HCG may a l s o be used i n t r e a t m e n t of p i t u i t a r y d w a r f i s m a t a dose of 1000 t o 5000 USP u n i t s t h r e e times weekly. In the fe m a l e , HCG i s a d m i n i s t e r e d d u r i n g the second phase of the m e n s t r u a l c y c l e t o m a i n t a i n the f u n c t i o n a l i n t e g r i t y of t h e co r p u s luteum and t o s t i m u l a t e p r o g e s t e r o n e s e c r e t i o n . Human c h o r i o n i c g o n a d o t r o p i n i s i n d i c a t e d f o r the t r e a t m e n t of h a b i t u a l a b o r t i o n a t a dosage of 1000 t o 2000 USP u n i t s one or more times d a i l y u n t i l the danger of a b o r t i o n has passed. Both f u n c t i o n a l s t e r i l i t y and i n f r e q u e n t s c a n t y b l e e d i n g d i s o r d e r s ( o l i g o m e n o r r h e a and amenorrhea) are i n d i c a t i o n s f o r HCG t r e a t m e n t . The -11-suggested dosage i s 500 t o 1000 USP u n i t s of HCG d a i l y from th e 1 5 t h t o the 24th day or 1500 USP u n i t s on the 1 6 t h , 18th and 20th day of the m e n s t r u a l c y c l e . 1.3 Chemistry of HCG Human c h o r i o n i c g o n a d o t r o p i n c o n s i s t s of a p e p t i d e backbone w i t h a t t a c h e d c a r b o h y d r a t e s i d e c h a i n s and i s s i m i l a r t o the g l y c o p r o t e i n g o n a d o t r o p i c hormones of p i t u i t a r y o r i g i n (Tyrey 1982) . The p e p t i d e backbone c o n s i s t s o f two n o n i d e n t i c a l s u b u n i t s t h a t a re u n i t e d n o n - c o v a l e n t l y by hydrogen bonding or charge i n t e r a c t i o n s s i n c e they r e a d i l y d i s s o c i a t e i n 8 M u r e a (Swaminathan 1970, Tyrey 1982) . The s u b u n i t s of HCG were d e s i g n a t e d a l p h a and b e t a . The a l p h a - s u b u n i t s of HCG, human LH, FSH and TSH (another g l y c o p r o t e i n hormone produced i n the a n t e r i o r p i t u i t a r y ) , a re e s s e n t i a l l y i d e n t i c a l (Morgan 1975, Sairam 1972; 1973, Shome 1974a; 1974b) w h i l e t h e b e t a - s u b u n i t s show s i g n i f i c a n t d i f f e r e n c e s . 1.3.1 Alpha and Beta S u b u n i t s o f HCG H e t e r o g e n e i t y of the a m i n o - a c i d t e r m i n a l of the a l p h a c h a i n v a r i e s t h e t o t a l number of a m i n o - a c i d r e s i d u e s from 89 -12-t o 92- The 92 a m i n o - a c i d sequence f o r the a l p h a - s u b u n i t shown i n F i g u r e 1 r e p r e s e n t s a p p r o x i m a t e l y 60% of the t o t a l (Morgan 197.5) . Around 30% d i d n o t c o n t a i n t h e i n i t i a l t h r e e a m i n o - t e r m i n a l r e s i d u e s w h i l e 10% l a c k e d o n l y the f i r s t two r e s i d u e s (Morgan 1975) . S t u d i e s on the b i o s y n t h e s i s of HCG i n d i c a t e t h a t t h i s h e t e r o g e n e i t y i s due t o d e g r a d a t i o n of the hormone i n plasma or u r i n e ( B i r k e n 1980) . D i f f e r e n c e s i n the a m i n o - a c i d sequences of the b e t a - s u b u n i t s of human g l y c o p r o t e i n hormones are thought t o g i v e the unique b i o l o g i c a l a c t i v i t i e s t o the i n t a c t hormone. Thus the b e t a - s u b u n i t i s c a l l e d the hormone s p e c i f i c s u b u n i t . D e s p i t e t h e s e i m p o r t a n t d i f f e r e n c e s , some homology i n s t r u c t u r e i s r e t a i n e d (Shome 1973). The b e t a - s u b u n i t s of human LH and HCG, which have s i m i l a r b i o l o g i c a l a c t i o n s , show g r e a t e r homologies i n t h e i r a m i n o - a c i d sequences than o t h e r g l y c o p r o t e i n hormones (Shome 1973, Tyrey 1982). The b e t a - s u b u n i t of human LH c o n s i s t s o f 115 a m i n o - a c i d r e s i d u e s of which 89 share i d e n t i c a l a m i n o - t e r m i n a l p o s i t i o n s i n KCG-beta (Shome 1973). However, HCG-beta has an a d d i t i o n a l 30 amino a c i d r e s i d u e s a t the c a r b o x y - t e r m i n a l t h a t a re not found i n the b e t a - s u b u n i t of human LH ( B i r k e n 1977, Shome 1973). Two p r i m a r y s t r u c t u r e s f o r HCG have been p u b l i s h e d ( B i r k e n 1977, C a r l s e n 1973, Morgan 1975). The p r o p o s a l by C a r l s e n (1973) d i f f e r s from o t h e r r e s u l t s i n the a m i n o - a c i d sequence o f the c a r b o x y l - t e r m i n u s o f the b e t a - s u b u n i t and i n -13-FIGURE 1 THE AMINO-ACID SEQUENCE OF THE ALPHA-SUBUNIT OF HCG 10 H 2 N - A l a - P r o - A s p - V a l - G i n - A s p - C y s - P r o - G l u - C y s - T h r - L e u - G l n -20 G l u - A s p - P r o - P h e - P h e - S e r - G l n - P r o - G l y - A l a - P r o - I l e - L e u -30 Gln- C y s - M e t - G l y - C y s - C y s - P h e - S e r - A r g - A l a - T y r - P r o - T h r -40 50 CHO Pro - L e u - A r g - S e r - L y s - L y s - T h r - M e t - L e u - V a l - G l n - L y s - A s n -60 V a l - T h r - S e r - G l u - S e r - T h r - C y s - C y s - V a l - A l a - L y s - S e r - T y r -70 CHO A s n - A r g - V a l - T h r - V a l - M e t - G l y - G l y - P h e - L y s - V a l - G l u - A s n -80 90 H i s - T h r - A l a - C y s - H i s - C y s - S e r - T h r - C y s - T h r - T h r - H i s - L y s -Ser-COOH The c a r b o h y d r a t e s i d e c h a i n s a r e a t t a c h e d t o a m i n o - a c i d r e s i d u e s i n d i c a t e d by CHO (Tyrey 1982) . -14-the s i t e s of c a r b o h y d r a t e attachment t o s e r i n e r e s i d u e s . These disa g r e e m e n t s have been r e s o l v e d and the a c c e p t e d a r n i n o - a c i d sequence of the b e t a - s u b u n i t i s shown i n F i g u r e 2. P o s t t r a n s l a t i o n a l m o d i f i c a t i o n of the c a r b o x y - t e r m i n a l r e g i o n of the b e t a - s u b u n i t has been obser v e d (Lentz 1984) b e f o r e t h i s mature form of the b e t a - s u b u n i t i s produced. M o l e c u l a r weight d e t e r m i n a t i o n s f o r HCG and i t s s u b u n i t s from normal p l a c e n t a l t i s s u e produce c o n f l i c t i n g r e s u l t s . The c a l c u l a t e d m o l e c u l a r w e i g h t f o r HCG i s a p p r o x i m a t e l y 37000 Da and t h a t of the a l p h a - s u b u n i t i s 15000 Da and 22000 Da f o r the b e t a - s u b u n i t (Tyrey 1982) . U s i n g e l e c t r o p h o r e s i s and s i z e e x c l u s i o n chromatography t o determine m o l e c u l a r w e i g h t , h i g h e r v a l u e s f o r HCG and the i n d i v i d u a l s u b u n i t s were found (Bahl 1969a, F e i n 1980, P a l a 1973) . V a i t u k a i t i s (1974) i d e n t i f i e d two immunoreactive s p e c i e s of the a l p h a - s u b u n i t i n p l a c e n t a l e x t r a c t s , one w i t h a m o l e c u l a r w e i g h t s i m i l a r t o u r i n a r y HCG-alpha, and a n o t h e r w i t h a l a r g e r m o l e c u l a r w e i g h t . The l a r g e form of HCG-alpha (29000 Da compared t o 22000 Da f o r u r i n a r y HCG-alpha de t e r m i n e d u s i n g Sephadex G-100) was a l s o found i n the s e r a of pregnant p a t i e n t s ( F e i n 1980) but not i n the media of c u l t u r e d normal p l a c e n t a (Maruo 1976). More r e c e n t l y P o s i l l i c o (1983) o b s e r v e d t h a t a l a r g e form o f HCG-alpha was p r e s e n t i n normal p l a c e n t a l t i s s u e and s e c r e t e d i n t o the -15-FIGURE 2 THE AMINO-ACID SEQUENCE OF THE BETA-SUBUNIT OF HCG 10 CHO H 2 N - S e r - L y s - G l u - P r o - L e u - A r g - P r o - A r g - C y s - A r g - P r o - I l e - A s n -20 A l a - T h r - L e u - A l a - V a l - G l u - L y s - G l u - G l y - C y s - P r o - V a l - C y s -CHO I l e - T h r - V a l - A s n - T h r - T h r - I l e - C y s - A l a - G l y - T y r - C y s - P r o -40 50 T h r - M e t - T h r - A r g - V a l - L e u - G l n - G l y - V a l - L e u - P r o - A l a - L e u -60 P r o - G l n - V a l - V a l - C y s - A s n - T y r - A r g - A s p - V a l - A r g - P h e - G l u -70 S e r - I l e - A r g - L e u - P r o - G l y - C y s - P r o - A r g - G l y - V a l - A s n - P r o -80 90 V a l - V a l - S e r - T y r - A l a - V a l - A l a - L e u - S e r - C y s - G l n - C y s - A l a -100 Le u - C y s - A r g - A r g - S e r - T h r - T h r - A s p - C y s - G l y - G l y - P r o - L y s -110 Asp-His-Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Phe-GIn-Asp-120 CHO CHO 130 S e r - S e r - S e r - S e r - L y s - A l a - P r o - P r o - P r o - S e r - L e u - P r o - S e r -CHO CHO 140 P r o - S e r - A r g - L e u - P r o - G l y - P r o - S e r - A s p - T h r - P r o - I l e - L e u -Pro-Gln-COOH The c a r b o h y d r a t e s i d e c h a i n s a re a t t a c h e d t o a m i n o - a c i d r e s i d u e s i n d i c a t e d by CHO (Tyrey 1982) . -16-c u l t u r e medium. Other workers observed l a r g e HCG-alpha i n c u l t u r e media ( B e n v e n i s t e 1979, Dean 1980) but not i n c e l l e x t r a c t s (Ruddon 1979, Dean 1980). L a r g e r immunologic t y p e s of HCG and HCG-beta have a l s o been obser v e d (Maruo 1976). I n c r e a s e d g l y c o s y l a t i o n of HCG ( F e i n 1980, L e n t z 1984) may, i n p a r t , account f o r the l a r g e r m o l e c u l a r w e i g h t forms. These l a r g e t y p e s of HCG and i t s s u b u n i t s have been proposed as the e a r l i e s t d e t e c t a b l e b i o s y n t h e t i c forms and t h a t they may then be c o n v e r t e d t o the s m a l l e r m o l e c u l a r s p e c i e s (Maruo 1976). The r e s u l t s from s y n t h e s i s and s e c r e t i o n s t u d i e s (Dean 1980) suggest t h a t the s m a l l i n t r a c e l l u l a r a l p h a - s u b u n i t i s a p r e c u r s o r of the l a r g e r s e c r e t e d a l p h a - s u b u n i t . To d a t e , the r o l e of t h e s e forms of HCG and i t s s u b u n i t s i n the b i o s y n t h e s i s and f u n c t i o n of HCG has not been d e t e r m i n e d . A s s o c i a t i o n of the s u b u n i t s t o form i n t a c t HCG was a c c o m p l i s h e d w i t h h e a t i n g i n a p h o s p h a t e b u f f e r a t 34°C or 37°C ( S t r i c k l a n d 1982, T a l i a d o u r o s 1982). The r a t e of r e a s s o c i a t i o n i s d e t e r m i n e d by the b e t a - s u b u n i t ( S t r i c k l a n d 1982). A s s o c i a t i o n of HCG s u b u n i t s appeared t o be second -1 -1 o r d e r w i t h a r a t e c o n s t a n t of 70 M min . F i r s t the s u b u n i t s form a l o o s e complex and then f o l d t o g i v e the a c t i v e hormone. The e q u i l i b r i u m d i s s o c i a t i o n r a t e c o n s t a n t -7 f o r the s u b u n i t s was found t o be 6.0 x 10 M f o r HCG ( S t r i c k l a n d 1982). S t u d y i n g the e f f e c t of s u b u n i t - 1 7 -c o n c e n t r a t i o n on r e c o m b i n a t i o n , Ingham (1976) found t h a t a s u b u n i t c o n c e n t r a t i o n of 0.0015 mM and 0.0054 mM gave 37% and 90% r e c o m b i n a t i o n r e s p e c t i v e l y . U s i n g a s u b u n i t c o n c e n t r a t i o n from 0.016 mM t o 0.143 mM, complete r e c o m b i n a t i o n was o b t a i n e d (Ingham 1976). D i s s o c i a t i o n of HCG i n t o i t s s u b u n i t s has been i n d u c e d by low pH (Bahl 1973), d e n a t u r a n t s (Bahl 1973, S c h l a f f 1976, Swaminathan 1970, Tyrey 1982) and h i g h temperature ( S t r i c k l a n d 1982). E q u i l i b r i u m between HCG and i t s s u b u n i t s by a s s o c i a t i o n and d i s s o c i a t i o n i s a c h i e v e d s l o w l y and the p l a t e a u l e v e l s formed a r e c o n c e n t r a t i o n - d e p e n d e n t ( S t r i c k l a n d 1982) . 1.3.2 C a r b o h y d r a t e Content A p p r o x i m a t e l y 30% of the m o l e c u l a r w e i g h t o f HCG i s due t o c a r b o h y d r a t e . Human c h o r i o n i c g o n a d o t r o p i n i s e s p e c i a l l y r i c h i n the s i a l i c a c i d N - a c e t y l n e u r a m i n i c a c i d (NANA) and g a l a c t o s e found i n t h e o l i g o s a c c h a r i d e c h a i n s . B a h l (1969b) d e s c r i b e d two t y p e s of c a r b o h y d r a t e s i d e c h a i n s l i n k e d t o the p e p t i d e backbone. S e q u e n t i a l c l e a v a g e of the monosaccharides w i t h s p e c i f i c g l y c o s i d a s e s was used t o determine the s t r u c t u r e of the c a r b o h y d r a t e c h a i n s t h a t a re composed of NANA, f u c o s e ( F u c ) , g a l a c t o s e ( G a l ) , mannose (Man), N - a c e t y l g l u c o s a m i n e (GluNAc) and N - a c e t y l g a l a c t o s a m i n e (GalNAc) (Bahl 1969b). Human -18-c h o r i o n i c g o n a d o t r o p i n appears t o have two t y p e s of c a r b o h y d r a t e - p r o t e i n l i n k a g e s , N - a c e t y l g l u c o s a m i n y 1 a s p a r a g i n e ( N - g l y c o s i d i c ) and N - a c e t y l g a l a c t o s a m i n y l s e r i n e ( O - g l y c o s i d i c ) (Bahl 1969b). The p o s i t i o n of c a r b o h y d r a t e l i n k a g e s on the p r o t e i n backbone are g i v e n i n F i g u r e s 1 and 2 (Tyrey 1982). A l l the N - g l y c o s i d i c c a r b o h y d r a t e u n i t s appear t o have the same b a s i c s t r u c t u r e e x c e p t t h a t f u c o s e i s p r e s e n t o n l y i n the two g l y c o p e p t i d e s from the b e t a - s u b u n i t of HCG ( K e s s l e r 1979a). The b e t a - s u b u n i t a l s o c o n t a i n s f o u r O - g l y c o s i d i c c a r b o h y d r a t e c h a i n s a t t a c h e d t o the p r o t e i n c h a i n as shown i n F i g u r e 2 (Cole 1985, K e s s l e r 1979b). These O - g l y c o s i d i c c a r b o h y d r a t e c h a i n s of p u r i f i e d u r i n a r y HCG were p r e v i o u s l y r e p o r t e d ( K e s s l e r 1979b) as b e i n g a t e t r a s a c c h a r i d e . However, C o l e (1985) i s o l a t e d a m i x t u r e o f d i - , t r i - , t e t r a - and h e x a s a c c h a r i d e s where the t e t r a s a c c h a r i d e proposed ac c o u n t s f o r o n l y 34% ( F i g u r e 3 ) . Heterogeneous forms of HCG-alpha have been found i n c h o r i o c a r c i n o m a c e l l l i n e s . R e c e n t l y , C o l e (1984a) found m a l i g n a n t t r o p h o b l a s t c e l l s produced two d i f f e r e n t a l p h a - s u b u n i t s ; a f r e e a l p h a - s u b u n i t and an a l p h a - s u b u n i t combined t o HCG-beta. These a l p h a - s u b u n i t s d i f f e r i n m o l e c u l a r w e i g h t , the f r e e a l p h a b e i n g l a r g e r . A l s o , the f r e e a l p h a - s u b u n i t was unable t o a s s o c i a t e w i t h the b e t a - s u b u n i t t o form i n t a c t HCG. The f r e e s u b u n i t was found t o c o n t a i n an O - l i n k e d o l i g o s a c c h a r i d e , p r o b a b l y a t t a c h e d t o FIGURE 3 THE STRUCTURES OF THE N-GLYCOSIDIC AND O-GLYCOSIDIC CARBOHYDRATE UNITS OF HCG N-GLYCOSIDIC UNIT NANA-Gal-GluNAc-Man-Man-GluNAc-GluNAc-Asn I I NANA-Gal-GluNAc-Man (Fuc) O-GLYCOSIDIC UNIT NANA-Gal-GalNAc-Ser 43% NANA-GalNAc-Ser 10% NANA-Gal-GalNAc-Ser 34% NANA-Gal-GalNAc I I NANA NANA-Gal-GalNAc-Ser 13% The N - g l y c o s i d i c u n i t s are a t t a c h e d t o a s p a r a g i n e r e s i d u e s a t p o s i t i o n s 52 and 78 i n the a l p h a - s u b u n i t , and p o s i t i o n s 13 and 30 i n the b e t a - s u b u n i t . Only the N - g l y c o s i d i c u n i t s found i n the b e t a s u b u n i t c o n t a i n f u c o s e The O - g l y c o s i d i c u n i t s a re p r e s e n t o n l y i n the b e t a s u b u n i t a t t a c h e d t o s e r i n e a t p o s i t i o n s 1 127, 132, and 138 (Cole 1985, Tyrey 1982). -20-a t h r e o n i n e r e s i d u e , which was not p r e s e n t i n the combined s u b u n i t (Cole 1984a). Others (Jones-Brown 1984, M i z u o c h i 1983, P e t e r s 1984) have a l s o found t h a t c h o r i o c a r c i n o m a c e l l s produce f r e e HCG-alpha t h a t has a h i g h e r c a r b o h y d r a t e c o n t e n t than combined HCG-alpha and u r i n a r y HCG-alpha. M i z u o c h i (1983) suggested t h a t t h i s v a r i a t i o n was i n NANA and f u c o s e c o n t e n t , as w e l l as complex b i a n t e n n a r y type sugar c h a i n s . F u r t h e r m o r e , c a r b o x y - t e r m i n a l fragments of HGC-beta and d e s i a l y l a t e d forms of HCG have been d e t e c t e d i n the u r i n e from c h o r i o c a r c i n o m a p a t i e n t s but not from normal pregnancy p a t i e n t s (Amr 1984) . These o b s e r v a t i o n s suggest t h a t HCG and i t s i n d i v i d u a l s u b u n i t s produced by abnormal c e l l s are c h e m i c a l l y d i f f e r e n t from t h a t produced by normal c e l l s w hich may enable more s p e c i f i c d i a g n o s i s of d i s e a s e s t a t e s . 1.3.3 S t r u c t u r e A c t i v i t y R e l a t i o n s h i p s For HCG In b o t h t h e t e s t e s and o v a r i e s , HCG and LH are thought t o b i n d the same r e c e p t o r and e l i c i t the same b i o l o g i c a l r e s p o n s e s ( B u e t t n e r 1984). T h i s view i s s u p p o r t e d by b i n d i n g s t u d i e s t h a t show the b i n d i n g of i o d i n e - 1 2 5 l a b e l l e d HCG and human and o v i n e LH may be p r e v e n t e d by HCG or LH from s e v e r a l s p e c i e s ( B u e t t n e r 1984). Ranta (1985) found t h a t d e g l y c o s y l a t e d HCG and LH b i n d the same r e c e p t o r . -21-D e g l y c o s y l a t e d HCG has s i g n i f i c a n t e f f e c t s on FSH a c t i o n i n r a t g r a n u l o s a c e l l s , namely r e d u c t i o n of F S H - s t i m u l a t e d c y c l i c AMP and p r o g e s t e r o n e p r o d u c t i o n and enhanced c y c l i c GMP a c c u m u l a t i o n , i n d i c a t i n g i t a l s o i n t e r a c t s w i t h FSH r e c e p t o r s . S p a t i a l r e l a t i o n s h i p s of the HCG s u b u n i t s i n the assembly of the HCG-receptor complex was s t u d i e d (Hwang 1984) u s i n g l u t e i n i z e d r a t o v a r y . Iodine-125 l a b e l l i n g i n d i c a t e d the a l p h a - s u b u n i t can c r o s s - l i n k w i t h a l l f o u r s u b u n i t s of the HCG r e c e p t o r w h i l e the b e t a - s u b u n i t o n l y c r o s s - l i n k s w i t h one. The model proposed by Hwang (1984) f o r the hormone-receptor complex under e q u i l i b r i u m b i n d i n g c o n d i t i o n s shows the b e t a - s u b u n i t bound t o one r e c e p t o r s u b u n i t w i t h t h e a l p h a - s u b u n i t exposed a t the s u r f a c e o f the complex, thus a l l o w i n g the a l p h a - s u b u n i t t o b i n d a l l f o u r s u b u n i t s of the r e c e p t o r . I s o l a t e d s u b u n i t s of HCG l a c k s i g n i f i c a n t b i o l o g i c a l a c t i v i t y but i n c u b a t i o n of s e p a r a t e d s u b u n i t s l e a d s t o spontaneous r e a s s o c i a t i o n and a r e t u r n of c o n s i d e r a b l e b i o l o g i c a l a c t i v i t y (Morgan 1971, Swaminathan 1970). Low s t e r o i d o g e n i c a c t i v i t y w i t h r e s p e c t t o p u r i f i e d HCG (0.01% t o 0.06%) has been o b s e r v e d f o r HCG-beta w i t h the r a t L e y d i g c e l l a ssay (Moudgal 1982) . B i o l o g i c a l a c t i v i t y i s d e c r e a s e d s i g n i f i c a n t l y when amino a c i d s a r e removed from the c a r b o x y - t e r m i n a l r e g i o n of -22-the a l p h a - s u b u n i t of HCG (Merz 1979). Over h a l f o f the b i o l o g i c a l a c t i v i t y i s l o s t when s e r i n e 92 i s removed. F u r t h e r removal of amino a c i d s has no e f f e c t on the b i o l o g i c a l a c t i v i t y u n t i l t y r o s i n e s 88 and 89 are removed. The r e c o m b i n a t i o n of d e s - ( 8 8 - 9 2 ) - a l p h a - s u b u n i t w i t h n a t i v e b e t a - s u b u n i t g i v e s a p r o d u c t w i t h no s i g n i f i c a n t b i o l o g i c a l a c t i v i t y (Merz 1979). The i m m u n o l o g i c a l a c t i v i t y o f d e s - ( 8 8 - 9 2 ) - a l p h a s u b u n i t and the n a t i v e a l p h a - s u b u n i t of HCG were i d e n t i c a l (Merz 1985a), however, i n d i c a t i n g t h a t the a n t i g e n i c d e t e r m i n a n t s of the a l p h a - s u b u n i t were not a f f e c t e d by removal of t h e s e a m i n o - a c i d r e s i d u e s . Comparing t h e r e c o m b i n a t i o n p r o d u c t of d e s - ( 8 8 - 9 2 ) - a l p h a s u b u n i t and n a t i v e b e t a - s u b u n i t w i t h n a t i v e HCG, Merz (1985a) r e p o r t s t h a t the m o d i f i e d hormone has almost no i m m u n o r e a c t i v i t y . T h i s i n d i c a t e s t h a t an a n t i g e n i c d e t e r m i n a n t w h i c h i s p r e s e n t i n n a t i v e HCG b u t not i n the i s o l a t e d s u b u n i t s was i n c o m p l e t e l y e x p r e s s e d i n the m o d i f i e d hormone (Merz 1985a). P r e v i o u s work showed a d i s t i n c t c o n f o r m a t i o n a l d i f f e r e n c e between d e s - ( 8 8 - 9 2 ) - a l p h a s u b u n i t and t h e n a t i v e s u b u n i t (Merz 1979 ; 1985a) . M o d i f i e d HCG a l s o shows a d e c r e a s e i n a b i l i t y t o s t i m u l a t e t e s t i c u l a r a d e n y l a t e c y c l a s e w i t h p r o g r e s s i v e l o s s of a m i n o - a c i d r e s i d u e s from the C-terminus of the a l p h a - s u b u n i t (Merz 1985b). T h i s d e c r e a s e i n s t i m u l a t o r y e f f e c t c o r r e l a t e s w i t h a d e c r e a s e i n a b i l i t y t o b i n d t e s t i c u l a r LH/HCG r e c e p t o r s - 2 3 -(Merz 1985b). T o g e t h e r , t h e s e r e s u l t s i n d i c a t e t h a t t h i s a l t e r a t i o n i n the a l p h a - s u b u n i t , removal of a m i n o - a c i d r e s i d u e s 88 t o 92, causes a d e f i c i e n c y i n s u b u n i t i n t e r a c t i o n and c o n f o r m a t i o n a l changes i n the m o d i f i e d hormone which are p r o b a b l y r e s p o n s i b l e f o r the l o s s i n r e c e p t o r - b i n d i n g a b i l i t y (Merz 1985a). Removal of the l a s t t h r e e amino a c i d r e s i d u e s from the c a r b o x y - t e r m i n a l of the b e t a - s u b u n i t has no s i g n i f i c a n t e f f e c t on b i o l o g i c a l a c t i v i t y o f the r e c o m b i n a t i o n p r o d u c t w i t h n a t i v e a l p h a - s u b u n i t (Merz 1979). The e f f e c t of f u r t h e r removal of a m i n o - a c i d r e s i d u e s from the c a r b o x y - t e r m i n a l of HCG-beta has not been r e p o r t e d . H y b r i d s formed from the r e c o m b i n a t i o n of a l p h a and b e t a s u b u n i t s of d i f f e r e n t human g l y c o p r o t e i n s r e c o v e r the b i o l o g i c a l a c t i v i t y a p p r o p r i a t e t o the b e t a - s u b u n i t (Tyrey 1982) e m p h a s i s i n g the r o l e o f the b e t a - s u b u n i t i n hormone a c t i v i t y . The r o l e o f t h e c a r b o h y d r a t e s t r u c t u r e s o f each s u b u n i t i n the b i o l o g i c a l a c t i v i t y of HCG was r e c e n t l y i n v e s t i g a t e d ( S h i m o h i g a s h i 1982). An i n v i v o b i o a s s a y showed b e t a - m o d i f i e d recombinants were c o m p l e t e l y i n a c t i v e and a l p h a - m o d i f i e d recombinants r e t a i n e d 2% t o 10% of t h e potency of HCG ( S h i m o h i g a s h i 1982). T h e r e f o r e , the c a r b o h y d r a t e s t r u c t u r e s i n the b e t a - s u b u n i t are thought t o p l a y a dominant r o l e i n i n v i v o b i o l o g i c a l a c t i v i t y . -24-I n t r i n s i c h e t e r o g e n e i t y i n the c a r b o h y d r a t e c o n t e n t of e a r l y pregnancy HCG has been r e p o r t e d ( B e l l 1969). R e c e n t l y , the s e r i n e - l i n k e d sugar c h a i n s found i n the b e t a - s u b u n i t of u r i n a r y HCG were shown t o be a m i x t u r e of d i - , t r i - , t e t r a - and h e x a s a c c h a r i d e s (Cole 1985). The amino a c i d c o m p o s i t i o n , however, appears t o be r e l a t i v e l y c o n s t a n t s u g g e s t i n g t h a t the p o l y p e p t i d e p o r t i o n of the s t r u c t u r e i s homogeneous ( B e l l 1969). I t has been demonstrated t h a t HCG c o n t a i n s s e v e r a l b i o l o g i c a l l y a c t i v e components ( G r a e s s l i n 1972, Hamashige 1967, Van H e l l 1968). The most p o t e n t p r e p a r a t i o n (18800 IU/mg) s t u d i e d by Van H e l l (1968) c o n s i s t s of t h r e e components t h a t d i f f e r from each o t h e r i n b i o l o g i c a l p o t e n c y , e l e c t r o p h o r e t i c m o b i l i t y and s i a l i c a c i d c o n t e n t . The e f f e c t s of p r o g r e s s i v e removal of s i a l i c a c i d r e s i d u e s on the i m m u n o l o g i c a l and b i o l o g i c a l a c t i v i t y o f HCG has been r e p o r t e d (Van H a l l 1971). D e s i a l y l a t i o n of HCG by t r e a t m e n t w i t h v a r y i n g amounts o f C l o s t r i d i u m p e r f r i n g e s n e u r a m i n i d a s e d i d not a f f e c t i m m u n o l o g i c a l a c t i v i t y as measured by radioimmunoassay (Van H a l l 1971). The o v a r i a n a s c o r b i c a c i d d e p l e t i o n (OAAD) and v e n t r a l p r o s t a t e w e i g h t (VPW) a s s a y s i n d i c a t e d t h a t p r o g r e s s i v e d e s i a l y l a t i o n (up t o 25%) r e s u l t s i n about 75% r e d u c t i o n of b i o l o g i c a l a c t i v i t y . D e s i a l y l a t i o n from 25% t o 62% f u r t h e r reduced b i o l o g i c a l a c t i v i t y . T h i s d e c r e a s e i n a c t i v i t y was more marked when -25-measured by the VPW assay than by the OAAD assay (Van H a l l 1971). Complete d e s i a l y l a t i o n d i d not a f f e c t b i o l o g i c a l p o tency f u r t h e r (Van H a l l 1971) . W h i l e s t u d y i n g HCG p r e p a r a t i o n s w i t h a d i f f e r i n g p u r i t y and b i o l o g i c a l p o t e n c y f o r c a r b o h y d r a t e c o m p o s i t i o n (Goverde 1968), a mat h e m a t i c a l r e l a t i o n s h i p between the b i o l o g i c a l p o t e n c y and t h e NANA c o n t e n t was o b s e r v e d . However, Goverde e t . a l . (1968) f e l t t h a t the p r o s p e c t s of d e v e l o p i n g a c h e m i c a l assay f o r HCG based on the d e t e r m i n a t i o n of NANA were not e n c o u r a g i n g . These p r e p a r a t i o n s of HCG which d i f f e r i n e l e c t r o p h o r e t i c m o b i l i t i e s , NANA c o n t e n t and i n b i o l o g i c a l potency appear t o be immunochemically i d e n t i c a l (Schuurs 1968) . R e c e n t l y , Imamura (1985) found o t h e r s u b s t a n c e s i n the u r i n e of some p a t i e n t s w i t h h y d a t i d i f o r m moles t h a t had apparent HCG i m m u n o r e a c t i v i t y but l a c k e d b i o l o g i c a l a c t i v i t y . The r e s u l t s g i v e n here i n d i c a t e t h a t i t i s not e s s e n t i a l f o r immunochemical and b i o l o g i c a l e s t i m a t i o n s of HCG t o c o r r e l a t e w i t h each o t h e r . S i n c e d e g l y c o s y l a t e d HCG r e t a i n s the a b i l i t y t o b i n d HCG r e c e p t o r s but e x h i b i t s reduced a c t i v i t y , i t a c t s as an a n t a g o n i s t t o HCG (Rebois 1984). R a b b i t a n t i - H C G a n t i b o d i e s were found t o r e s t o r e some a c t i v i t y t o d e g l y c o s y l a t e d HCG a f t e r i n c u b a t i o n w i t h a n t a g o n i s t bound t o membranes or c e l l s ( R e b o i s 1984) . N e i t h e r t h e a n t i b o d i e s a l o n e nor a d d i t i o n of the a n t i b o d i e s b e f o r e d e g l y c o s y l a t e d HCG (Rebois 1984) -26-produced any a c t i v i t y . Rebois (1984) p o s t u l a t e s t h a t d e g l y c o s y l a t i o n of HCG produces a s t r u c t u r a l form of the hormone t h a t behaves as an a n t a g o n i s t and t h a t a n t i b o d i e s t o HCG c o n v e r t the a n t a g o n i s t t o an a g o n i s t form. A l k y l a t i o n of f r e e s u l f i d e groups and r e d u c t i o n and a l k y l a t i o n of d i s u l f i d e groups i n r a t i n t e r s t i t i a l c e l l r e c e p t o r s has a l s o been r e p o r t e d (Dufau 1974). These s t u d i e s i n d i c a t e d t h a t f r e e s u l f i d e groups of the r e c e p t o r were not r e q u i r e d f o r s p e c i f i c g o n a d o t r o p i n b i n d i n g , whereas the d i s u l f i d e bonds formed an i m p o r t a n t component of the r e c e p t o r and were e s s e n t i a l f o r h o r m o n e - r e c e ptor i n t e r a c t i o n (Dufau 1974) . 1.3.4 D i s u l f i d e Bonds of HCG R e d u c t i o n of the d i s u l f i d e bonds i n the p e p t i d e backbone o f HCG w i t h c y s t e i n e r e s u l t s i n i n a c t i v a t i o n o f the hormone (Bahl 1973). U s i n g a lower c o n c e n t r a t i o n of c y s t e i n e (40 f o l d ) , F r a e n k e l - C o n r a t (1940) found t h a t a c t i v i t y was not l o s t , i n d i c a t i n g t h a t the d i s u l f i d e bonds i m m e d i a t e l y a c c e s s i b l e t o the r e a g e n t are not e s s e n t i a l f o r a c t i v i t y . Human c h o r i o n i c g o n a d o t r o p i n i s a l s o i n a c t i v a t e d by u r e a (Bahl 1973, Swaminathan 1970) which i s presumably due t o d i s s o c i a t i o n of HCG i n t o i t s s u b u n i t s . F r a e n k e l - C o n r a t (1940) found t h a t , f o r HCG and o t h e r -27-g o n a d o t r o p i n s , c y s t e i n e i n a c t i v a t i o n was much more r a p i d when urea was added. T h i s o b s e r v a t i o n i n d i c a t e s t h a t d e n a t u r a t i o n of the g l y c o p r o t e i n w i t h urea causes an i n c r e a s e i n the r e a c t i v i t y o f d i s u l f i d e bonds, p r o b a b l y due t o an i n c r e a s e i n a c c e s s i b i l i t y . In an attempt t o l o c a t e the i n d i v i d u a l d i s u l f i d e bonds i n the s u b u n i t s of HCG, M o r i (1977) examined the e f f e c t of d i t h i o t h r e i t o l (DTT) t r e a t m e n t on n a t i v e HCG. F i v e d i s u l f i d e bonds i n the a l p h a - s u b u n i t and s i x i n the b e t a - s u b u n i t have been proposed f o r HCG (Mori 1977) . A t e n - f o l d molar exc e s s of DTT r e l a t i v e t o the q u a n t i t y of hormone, r e d u c e d two o f t h e e l e v e n d i s u l f i d e bonds. S i n c e a r e o x i d i z e d p r o d u c t and a S-carboxymethy1 d e r i v a t i v e of the DTT reduced HCG r e t a i n e d f u l l b i o l o g i c a l a c t i v i t y , t h e s e d i s u l f i d e bonds are l i k e l y s i t u a t e d i n the a l p h a - s u b u n i t o f HCG (Mori 1977). A t h i r d d i s u l f i d e bond, l o c a t e d i n the b e t a - s u b u n i t , was reduced u s i n g a 4 0 - f o l d molar exc e s s of DTT. R e d u c t i o n of a f o u r t h d i s u l f i d e bond, p r o b a b l y i n t h e b e t a - s u b u n i t , by a 1 0 0 - f o l d excess of DTT r e s u l t e d i n m o d i f i c a t i o n s i n the c o n f o r m a t i o n of HCG (Mori 1977) as shown by sodium d o d e c y l s u l f a t e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . I n f o r m a t i o n r e g a r d i n g the l o c a t i o n of d i s u l f i d e bonds i n t h e p u r i f i e d b e t a - s u b u n i t was r e p o r t e d by Swaminathan (1978). These r e s u l t s i n d i c a t e t h a t a t l e a s t two d i s u l f i d e b r i d g e s connect c y s t e i n e r e s i d u e s 23, 26, 72 -28-and 110 of the b e t a - s u b u n i t (Swaminathan 1978). To d a t e , the complete d i s u l f i d e b r i d g e s t r u c t u r e of HCG i s s t i l l i n d i s p u t e ( B i r k e n 1984) . Holmgren (1976) r e p o r t e d the enzymic r e d u c t i o n of d i s u l f i d e bonds i n HCG and i t s s u b u n i t s w i t h t h i o r e d o x i n and t h i o r e d o x i n r e d u c t a s e from E s c h e r i c h i a c o l i . R e d u c t i o n of t h e d i s u l f i d e bonds i n HCG by t h i o r e d o x i n was much s l o w e r than t h a t of the i n d i v i d u a l s u b u n i t s (Holmgren 1976). P a r t i a l r e d u c t i o n of the a l p h a - s u b u n i t w i t h t h i o r e d o x i n f o l l o w e d by S - c a r b o x y m e t h y l a t i o n i n d i c a t e d t h a t a l l the d i s u l f i d e bonds i n the a l p h a - s u b u n i t were s u r f a c e o r i e n t e d and e q u a l l y r e a c t i v e (Holmgren 1976). 1.3.5 Immunochemistry A n t i s e r a s p e c i f i c f o r HCG has been produced by two immunogens ( B i r k e n 1980) . I n i t i a l l y HCG-beta was used t o g e n e r a t e a n t i s e r a ( V a i t u k a i t i s 1972) . Then, use of a p o r t i o n of the c a r b o x y l - t e r m i n a l unique t o HCG-beta was r e p o r t e d by Louvet (1974) and Chen (1976) . P a r t i a l l y reduced and a l k y l a t e d HCG-beta a l s o produce a n t i s e r a w i t h good s p e c i f i c i t y f o r HCG ( B i r k e n 1980). Problems have been found w i t h the a n t i s e r a g e n e r a t e d t o a l l of t h e s e immunogens. B i r k e n (1980) r e p o r t s t h a t a n t i s e r a p r o d u c e d by u s i n g HCG-beta r a r e l y e x i b i t e d good s p e c i f i c i t y and -29-s e n s i t i v i t y f o r HCG. A l s o , s i g n i f i c a n t c r o s s - r e a c t i v i t y w i t h human LH-beta and a n t i s e r a from HCG-beta has been found ( B i r k e n 1980). These a n t i s e r a f r e q u e n t l y r e a c t b e t t e r w i t h the b e t a - s u b u n i t than w i t h i n t a c t HCG, r e s u l t i n g i n a lower s e n s i t i v i t y t o HCG (Swaminathan 1978) . A n t i s e r a made from the c a r b o x y l - t e r m i n a l p e p t i d e of HCG-beta a r e always s p e c i f i c f o r HCG i f the p e p t i d e used i s f r e e of i m p u r i t i e s f r om the r e s t o f the HCG m o l e c u l e . U s u a l l y t h e s e a n t i b o d i e s have a lower a f f i n i t y and t h e r e f o r e a lower s e n s i t i v i t y t o HCG than a n t i s e r a r a i s e d a g a i n s t the whole b e t a - s u b u n i t ( B i r k e n 1980). P o l y c l o n a l (Chen 1976, V a i t u k a i t i s 1972) and monoclonal (Rahamim 1984, S h a p i r o 1984, Wu 1983) a n t i b o d i e s have been r a i s e d a g a i n s t b o t h the b e t a - s u b u n i t and a H C G - b e t a - c a r b o x y l - t e r m i n a 1 p e p t i d e t h a t do n o t s i g n i f i c a n t l y r e a c t t o p h y s i o l o g i c a l l e v e l s of o t h e r g o n a d o t r o p i n s (Chen 1976, V a i t u k a i t i s 1972) thus a l l o w i n g the development of s p e c i f i c immunoassays. The c a r b o h y d r a t e moiety of HCG may o n l y be e s s e n t i a l f o r the b i o l o g i c a l a c t i v i t y (Mori 1970) s i n c e i t does not appear t o be i n v o l v e d i n the a n t i g e n i c s t r u c t u r e . The p r o t e i n moiety of HCG, however, i s i m p o r t a n t f o r b o t h the a n t i g e n i c and b i o l o g i c a l a c t i v i t y of HCG ( M o r i 1970). A l s o , b o t h a c t i v i t i e s appear t o r e q u i r e an o r d e r e d c o n f o r m a t i o n of the p r o t e i n backbone (Mori 1970) . Swaminathan (1978) c o n c l u d e d t h a t a major a n t i g e n i c s i t e of HCG-beta i s the -30-r e g i o n of r e s i d u e s 21 t o 23 w i t h a d i s u l f i d e bond c o n n e c t i n g c y s t e i n e 23 or 26 w i t h c y s t e i n e a t p o s i t i o n 72 or 110. Berger (1984) demonstrated t h a t the a n t i g e n i c d e t e r m i n a n t s of HCG are i n v o l v e d i n the e x p r e s s i o n of b i o l o g i c a l a c t i v i t y . M o noclonal a n t i b o d i e s t o HCG were produced and t h e i r a b i l i t y t o n e u t r a l i z e the b i o l o g i c a l a c t i v i t y of HCG, as measured by a r a t b i o a s s a y , was d e t e r m i n e d (Berger 1984). U n e x p e c t e d l y , the a b i l i t y t o n e u t r a l i z e HCG was not r e s t r i c t e d t o the monoclonal a n t i b o d i e s t h a t r e c o g n i z e d HCG-beta (Berger 1984). A l s o , no c o r r e l a t i o n was found between a f f i n i t y or s p e c i f i c i t y and a b i l i t y t o n e u t r a l i z e b i o l o g i c a l a c t i v i t y (Berger 1984) . Another s t u d y t o l o c a t e the a n t i g e n i c d e t e r m i n a n t s of HCG i n v o l v e d p r o d u c i n g a monoclonal a n t i b o d y ( B e l l e t 1984) u s i n g a s y n t h e t i c p e p t i d e analogous t o the 109 t o 145 segment of the b e t a - s u b u n i t as a n t i g e n . T h i s monoclonal a n t i b o d y was s p e c i f i c f o r the f r e e n a t i v e b e t a - s u b u n i t . S i n c e monoclonal a n t i b o d i e s produced u s i n g p e p t i d e s analogous t o the 118 t o 145 segment r e c o g n i z e b o t h the b e t a - s u b u n i t and i n t a c t HCG, t h e r e c o g n i t i o n of an a n t i g e n i c d e t e r m i n a n t i n the 109 t o 118 r e g i o n of the b e t a - s u b u n i t may e x p l a i n the s p e c i f i c i t y of t h i s monoclonal a n t i b o d y f o r the f r e e n a t i v e s u b u n i t ( B e l l e t 1984). R e c e n t l y , however, monoclonal a n t i b o d i e s t o a s y n t h e t i c p e p t i d e analogous t o the 109 t o 145 segment of the b e t a - s u b u n i t of HCG (Caraux -31-1985) were found t o r e a c t w i t h b o t h f r e e b e t a - s u b u n i t and i n t a c t HCG. Comparison of C o r y n e b a c t e r i u m parvum w i t h Freund's complete a d j u v a n t as a p o t e n t i a t o r of t h e immune r e s p o n s e t o HCG-beta l i n k e d t o t e t a n u s t o x o i d (Covey 1984) i n d i c a t e d t h a t h i g h e r a n t i b o d y t i t e r s were produced w i t h Freund's complete a d j u v a n t . The a b i l i t y of a n t i s e r a t o HCG-beta-tetanus t o x o i d t o n e u t r a l i z e the b i o l o g i c a l a c t i v i t y of HCG was s t u d i e d u s i n g the r a t u t e r i n e w e i g h t a s s a y . No s i g n i f i c a n t d i f f e r e n c e was found between a n t i s e r a p o t e n t i a t e d by C. parvum and Freund's complete a d j u v a n t (Covey 1984). I t may be advantageous t o use C. parvum as a d j u v a n t when o n l y n e u t r a l i z a t i o n of b i o l o g i c a l a c t i v i t y i s d e s i r e d s i n c e i t does not cause development o f the s e v e r e granulomatous l e s i o n s c h a r a c t e r i s t i c of Freund's complete a d j u v a n t (Covey 1984) . Immunization w i t h HCG c o n j u g a t e d t o 2 , 4 - d i n i t r o -b e n z e n e s u l f o n i c a c i d (DNP) produced a h i g h e r t i t e r t han HCG-tetanus t o x o i d (Mangano 1983). However, HCG-DNP l i n k e d t o t e t a n u s t o x o i d and HCG-DNP showed the same a n t i g e n i c i t y as d e t e r m i n e d by a l a t e x a g g l u t i n a t i o n a s s a y (Mangano 1983). T h e r e f o r e , the maximum a n t i g e n i c i t y was found w i t h the HCG-DNP c o n j u g a t e and no f u r t h e r change was found w i t h a d d i t i o n a l l i n k i n g t o t e t a n u s t o x o i d . A n t i s e r u m t o HCG w i t h h i g h a f f i n i t y and low -32-c r o s s - r e a c t i v i t y w i t h human LH and the s u b u n i t s of HCG was o b t a i n e d from a p a t i e n t who produced a n t i b o d i e s d u r i n g HCG t r e a t m e n t (Thau 1983). However, p r o l o n g e d a d m i n i s t r a t i o n of HCG t o rhesus monkeys produced a n t i b o d i e s w h i c h were found t o c r o s s - r e a c t w i t h o t h e r human g o n a d o t r o p i n s but not w i t h macaque hormones (O t t o b r e 1985). S e v e r a l monoclonal a n t i b o d i e s t o HCG-beta t h a t r e c o g n i z e HCG-beta i n a double a n t i b o d y RIA have been produced (Rahamim 1984). However, the s e a n t i b o d i e s d i d not show a g g l u t i n a t i o n r e a c t i o n s i n a h e m a g g l u t i n a t i o n i n h i b i t i o n a s s a y . With d u a l s c r e e n i n g p r o c e d u r e s , Rahamim (1984) r e p o r t s f i n d i n g a monoclonal a n t i b o d y s p e c i f i c t o HCG-beta t h a t i s c a p a b l e of c a u s i n g an a g g l u t i n a t i o n r e a c t i o n . Hence the s e a n t i b o d i e s are u s e f u l f o r the q u a l i t a t i v e d e t e c t i o n and q u a n t i t a t i v e d e t e r m i n a t i o n of HCG by b o t h RIA and h e m a g g l u t i n a t i o n i n h i b i t i o n methods (Rahamim 1984) . Mon o c l o n a l a n t i b o d i e s d i r e c t e d a g a i n s t HCG-alpha have been used i n t w o - s i t e immunoassays (S h i m i z u 1982, Wada 1982). These a s s a y s r e l y on anti-HCG-beta a n t i b o d i e s f o r s p e c i f i c i t y . Because of the s t r u c t u r a l l y and immunochemically s i m i l a r n a t u r e of the a l p h a - s u b u n i t s o f the g l y c o p r o t e i n hormones, Wada (1982) was a b l e t o d e v e l o p a s s a y s f o r HCG, LH, and TSH u s i n g the same a n t i - a l p h a monoclonal a n t i b o d y . T h i s l a c k of s p e c i f i c i t y of -33-a n t i - H C G - a l p h a monoclonal a n t i b o d i e s (Bosch 1984) must be c o n s i d e r e d when used i n d e v e l o p i n g immunoassays f o r HCG. In the s e a r c h f o r a n t i s e r a t o produce h i g h l y s p e c i f i c and s e n s i t i v e HCG a s s a y s , emphasis l a y i n s t u d y i n g the area s of c o n t r a s t between HCG and o t h e r g l y c o p r o t e i n s . T h e r e f o r e l i t t l e i n f o r m a t i o n on the immunogenic d e t e r m i n a n t s of the a l p h a - s u b u n i t i s a v a i l a b l e . 1.3.6 B i o s y n t h e s i s of HCG B i o s y n t h e s i s o f HCG o c c u r s i n the s y n c y t i o -t r o p h o b l a s t i c c e l l s of the p l a c e n t a ( C a n f i e l d 1971). C y t o t r o p h o b l a s t i c c e l l s a re a l s o thought t o p l a y a r o l e i n the s y n t h e s i s and/or s t o r a g e of HCG. Immunohistochemical t e c h n i q u e s u s i n g a n t i b o d i e s s p e c i f i c t o HCG, HCG-alpha and HCG-beta show HCG and HCG-beta are l o c a l i z e d i n s y n c y t i o t r o p h o b l a s t s w h i l e HCG-alpha i s l o c a t e d i n b o t h s y n c y t i o t r o p h o b l a s t s and c y t o t r o p h o b l a s t s (Tojo 1982). An excess of f r e e a l p h a - s u b u n i t e x i s t s i n serum of pregnant women and i n the p l a c e n t a whereas f r e e b e t a - s u b u n i t i s u s u a l y not p r e s e n t or i s found i n e x t r e m e l y low c o n c e n t r a t i o n s (Tojo 1982) s u g g e s t i n g an unbalanced s y n t h e s i s of the a l p h a and b e t a s u b u n i t s . R e c e n t l y , second t r i m e s t e r human f e t a l k i d n e y and l i v e r t i s s u e s have a l s o been found t o s e c r e t e HCG ( G o l d s m i t h 1983). - 3 4 -I. 4 Pharmacokinetics of HCG and i t s Subunits Serum HCG c o n c e n t r a t i o n s i n pos t partum p a t i e n t s e x h i b i t b i p h a s i c e l i m i n a t i o n w i t h an i n i t i a l h a l f l i f e of I I . 2 hours f o l l o w e d by a slo w e r h a l f l i f e o f 23.4 hours (Yen 1968). Using serum samples up t o 120 hours a f t e r d e l i v e r y (Mann 1980) , the h a l f l i f e of HCG was c a l c u l a t e d t o be 10 t o 34 h o u r s . The r e n a l c l e a r a n c e of HCG i n thes e p a t i e n t s was 0.95 mL/min (Yen 1968) . Col e (1984b) r e p o r t e d t he time c o u r s e o f f r e e a l p h a - s u b u n i t , f r e e b e t a - s u b u n i t and i n t a c t HCG i n s e r a d u r i n g pregnancy. Human c h o r i o n i c g o n a d o t r o p i n l e v e l s i n pregnancy s e r a peaked a t about t e n weeks a f t e r t he l a s t m e n s t r u a l p e r i o d , and d e c l i n e d t h e r e a f t e r (Cole 1984b). As HCG l e v e l s d e c l i n e d , f r e e a l p h a l e v e l s r o s e . A s i g n i f i c a n t l y h i g h e r a l p h a - s u b u n i t l e v e l i n the t e n t h t o t h i r t e e n t h week of pregnancy has been a s s o c i a t e d w i t h a h i g h e r i n c i d e n c e of t r o p h o b l a s t i c d i s e a s e (Elegbe 1984) . The i n c r e a s e d l e v e l o f a l p h a - s u b u n i t may i n d i c a t e a p o p u l a t i o n of t r o p h o b l a s t i c c e l l s a t a g r e a t e r r i s k of d e v e l o p i n g malignancy (Elegbe 1984). By the t h i r d t r i m e s t e r , 30% t o 40% of the t o t a l serum a l p h a - s u b u n i t was f r e e . At the time of the peak i n i n t a c t HCG l e v e l s and t h e r e a f t e r , o n l y a s m a l l p r o p o r t i o n (2.4% t o 3.6%) of the t o t a l b e t a - s u b u n i t was i n the f r e e s t a t e (Cole -35-1984b). However, e a r l y i n p r e g n a n c y , 4 t o 6 weeks a f t e r the l a s t m e n s t r u a l p e r i o d , about 16% f r e e b e t a - s u b u n i t was observed (Cole 1984b). The d i s t r i b u t i o n , metabolism and e x c r e t i o n of p u r i f i e d HCG and i t s s u b u n i t s i n normal human s u b j e c t s has r e c e n t l y been r e p o r t e d (Wehmann 1984). U n l a b e l l e d p u r i f i e d s u b s t a n c e s were a d m i n i s t e r e d by s i n g l e r a p i d i n t r a v e n o u s i n j e c t i o n and c o n t i n u o u s i n t r a v e n o u s i n j e c t i o n and bo t h serum and u r i n e samples were assayed u s i n g RIA t e c h n i q u e s . The i n i t i a l a pparent volume of d i s t r i b u t i o n was the same, 1780 mL/m2, f o r i n t a c t HCG and i t s a l p h a - and b e t a - s u b u n i t s (Wehmann 1984) . E l i m i n a t i o n from serum f i t a two-compartment model w i t h the h a l f - l i f e f o r b o t h phases f o r HCG (6 hours and 36 hours) b e i n g t e n - f o l d g r e a t e r than t h o s e of HCG-beta (0.68 hour and 3.9 hours) and t h i r t y - f o l d g r e a t e r than those of HCG-alpha (0.22 hour and 1.3 hours) (Wehmann 1984). The h a l f l i f e v a l u e s r e p o r t e d h e r e and t h o s e p r e v i o u s l y d e t e r m i n e d f o r HCG are s i m i l a r . However, d i f f e r e n c e s may be due t o v a r i a t i o n i n the p a t i e n t s as w e l l as s p e c i f i c i t y of the a n a l y t i c a l methods used and p u r i t y of the HCG a d m i n i s t e r e d . From c o n t i n u o u s i n f u s i o n s t u d i e s , b o t h t o t a l body and r e n a l c l e a r a n c e r a t e s were c a l c u l a t e d . The t o t a l body 2 c l e a r a n c e r a t e o f HCG (1.9 mL/min/m ) was low w i t h r e s p e c t 2 to t h a t f o r the a l p h a (49.7 mL/min/m ) and b e t a (19.0 -36-mL/min/m ) s u b u n i t s (Wehmann 1984). However, the r e n a l 2 c l e a r a n c e of the s u b u n i t s (0.16 and 0.13 mL/min/m f o r a l p h a and b e t a r e s p e c t i v e l y ) was two t o t h r e e t i m e s lower 2 than t h a t f o r i n t a c t hormone (0.40 mL/min/m ) (Wehmann 1984). Thus, r e n a l c l e a r a n c e of the s u b u n i t s a c c o u n t s f o r l e s s than 1% of the t o t a l body c l e a r a n c e , w h i l e i t r e p r e s e n t s almost 25% f o r HCG (Wehmann 1984) . A f t e r a d m i n i s t r a t i o n of the b e t a - s u b u n i t , two components were e x c r e t e d i n t h e u r i n e : i n t a c t HCG-beta and a fragment of HCG-beta which l a c k s the c a r b o x y - t e r m i n a l . p e p t i d e immunologic d e t e r m i n a n t (Wehmann 1984). No e v i d e n c e , however, was found ( V a i t u k a i t i s 1984, Wehmann 1984) t o s u p p o r t the t h e o r y t h a t HCG i s m e t a b o l i z e d by d i s s o c i a t i o n of i t s s u b u n i t s as an i n i t i a l s t e p . V a i t u k a i t i s (1984) found t h a t HCG r a p i d l y d i s s o c i a t e s from i t s r e c e p t o r a f t e r i n t e r n a l i z a t i o n . Free HCG can be i n t e r n a l i z e d and i s a c t u a l l y c o n c e n t r a t e d w i t h i n c e l l s . I n t r a c e l l u l a r m etabolism o f HCG appears t o s t a r t w i t h m o d i f i c a t i o n of the o l i g o s a c c h a r i d e s i d e c h a i n s f o l l o w e d by d e g r a d a t i o n of the p r o t e i n p o r t i o n ( V a i t u k a i t i s 1984) . I n d i r e c t e v i d e n c e f o r more r a p i d i n t r a c e l l u l a r m e t a b o l i s m of HCG-alpha than HCG-beta was a l s o r e p o r t e d ( V a i t u k a i t i s 1984) which may r e f l e c t the lower c a r b o h y d r a t e c o n t e n t of HCG-alpha. -37-1.5 Physicochemical P r o p e r t i e s of HCG E a r l y r e p o r t s i n d i c a t e d the m o l e c u l a r w e i g h t of HCG t o be 100000 Da ( L i 1949) but more r e c e n t l y the m o l e c u l a r w e i g h t has been c a l c u l a t e d , based on c o m p o s i t i o n , t o be a p p r o x i m a t e l y 37000 Da; 15000 Da f o r the a l p h a - s u b u n i t and 22000 Da f o r the b e t a - s u b u n i t (Tyrey 1982). D e t e r m i n a t i o n s of m o l e c u l a r w e i g h t by s e d i m e n t a t i o n e q u i l i b r i u m ( B a h l 1973) y i e l d s a v a l u e o f 40660 +_ 3000 Da. E l e c t r o p h o r e s i s ( P a l a 1973) e x p e r i m e n t s g i v e a m o l e c u l a r weight of a p p r o x i m a t e l y 35000 Da f o r the slow moving f r a c t i o n and 27000 Da f o r a f a s t e r moving f r a c t i o n g i v i n g a t o t a l of about 62000 Da f o r i n t a c t HCG. The s e d i m e n t a t i o n c o e f f i c i e n t v a l u e s r e p o r t e d v a r y from 2.7 (Got 1960) t o 2.89 (Bahl 1973) w i t h some f i n d i n g s as h i g h as 4.3 ( L i 1949). The p a r t i a l s p e c i f i c volume of HCG i s about 0.7 (Got 1960, L i 1949). For u l t r a v i o l e t absorbance, a maximum was obser v e d a t 278 nm w i t h HCG (Got 1960) . Human c h o r i o n i c g o n a d o t r o p i n has an e x t i n c t i o n c o e f f i c i e n t (1 cm path l e n g t h ) of 0.425 f o r a 1 mg/mL s o l u t i o n (Got 1960) and 3.89 f o r a 1% s o l u t i o n (Bahl 1973). The e x t i n c t i o n c o e f f i c i e n t s f o r p u r i f i e d HCG (1.41 x 1 0 4 M~ 1cm~ 1), HCG-alpha (0.64 x 1 0 4 - 1 - 1 4 -1 -1 M cm ) and HCG-beta (0.56 x 10 M cm ) were r e c e n t l y r e p o r t e d ( B i r k e n 1984). -38-S t r u c t u r a l c h a r a c t e r i s t i c s o f HCG and i t s s u b u n i t s have been e l u c i d a t e d from u l t r a v i o l e t c i r c u l a r d i c h r o i s m (CD) s t u d i e s ( H i l g e n f e l d t 1974). In aqueous s o l u t i o n s , HCG and i t s s u b u n i t s e x h i b i t m a i n l y random c h a i n s t r u c t u r e w i t h o n l y s l i g h t v a r i a t i o n s i n the CD spectrum u s i n g a pH range from 2.0 t o 11.0 ( H i l g e n f e l d t 1974). A l s o , d i l u t i o n of HCG from 0.2% t o 0.002% does not a f f e c t the CD spectrum. High c o n c e n t r a t i o n s ( g r e a t e r than 60%) of h e x a f l u o r o p r o p a n o l c o n v e r t HCG and the i n d i v i d u a l s u b u n i t s t o an o r d e r e d s t r u c t u r e t h a t c o n s i s t s m a i n l y of a l p h a - h e l i x components ( H i l g e n f e l d t 1974). The secondary s t r u c t u r e s of g l y c o p r o t e i n hormone s u b u n i t s , i n c l u d i n g HCG, has a l s o been p r e d i c t e d ( G a r n i e r 1984). The a l p h a - s u b u n i t c o n s i s t s m a i n l y of b e t a - s h e e t and b e t a - t u r n w i t h some c o i l w h i l e the b e t a - s u b u n i t c o n s i s t s m a i n l y of b e t a - s h e e t ( G a r n i e r 1984). E x a m i n a t i o n of the s t a b i l i t y of HCG and i t s a l p h a - s u b u n i t i n human b l o o d (Rao 1983) i n d i c a t e s t h a t they are b o t h s t a b l e i n u n f r o z e n whole b l o o d , plasma and serum f o r a t l e a s t s i x days. F r o z e n plasma and serum samples were s t a b l e f o r a t l e a s t s i x months (Rao 1983). No e f f e c t was observe d when samples were r e p e a t e d l y f r o z e n (Rao 1983). A l s o , s e p a r a t i o n of plasma or serum from whole b l o o d i s not r e q u i r e d f o r 12 hours (Rao 1983) . Hemolysed samples showed a 20% t o 30% de c r e a s e i n the c o n c e n t r a t i o n of HCG and i t s a l p h a - s u b u n i t (Rao 1983), w h i c h may be from sample d i l u t i o n . -39-•1.6 Methods of S e p a r a t i o n And A n a l y s i s of HCG Methods of s e p a r a t i o n and q u a n t i t a t i o n o f HCG have been deve l o p e d t o monitor t h e r a p y , d i a g n o s e v a r i o u s d i s e a s e s t a t e s and diag n o s e and monitor pregnancy. Serum l e v e l s of HCG-beta have been used t o a s c e r t a i n g e s t a t i o n a l age (Lagrew 1983) and may be u s e f u l i n p r e d i c t i n g the development of severe p r e - e c l a m p s i a ( S a i d 1984). D e t e r m i n a t i o n of f e t a l sex by a n a l y s i s of HCG i n serum and a m n i o t i c f l u i d has a l s o been r e p o r t e d (Bremme 1982, S a i d 1984). S i n g l e random HCG serum d e t e r m i n a t i o n s were of minimum v a l u e i n p r e d i c t i n g f a i l u r e of pregnancy ( K a u p p i l a 1984) . M u l t i p l e HCG serum l e v e l s were of some b e n e f i t as a p r o g n o s t i c t o o l f o r d e t e r m i n i n g f e t a l outcome (Bremme 1983). D i a g n o s i s of e c t o p i c pregnancy p r i o r t o o p e r a t i o n can be a c h i e v e d w i t h n e a r l y 100% a c c u r a c y u s i n g an HCG-beta RIA as a s c r e e n i n g agent p l u s t h e s e l e c t i v e use of p e l v i c u l t r a s o u n d (Bryson 1983, Goh 1984). S e n s i t i v e and s p e c i f i c serum a s s a y s f o r HCG have a l s o been used w h i l e s t u d y i n g the mechanism of a c t i o n of i n t r a - u t e r i n e c o n t r a c e p t i v e d e v i c e s (Dhont 1984) . The low serum and u r i n a r y l e v e l s of HCG i n the e a r l y and l a t e s t a g e s of pregnancy have prompted the development of e x t r e m e l y s e n s i t i v e a ssay p r o c e d u r e s . Human c h o r i o n i c g o n a d o t r o p i n may be a n a l y s e d by s e v e r a l s e n s i t i v e methods such as immunoassay t e c h n i q u e s i n c l u d i n g radioimmunoassay - 4 0 -(RIA) ( A y a l a 1978, B r a u n s t e i n 1983, F i l s t e i n 1983, Forbes 1981, Hussa 1984, Kosasa 1973, Lagrew 1983, Mann 1980, M i d g l e y 1966, Rutanen 1980, Schroeder 1983, S h i m i z u 1982? 1984, S i i t e r i 1981, Stenman 1982, Than 1982, Turkes 1984, Wu 1983), r a d i o r e c e p t o r assay (RRA) (Bryson 1983, Forbes 1981, Saxena 1974), enzyme immunoassays ( F u r u h a s h i 1982, Kondo 1984, Wada 1982, Yorde 1976), an immunoradiometric assay (Armstrong 1984, S h a p i r o 1984), s o l p a r t i c l e immunoassay (SPIA) ( L e u v e r i n g 1983), a f l u o r o i m m u n o m e t r i c a s s a y (Stenman 1983) and a h e m a g g l u t i n a t i o n i n h i b i t i o n a ssay (Rahamim 1984) . B i o l o g i c a l methods f o r d e t e r m i n i n g the a c t i v i t y of HCG samples have a l s o been d e v e l o p e d ( C a n f i e l d 1971, C z e k a l a 1981, D a v i e s 1982, G u r i n 1939, McArthur 1952, Morgan 1971, Turner 1971) . Methods used i n the s e p a r a t i o n of HCG and p r o t e i n s i n g e n e r a l i n c l u d e v a r i o u s column c h r o m a t o g r a p h i c a s s a y s ( A l b e r t 1956, A y a l a 1978, B a h l 1969a, C a n f i e l d 1971, Katzman 1943, Merz 1974, S h i m i z u 1984, T a l i a d o u r o s 1982, Van H a l l 1971, Van H e l l 1968), e l e c t r o p h o r e s i s ( G r a e s s l i n 1972, K a p a d i a 1981, M a f f e z z o l i 1972, Morgan 1971, Q a z i 1974, Weber 1969), and high-performance l i q u i d chromatography (HPLC) ( F o r a s t i e r i 1982, Moudgal 1982, Putterman 1982, S h i m o h i g a s h i 1982; 1983) . A f t e r i s o l a t i o n , d e t e c t i o n of p r o t e i n s i s n o r m a l l y a c h i e v e d by u l t r a - v i o l e t a b s o r p t i o n a t 280 nm (which measures a r o m a t i c r e s i d u e s ) ( P a r i k h 1974) or 214 nm (which m o n i t o r s the p e p t i d e bond). S o l v e n t e x t r a c t i o n - 4 1 -t e c h n i c u e s have a l s o been used i n the removal and p u r i f i c a t i o n of HCG from u r i n e samples ( G u r i n 1939). None o f t h e methods d e v e l o p e d t o d a t e can compete w i t h the RIA i n terms of p r e c i s i o n , s e n s i t i v i t y , and c o s t t h e r e f o r e t h i s method i s used c l i n i c a l l y . 1.6.1 Immunoassays Radioimmunoassay t e c h n i q u e s have a l s o been deve l o p e d f o r the a n a l y s i s of o t h e r endogenous compounds and drugs ( B u t l e r 1978, E r t i n g s h a u s e n 1975, Lenz 1975, Leung 1975). T h i s assay method i s based on the c o m p e t i t i v e b i n d i n g of r a d i o a c t i v e and n o n - r a d i o a c t i v e compounds f o r a s p e c i f i c a n t i b o d y such t h a t the amount of n o n - r a d i o a c t i v e compound p r e s e n t d e t e r m i n e s how much r a d i o a c t i v i t y b i n d s the a n t i b o d y . V a r i o u s methods f o r s e p a r a t i o n of antibody-bound and f r e e (unbound) r a d i o a c t i v i t y (by e i t h e r removing f r e e compound o r by r e moving bound compound) a r e a v a i l a b l e . They i n c l u d e the use of c h a r c o a l s e p a r a t i o n , double a n t i b o d y s e p a r a t i o n , p o l y e t h y l e n e g l y c o l s e p a r a t i o n and s o l i d - p h a s e RIA methods ( B u t l e r 1978). The r a d i o a c t i v i t y r e m a i n i n g i s counted and the c o n c e n t r a t i o n of compound i n an unknown ( p a t i e n t ) sample i s o b t a i n e d by comparison of the unknown sample r a d i o a c t i v i t y t o t h a t o b t a i n e d from samples of known c o n c e n t r a t i o n . For HCG immunoassays p o l y e t h y l e n e g l y c o l -42-(Stenman 1982) and double a n t i b o d y t e c h n i q u e s (Mann 1980) have been used f o r s e p a r a t i o n of f r e e and bound r a d i o a c t i v i t y . P o l y e t h y l e n e g l y c o l p r e c i p i t a t e s antibody-bound r a d i o a c t i v i t y , which can be measured a f t e r c e n t r i f u g a t i o n and removal of the s u p e r n a t a n t f l u i d ( B u t l e r 1978). The double a n t i b o d y method i n v o l v e s a d d i t i o n of an a n t i s e r u m t o the immunoglobulin of the s p e c i e s from which the d r u g - s p e c i f i c ( H C G - s p e c i f i c ) a n t i b o d y was o b t a i n e d . The second a n t i b o d y causes p r e c i p i t a t i o n of the d r u g - s p e c i f i c a n t i b o d y and bound drug ( B u t l e r 1978) . A g a i n , the bound r a d i o a c t i v i t y can be counted a f t e r c e n t r i f u g a t i o n and removal of the s u p e r n a t a n t f l u i d c o n t a i n i n g the f r e e r a d i o a c t i v i t y . E r r o n e o u s or d i s c o r d a n t r e s u l t s have been o b s e r v e d w i t h commercial r e a g e n t s f o r the q u a l i t a t i v e and q u a n t i t a t i v e d e t e r m i n a t i o n o f HCG and HCG-beta i n serum samples ( F i l s t e i n 1983, Hussa 1984; 1985, Thomas 1985). V a r i a t i o n s i n p r e c i p i t a t i n g agent used and s p e c i f i c i t y of t h e a n t i b o d y f o r f r e e b e t a - s u b u n i t , i n t a c t HCG or b o t h a r e c o n s i d e r e d t o be a source f o r t h e s e d i f f e r i n g r e s u l t s (Hussa 1984) a l o n g w i t h i n t e r f e r e n c e from l u t e i n i z i n g hormone b e t a - s u b u n i t (Thomas 1985). The presence of a l t e r e d forms of HCG and i t s s u b u n i t s may a l s o c o n t r i b u t e t o d i s c o r d a n t r e s u l t s (Hussa 1985). U s u a l l y serum and plasma samples are used f o r HCG but p r o c e d u r e s u s i n g s a l i v a (Turkes 1984) and u r i n e ( A y a l a -43-1978, S h i m i z u 1984) have a l s o been d e v e l o p e d . A n t i b o d i e s s p e c i f i c f o r HCG are r e q u i r e d f o r t h e s e i m m u n o l o g i c a l a s s a y s . Due t o e x t e n s i v e s i m i l a r i t i e s t o o t h e r g l y c o p r o t e i n hormones making s p e c i f i c a n t i s e r a t o HCG i s d i f f i c u l t . One s o l u t i o n t o t h i s problem has been t o u t i l i z e the b e t a - s u b u n i t as immunogen t o r a i s e a n t i s e r a t o t h a t p o r t i o n of the b e t a - s u b u n i t t h a t i s unique t o HCG ( S i i t e r i 1981, Than 1982, Tyrey 1982). A n t i b o d i e s t o HCG-alpha have a l s o been dev e l o p e d ( S h i m i z u 1982) . Immunogenic c o n j u g a t e s of t h e s e p e p t i d e s w i t h ovalbumin are formed u s i n g the c a r b o d i i m i d e t e c h n i q u e ( B i r k e n 1977) which c o n j u g a t e s the f r e e c a r b o x y l groups t o amino groups on the c a r r i e r m o l e c u l e ( B u t l e r 1978) . Mice and r a b b i t s have been used t o produce a n t i b o d i e s t o HCG and HCG-beta ( B i r k e n 1977, Forbes 1981, Tojo 1982). Both p o l y c l o n a l and monoclonal a n t i b o d i e s t o HCG have been used (Stenman 1982) . The p o l y c l o n a l a n t i b o d i e s used by most m a n u f a c t u r e r s of HCG RIA k i t s have a lower s p e c i f i c i t y than monoclonal a n t i b o d i e s (Schroeder 1983). Monoclonal a n t i b o d i e s s p e c i f i c f o r the b e t a - s u b u n i t are c o m m e r c i a l l y a v a i l a b l e (Monoclonal Ab, M i l e s L a b s , H y b r i t e c h ) (Wu 1983). A r a d i o r e c e p t o r immunoassay has been d e v e l o p e d (Saxena 1974) u s i n g the plasma membrane of b o v i n e corpus luteum as th e s o u r c e of HCG r e c e p t o r s . These r e c e p t o r s b i n d o n l y HCG, human LH and b o v i n e LH and show no s p e c i e s s p e c i f i c i t y -44-(Saxena 1974) . I t s advantage over the RIA was t h a t i t took l e s s time t o perfo r m but the c r o s s - r e a c t i v i t y w i t h human LH r e s u l t i n g i n f a l s e p o s i t i v e r e s u l t s g r e a t l y reduced i t s r e l i a b i l i t y . Thus the RIA of HCG was found t o be more r e l i a b l e t han the RRA (Bryson 1983, Forbes 1981). V a r i o u s enzyme immunoassays f o r HCG have been d e v e l o p e d ( F u r u h a s h i 1982, Wada 1982, Yorde 1976). C o n j u g a t i o n o f HCG to an enzyme i s r e q u i r e d such t h a t when the a n t i b o d y t o HCG b i n d s the enzyme-HCG complex, the enzyme becomes i n a c t i v e . Comparison of enzyme a c t i v i t y i n samples o f unknown HCG c o n c e n t r a t i o n w i t h known s t a n d a r d s i s used t o o b t a i n r e s u l t s . H o r s e r a d i s h p e r o x i d a s e (Wada 1982), a l k a l i n e phosphatase (Bock 1985) and b a c t e r i a l g a l a c t o s i d a s e ( F u r u h a s h i 1982) have been used w i t h HCG enzyme immunoassays. Commonly, t h e s e a s s a y s used a n t i b o d i e s t o the b e t a - s u b u n i t ( F u r u h a s h i 1982) or b o t h the a l p h a and b e t a s u b u n i t s o f HCG (Wada 1982) . Kondo (1984) r e p o r t s t h e development of a s e n s i t i v e and s p e c i f i c sandwich enzyme immunoassay f o r HCG u s i n g a n t i b o d i e s a g a i n s t the c a r b o x y 1 - t e r m i n a l p o r t i o n of the b e t a - s u b u n i t . In o r d e r t o improve the a n t i b o d y s p e c i f i c i t y f o r HCG (Kondo 1984), a n t i s e r a was passed t h r o u g h a column of human LH-Sepharose 4B b e f o r e use. H i g h l y s e n s i t i v e enzyme immunoassays f o r HCG a n a l y s i s (1 t o 20 IU/L) have a l s o been developed (Bock 1985, S t r o b e l 1985). -45-In a t w o - s i t e immunoradiometric a s s a y , S h i m i z u (1982) combined two monoclonal a n t i b o d i e s ; one d i r e c t e d a t a b i n d i n g s i t e unique t o the b e t a r e g i o n of the HCG m o l e c u l e , and t h e o t h e r d i r e c t e d a t a b i n d i n g s i t e u n i q u e t o the a l p h a r e g i o n . The r e q u i r e m e n t f o r two b i n d i n g s i t e s and the s p e c i f i c i t y of the monoclonal a n t i b o d i e s makes t h i s p r ocedure s p e c i f i c f o r o n l y i n t a c t HCG ( S h i m i z u 1982). S h a p i r o (1984) e v a l u a t e d an immunoradiometric assay t h a t employed two m o n o c l o n a l a n t i b o d i e s s p e c i f i c t o HCG; one a n t i b o d y embedded on a s o l i d - p h a s e medium and a second a n t i b o d y c o n t a i n i n g i o d i n e - 1 2 5 as r a d i o t r a c e r . One a n t i b o d y i s s p e c i f i c f o r the b e t a - s u b u n i t and the o t h e r a n t i b o d y r e a c t s a t a d i s t a n t s i t e such t h a t o n l y i n t a c t HCG i s sandwiched between them ( S h a p i r o 1984) . T h i s assay was found t o have s p e c i f i c i t y , a c c u r a c y and p r e c i s i o n s i m i l a r t o the RIA ( S h a p i r o 1984). A t w o - s i t e immunoradiometric assay u s i n g monoclonal a n t i b o d i e s t o e x t r a c t HCG from u r i n e has a l s o been de v e l o p e d (Armstrong 1984). U n t i l the s i g n i f i c a n c e of f r e e s u b u n i t s i n v a r i o u s n e o p l a s t i c d i s o r d e r s i s d e t e r m i n e d , o t h e r t e c h n i q u e s f o r q u a n t i t a t i o n of HCG s h o u l d a l s o be employed s i n c e the immunoradiometric assay i s s p e c i f i c f o r i n t a c t HCG ( S h a p i r o 1984). Another immunoradiometric assay ( R a t t l e 1984) w i t h two monoclonal a n t i b o d i e s d i r e c t e d a g a i n s t HCG has been d e v e l o p e d . T h i s assay uses a c a p t u r e monoclonal a n t i b o d y , -46-d i r e c t e d a g a i n s t a t h i r d s i t e on HCG, l a b e l l e d w i t h f l u o r e s c e i n i s o t h i o c y a n a t e ( F I T C ) . S e p a r a t i o n i s a c h i e v e d by i n c u b a t i o n w i t h a n t i - F I T C a n t i b o d i e s on magnetic p a r t i c l e s . In comparison t o s o l i d - p h a s e c a p t u r e a n t i b o d y a s s a y s , t h i s t e c h n i q u e enhances the r e a c t i o n k i n e t i c s and thus a l l o w s f a s t e r r e a c t i o n times ( R a t t l e 1984). The homogeneous s o l p a r t i c l e immunoassay of HCG ( L e u v e r i n g 1983) uses g o l d p a r t i c l e s c o a t e d w i t h the a n t i b o d y t o a g g l u t i n a t e w i t h t h e a n t i g e n . S p e c t r o p h o t o m e t r y and v i s u a l r e a d i n g , the d e t e c t i o n methods used w i t h t h e s e a s s a y s , a r e b a s e d on the o p t i c a l p r o p e r t i e s o f the d i s p e r s e d g o l d p a r t i c l e s ( L e u v e r i n g 1983). T h i s immunoassay i s not c o m m e r c i a l l y a v a i l a b l e a t p r e s e n t . A t w o - s i t e sandwich immunoassay u s i n g t i m e - r e s o l v e d f l u o r e s c e n c e ( e u r o p i u m - l a b e l ) and monoclonal a n t i b o d i e s has been r e p o r t e d by Stenman (1983). Sheep r e d b l o o d c e l l s c o a t e d w i t h a crude HCG p r e p a r a t i o n and monoclonal a n t i b o d i e s a g a i n s t HCG-beta were used t o d e v e l o p a h e m a g g l u t i n a t i o n i n h i b i t i o n a ssay (Rahamim 1984) . N e i t h e r of t h e s e a s s a y s are c o m m e r c i a l l y a v a i l a b l e . 1.6.2 E l e c t r o p h o r e s i s E l e c t r o p h o r e s i s methods f o r a n a l y s i s of HCG i n c l u d e sodium d o d e c y l s u l f a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s -47-(SDS-PAGE) (Morgan 1971, P a l a 1973, S c h l a f f 1976, Weber 1969), d i s c o n t i n u o u s (DISC) g e l e l e c t r o p h o r e s i s ( B e l l 1969, G r a e s s l i n 1972, Kapadia 1981, P a l a 1973, Q a z i 1974), i s o e l e c t r i c f o c u s i n g (IEF) (Gershey 1974, G r a e s s l i n 1971; 1972, M a f f e z z o l i 1972, Merz 1974, P a l a 1973, Q a z i 1974), c r o s s - e l e c t r o p h o r e s i s (Ohkawa 1966) and I m m u n o e l e c t r o p h o r e s i s ( G r a e s s l i n 1972, Hamashige 1967, Q a z i 1974) . SDS-PAGE of HCG w i t h 10% w/v a c r y l a m i d e ( P a l a 1973), a f t e r i n c u b a t i o n w i t h 4 M u r e a , 1% w/v SDS and 1% w/v 2 - m e r c a p t o e t h a n o l , shows two f r a c t i o n s ; a slow moving f r a c t i o n w i t h a m o l e c u l a r w e i ght of a p p r o x i m a t e l y 35000 Da and a f a s t moving f r a c t i o n a t a p p r o x i m a t e l y 27000 Da. U s i n g a DISC system w i t h 3.0% w/v a c r y l a m i d e f o r spacer g e l and 7.7% w/v a c r y l a m i d e f o r r u n n i n g g e l ( P a l a 1973), one f r a c t i o n a t 63000 Da was observed f o r HCG a f t e r i n c u b a t i o n i n 4 M u r e a and 1% w/v SDS. A l s o u s i n g a SDS-PAGE d i s c o n t i n u o u s system as r e p o r t e d by N e v i l l e (1971), the s u b u n i t s of HCG were c l e a r l y s e p a r a t e d from each o t h e r and from i n t a c t HCG ( S c h l a f f 1976). Re p o r t s i n the l i t e r a t u r e ( B e l l 1969, G r a e s s l i n 1972, Q a z i 1974) i n d i c a t e HCG g i v e s a s i n g l e , broad band w i t h DISC-PAGE ( w i t h o u t SDS). For p r o t e i n s DISC-PAGE u s u a l l y g i v e s v e r y s h a r p bands. However, f o r HCG ( B e l l 1969, G r a e s s l i n 1972, Swaminathan 1970) and o t h e r g l y c o p r o t e i n s (Cunningham 1966, Nueke 1961), -48-h e t e r o g e n e i t y observed i n the c a r b o h y d r a t e p o r t i o n c o n t r i b u t e s t o t h e b r o a d c h a r a c t e r of DISC bands. S i n c e the e l e c t r i c a l charge of g l y c o p r o t e i n s can be a l t e r e d by the q u a n t i t y of s i a l i c a c i d r e s i d u e s p r e s e n t , h e t e r o g e n e i t y i n the s i a l i c a c i d c o n t e n t o f HCG (Merz 1974) i s r e f l e c t e d by s l i g h t d i f f e r e n c e s i n e l e c t r o p h o r e t i c m o b i l i t y and thus broad bands w i t h DISC e l e c t r o p h o r e s i s . Other workers (Hammond 1971, Kapadia 1981, Kaplan 1972) have found DISC-PAGE of HCG a t a c i d or a l k a l i n e pH gave r i s e t o v a r i o u s zones r e p r e s e n t i n g d i f f e r e n t a g g r e g a t i o n s t a t e s . G r a e s s l i n (1971) s e p a r a t e d crude HCG (21 IU/mg) i n t o more than 30 p r o t e i n f r a c t i o n s e x h i b i t i n g s harp b a n d i n g i n the pH range 3 t o 10 w i t h g e l IEF. P o l y a c r y l a m i d e g e l IEF of HCG (2100 IU/mg) i n ' t h e pH range 3 t o 10 d i s p l a y e d f i v e s e p a r a t e d bands w i t h p i v a l u e s between 3.8 and 5.1 ( G r a e s s l i n 1972). A l l f i v e p r o t e i n bands p o s s e s s e d HCG a c t i v i t y ( G r a e s s l i n 1972) as de t e r m i n e d by b i o a s s a y (OAAD) and RIA. The g r e a t e s t b i o l o g i c a l a c t i v i t y was found i n the p r o t e i n band w i t h p i of 4.3 ( G r a e s s l i n 1972). Q a z i e t . a l . (1974) a l s o found the g r e a t e s t b i o l o g i c a l a c t i v i t y i n the p r o t e i n f r a c t i o n a t a p i of 4.3 u s i n g s u c r o s e d e n s i t y g r a d i e n t IEF. P o l y a c r y l a m i d e g e l IEF of HCG ( M a f f e z z o l i 1972) e x h i b i t e d a p i of about 3 as det e r m i n e d by i m m u n o r e a c t i v i t y . However, the method o f p o l y a c r y l a m i d e g e l IEF r e p o r t e d by P a l a (1973) i n d i c a t e s the peak of -49-i m m u n o r e a c t i v i t y f o r HCG i s a t a p i o f 4.90. Hammond (1971) found IEF of HCG from human c h o r i o c a r c i n o m a c e l l s was c h a r a c t e r i z e d by two bands w i t h p i v a l u e s o f 3 and 4.5. Merz (1974) found r e s u l t s t h a t were i n agreement w i t h e a r l i e r endeavors ( G r a e s s l i n 1972). F i v e homogeneous bands w i t h p i v a l u e s r a n g i n g from 4.0 t o 4.9 (Merz 1974) were i s o l a t e d u s i n g IEF on Sephadex t h i n l a y e r s . Gas chr o m a t o g r a p h i c a n a l y s i s (Merz 1974) of the amino a c i d c o m p o s i t i o n of thes e f r a c t i o n s a r e c l o s e w i t h l i t e r a t u r e v a l u e s . D e t e r m i n a t i o n of NANA c o n t e n t u s i n g a t h i o b a r b i t u r i c a c i d c o l o u r i m e t r i c assay f o r s i a l i c a c i d s (Warren 1959) and o t h e r c a r b o h y d r a t e s by gas chromatography (Merz 1974) i n d i c a t e d the major d i f f e r e n c e between the f r a c t i o n s o b t a i n e d by IEF was s i a l i c a c i d c o n t e n t . S i n c e removal o f s i a l i c a c i d e l i m i n a t e s e l e c t r o p h o r e t i c h e t e r o g e n i e t y o f HCG and i t s s u b u n i t s (Merz 1974) i t i s p o s t u l a t e d t h a t s i a l i c a c i d a l o n e i s r e s p o n s i b l e f o r the h e t e r o g e n e i t y o b s e r v e d . I t has been suggested ( G r a e s s l i n 1972) t h a t d i f f e r e n c e s i n s i a l i c a c i d c o n t e n t may be due t o an i n c o m p l e t e b i o s y n t h e s i s or t o p a r t i a l d e g r a d a t i o n by k i d n e y g l y c o s i d a s e s . A f t e r removal of s i a l i c a c i d (Warren 1959) , IEF i n a s u c r o s e g r a d i e n t (Gershey 1974) gave a p i v a l u e o f 10.0 f o r HCG w h i l e the p i v a l u e s f o r t h e a l p h a and b e t a s u b u n i t s were 9.5 and 10.5 r e s p e c t i v e l y . C r o s s - e l e c t r o p h o r e s i s i n agar g e l u s i n g r a b b i t -50-a n t i s e r u m a g a i n s t HCG (Ohkawa 1966) has been r e p o r t e d f o r q u a n t i t a t i o n of u r i n a r y HCG from pregnant women. Immu n o e l e c t r o p h o r e s i s ( G r a e s s l i n 1972, Hamashige 1967, Q a z i 1974) methods f o r HCG a n a l y s i s have a l s o been r e p o r t e d . 1.6.3 B i o l o g i c a l Assays V a r i o u s b i o l o q i c a l a s s a y s of HCG a c t i v i t y a re found i n the l i t e r a t u r e . They i n c l u d e the m o d i f i e d r a t v e n t r a l p r o s t a t e w e i g h t (VPW) assay (McArthur 1952, Morgan 1971), r a t i n t e r s t i t i a l c e l l t e s t o s t e r o n e assay ( C z e k a l a 1981), minimum e f f e c t i v e dose i n the p o s t partum r a b b i t ( G u r i n 1939), o v a r i a n a s c o r b i c a c i d d e p l e t i o n assay (OAAD) ( C a n f i e l d 1971) and the u t e r i n e w e i g h t a s s a y ( C a n f i e l d 1971, Morgan 1971) . Androgen s e c r e t i o n from HCG s t i m u l a t e d t e s t i c u l a r i n t e r s t i t i a l t i s s u e produces an i n c r e a s e i n the w e i g h t o f a c c e s s o r y sex organs i n t h e h y p o p h y s e c t o m i z e d male r a t . The m o d i f i e d r a t a s s a y i n v o l v e s i n j e c t i n g the p r e p a r a t i o n of HCG f o r f o u r days i n t o h y p o p h y s e c t o m i z e d male r a t s , t h e n w e i g h i n g the v e n t r a l p r o s t a t e a f t e r removal o f f a t and f i b r o u s t i s s u e (McArthur 1952). T h i s a s s a y has a l s o been used f o r FSH (McArthur 1952) and LH (Turner 1971). In the r a t i n t e r s t i t i a l c e l l t e s t o s t e r o n e a s s a y , t h e r e i s a p r o l o n g e d i n c r e a s e i n t e s t o s t e r o n e p r o d u c t i o n w i t h i n t e n minutes of HCG i n f u s i o n and measurement of the -51-t e s t o s t e r o n e l e v e l s i s c o r r e l a t e d w i t h the amount of HCG i n f u s e d (Davies 1982). P o s t partum r a b b i t does were i n j e c t e d w i t h the HCG sample t o be a s s a y e d . I n s p e c t i o n o f the o v a r i e s 24 t o 48 hours l a t e r f o r f r e s h l y o v u l a t e d f o l l i c l e s was used t o determine the minimum e f f e c t i v e dose per kg of r a b b i t body w e i g h t n e c e s s a r y t o cause o v u l a t i o n ( G u r i n 1939) . The u t e r i n e w e i g h t b i o a s s a y of HCG i n v o l v e s subcutaneous i n j e c t i o n o f the sample t o female mice or r a t s over a g i v e n p e r i o d of t i m e , removal of the u t e r i and s e p a r a t i o n of mesentery, f a t , o v a r i e s and o v i d u c t s . The i n t r a u t e r i n e f l u i d was then removed u s i n g f i l t e r paper b e f o r e the u t e r u s was weighed (Hobson 1983, USP 1980). U s i n g t h i s b i o a s s a y the r e s u l t s o b t a i n e d f o r monkey CG were s i g n i f i c a n t l y a f f e c t e d by the number of i n j e c t i o n s made, the i n t e r v a l between i n j e c t i o n s and the time of autopsy a f t e r the f i r s t i n j e c t i o n i n t o the mice (Hobson 1983) . R e s u l t s o b t a i n e d from t h e u t e r i n e w e i g h t a s s a y f o r HCG u s i n g mice or r a t s w i t h s l i g h t l y d i f f e r e n t p r o c e d u r e s may not be comparable. A r a t u t e r i n e w e i g h t b i o a s s a y i s the o f f i c i a l USP assay f o r HCG (USP 1980). S p e c i f i c i n s t r u c t i o n s ensure t h a t r e s u l t s from d i f f e r e n t a s s a y s w i t h t h i s method are comparable. Some problems w i t h the o f f i c i a l USP assay f o r HCG are the s a c r i f i c e of l a r g e numbers of a n i m a l s (minimum -52-of 60 female r a t s per a s s a y ) , the l e n g t h of time r e q u i r e d f o r each assay (5 days) t h a t may need t o be r e p e a t e d i f the r e s u l t s do not meet the s t a t i s t i c a l r e q i r e m e n t s and the l a c k of s p e c i f i c i t y of the a s s a y . A l s o , the use of a n i m a l s and the n e c e s s a r y a n i m a l c a r e f a c i l i t i e s make t h i s an e x p e n s i v e a s s a y . 1.6.4 Chromatograpic Methods Chromatography i s a method of s e p a r a t i o n t h a t depends on the d i f f e r e n t i a l d i s t r i b u t i o n of a m i x t u r e between two phases. The f o u r broad c l a s s i f i c a t i o n s of c h r o m a t o g r a p h i c s e p a r a t i o n are a d s o r p t i o n , p a r t i t i o n , i o n exchange and s i z e e x c l u s i o n . These c l a s s e s d i f f e r i n t h e n a t u r e of t h e f o r c e s t h a t d etermine the d i s t r i b u t i o n of the s o l u t e between the two phases. S e p a r a t i o n w i t h a d s o r p t i o n chromatography depends on the v a r i o u s degrees of a f f i n i t y of the s o l u t e s f o r t h e s o l i d s t a t i o n a r y phase. In p a r t i t i o n c hromatography the s t a t i o n a r y phase i s a l i q u i d and s e p a r a t i o n depends on the r e l a t i v e s o l u b i l i t y ( p a r t i t i o n ) o f the s o l u t e s i n the two phases. With i o n exchange chromatography, the most i m p o r t a n t s i n g l e f a c t o r i n s e p a r a t i o n i s exchange of i o n s . S i z e e x c l u s i o n s e p a r a t i o n s a r e p r i m a r i l y based on m o l e c u l a r s i z e and shape i n s o l u t i o n . The v a r i o u s ways of a p p l y i n g t h e s e c l a s s e s of c h r o m a t o g r a p h i c s e p a r a t i o n are paper, -53-t h i n - l a y e r , l i q u i d chromatography (both column and high-performance l i q u i d chromatography) and gas chromatography. L i q u i d c h r o m a t o g r a p h i c methods have been used f o r the p u r i f i c a t i o n and s e p a r a t i o n of HCG from b i o l o g i c a l samples and w i l l be d i s c u s s e d h e r e . 1.6.4.1 Column Chromatography of HCG Column c h r o m a t o g r a p h i c t e c h n i q u e s used f o r p u r i f i c a t i o n of HCG i n c l u d e s i z e e x c l u s i o n (SEC), i o n exchange (IEC) and a d s o r p t i o n . The s i z e e x c l u s i o n p a c k i n g s used i n c l u d e B i o - G e l ( p o l y a c r y l a m i d e g e l s ) ( C a n f i e l d 1971, M i d g l e y 1966) and Sephadex g e l s ( c r o s s - l i n k e d d e x t r a n ) (Bahl 1969a, C a n f i e l d 1971, T a i a d o u r o s 1982, Van H e l l 1978) of v a r y i n g pore s i z e s . Both a n i o n i c and c a t i o n i c exchange r e s i n s have been r e p o r t e d f o r p u r i f i c a t i o n o f HCG. The most common a n i o n exchangers are Dowex 1 (Bahl 1969b), d i e t h y l a m i n o e t h y l - S e p h a d e x (DEAE-Sephadex) (Bahl 1969a, C a n f i e l d 1971, Merz 1974) and D E A E - c e l l u l o s e (Van H a l l 1971). For c a t i o n e x c h a n g e r s , use of sulphopropy1-Sephadex (SP-Sephadex) (Merz 1974), sulphoethy1-Sephadex (SE-Sephadex) ( C a n f i e l d 1971) and carboxymethy1-Sephadex (CM-Sephadex) (Van H e l l 1968) have been r e p o r t e d i n the l i t e r a t u r e . Katzman (1943) d e s c r i b e s the use of P e r m u t i t f o r a d s o r p t i o n of HCG. V i t a c h a n g e ( S h i m i z u 1984) and k a o l i n -54-( A y a l a 1978) have a l s o been r e p o r t e d f o r HCG a d s o r p t i o n . C o m m e r c i a l l y a v a i l a b l e HCG (3200 IU/mg) was p u r i f i e d by column chromatography (Bahl 1969a) on DEAE-Sephadex A-50 f o l l o w e d by Sephadex G-100. F u r t h e r e x a m i n a t i o n (Bahl 1969a) showed the p r e p a r a t i o n t o be homogeneous by d i s c o n t i n u o u s and i m m u n o - e l e c t r o p h o r e s i s , u l t r a c e n t r i f u g a t i o n and end group a n a l y s i s . U s i n g the r a t VPW a s s a y (Bahl 1969a), the p r e p a r a t i o n had an a c t i v i t y of 12000 IU/mg. Gel f i l t r a t i o n on Sephadex G-150 i n d i c a t e d a m o l e c u l a r weight of 59000 +_ 4000 Da f o r HCG and 30000 + 2000 Da f o r reduced c a r b o x a m i d o m e t h y l a t e d HCG (Bahl 1969a) . S i m i l a r l y Van H a l l (1971) p u r i f i e d HCG u s i n g two t y p e s of c h r o m a t o g r a p h i c columns. I n i t i a l chromatography w i t h DEAE-Sephadex or D E A E - c e l l u l o s e was performed w i t h subsequent p u r i f i c a t i o n on CM-Sephadex (Van H a l l 1971) f o l l o w e d by d e t e r m i n a t i o n of b i o l o g i c a l and i m m u n o l o g i c a l p o t e n c y . T h i s method was used t o e v a l u a t e the a c t i v i t y of HCG a f t e r p r o g r e s s i v e d e s i a l y l a t i o n . Van H e l l (1968) d e v e l o p e d a method f o r p u r i f i c a t i o n of HCG u s i n g two c h r o m a t o g r a p h i c columns; CM-Sephadex C-25 w i t h f i n a l f r a c t i o n a t i o n on Sephadex G-100 or Sephadex G-200. Ch r o m a t o g r a p h i c a d s o r p t i o n o f u r i n a r y HCG onto P e r m u t i t has been i n v e s t i g a t e d . P e r m u t i t i s a s y n t h e t i c h y d r a t e d a l k a l i - a l u m i n u m s i l i c a t e w i t h an approximate c o m p o s i t i o n of N a _ o . A l O _ . 2 S i O _ . 6 H 0 (Merck 1952) t h a t can be -55-used as an a d s o r b e n t . More r e c e n t l y , S h i m i z u (1984) d e v e l o p e d an e x t r a c t i o n method f o r c o n c e n t r a t i n g HCG u r i n e samples u s i n g V i t a c h a n g e f o l l o w e d by a s e n s i t i v e RIA. V i t a c h a n g e , a c o m m e r c i a l l y a v a i l a b l e s i l i c a - b a s e d a d s o r b e n t , gave h i g h e r r e c o v e r y of HCG th a n t h e s t a n d a r d k a o l i n - a c e t o n e method ( A l b e r t 1956, A y a l a 1978, S h i m i z u 1984). 1.6.4.2 HPLC of HCG Comparing s i z e e x c l u s i o n HPLC t o methods f r e q u e n t l y used i n p r o t e i n a n a l y s i s , column SEC, u l t r a c e n t r i f u g a t i o n and e l e c t r o p h o r e s i s , i m p o r t a n t advantages a r e found. They i n c l u d e the speed of s e p a r a t i o n , which i s e s p e c i a l l y i m p o r t a n t when i s o l a t i n g u n s t a b l e p r o t e i n s , a r e a s o n a b l y s m a l l amount of p r o t e i n a d s o r p t i o n onto the column s t a t i o n a r y phase, s m a l l amounts of p r o t e i n s can be assayed and r e c o v e r y of p r o t e i n s i n a n a t i v e form can o f t e n be a c h i e v e d (Barden 1983, R i c h t e r 1983). M o l e c u l e s t h a t are too l a r g e t o p e n e t r a t e i n t o the pores of the s t a t i o n a r y phase w i l l e l u t e a t the v o i d volume w h i l e those t h a t can p e n e t r a t e a l l the pores w i l l e l u t e a t the t o t a l p e r m e a t i o n volume of the column. Between th e s e l i m i t s , m o l e c u l e s of i n t e r m e d i a t e s i z e e l u t e a c c o r d i n g t o t h e i r m o l e c u l a r s i z e i n the m o b i l e phase. However, hy d r o p h o b i c i n t e r a c t i o n w i t h bonded o r g a n i c groups, i o n exchange of b a s i c p r o t e i n s w i t h -56-s i l a n o l groups and i o n exchange of n e g a t i v e l y charged s o l u t e s can a l t e r t he e l u t i o n volume (Barden 1983). S a l t s have been added t o the mo b i l e phase b u f f e r i n o r d e r t o mi n i m i z e the e f f e c t s of the s t a t i o n a r y phase on p r o t e i n r e t e n t i o n (Barden 1983). The e l u t i o n volumes of p r o t e i n s v a r y as a f u n c t i o n o f i o n i c s t r e n g t h of the mo b i l e phase (Barden 1983). D e n a t u r a n t s such as u r e a , g u a n i d i n e h y d r o c h l o r i d e and SDS cause g l o b u l a r p r o t e i n s t o u n f o l d w i t h o u t a f f e c t i n g some s t a t i o n a r y phases used. T h i s u n f o l d i n g i n c r e a s e s the e q u i v a l e n t hydrodynamic r a d i u s ( S t o k e s ' r a d i u s ) of the p r o t e i n s , t h e r e f o r e i n c r e a s i n g t h e i r a p parent m o l e c u l a r weight (Barden 1983). For s i z e e x c l u s i o n HPLC a number of columns a re c o m m e r c i a l l y a v a i l a b l e . P r e s e n t l y , the Toya Soda M a n u f a c t u r i n g Company of Japan produce many SEC HPLC columns which a re then made a v a i l a b l e t h r o u g h o t h e r companies (such as LKB, Beckman In s t r u m e n t s I n c . , BioRad L a b o r a t o r i e s and Waters A s s o c i a t e s ) as w e l l as from Toya Soda. Many r e s e a r c h e r s a re u s i n g t h e s e columns as shown by r e c e n t r e p o r t s i n the l i t e r a t u r e . For example the TSK sw s e r i e s ( s i l i c a r e s i n , water s o l u b l e ) i s used f r e q u e n t l y f o r p r o t e i n s and o t h e r macromolecules (Barden 1983, Borrebaeck 1984, G i r a r d i 1983, Graeve 1983, Gupta 1983, Hunt 1983, Ka w a n i s h i 1983; 1984, Loo 1984, Maezawa 1983, R i c h t e r 1983; 1984, S e l l i t t i 1983, Wi e h l e 1984). Columns packed w i t h a -57-polymer based m a t e r i a l t h a t i s water s o l u b l e are a l s o used (TSK pw) (Hase 1984, R i c h t e r 1983; 1984). Both column types are a v a i l a b l e i n v a r i o u s pore s i z e s u s e f u l f o r s e p a r a t i o n of macromolecules. New c a t i o n (sulphopropy1) and anion exchangers (diethylaminoethy1) and hydrophobic ( b u t y l and phenyl) supports f o r HPLC have been d e r i v e d from these TSK s i z e e x c l u s i o n s t a t i o n a r y phases (Kato 1983a; 1983b; 1984a; 1984b) f o r macromolecules. High-performance l i q u i d chromatographic s e p a r a t i o n of HCG and i t s subunits was achieved u s i n g TSK-G 3000 sw ( F o r a s t i e r i 1982, Shimohigashi 1983), TSK-G 2000 sw (Shimohigashi 1982) , B i o - S i l TSK-125 and TSK-250 s i z e e x c l u s i o n and \i Bondapak C l g (Putterman 1982) , Vydac 218 TP1010 C l g (Parsons 1984) and C g (Moudgal 1982) reversed-phase columns. F o r a s t i e r i and Ingham ( F o r a s t e r i 1982) used HPLC a n a l y s i s to study the r e v e r s i b l e d i s s o c i a t i o n of the subunits of HCG at n e u t r a l pH. The e l u t i o n time was monitored by absorbance at 280 nm. Using a TSK-G 3000 sw column, 0.01 M phosphate b u f f e r (pH 7) and a flow r a t e of 1 mL/min., the u n d i s s o c i a t e d hormone e l u t e s at 14 minutes ( F o r a s t i e r i 1982) . A f t e r d i s s o c i a t i o n of the subunits the b e t a - s u b u n i t e l u t e s i n a p o s i t i o n almost i d e n t i c a l with t h a t of i n t a c t HCG. The al p h a - s u b u n i t appears at around 17.5 minutes. - 5 8 -S h i m o h i g a s h i and Chen (1982) s t u d i e d the e f f e c t s of a l t e r a t i o n of the c a r b o h y d r a t e s t r u c t u r e s i n b o t h s u b u n i t s on p o t e n c y . Absorbance a t 225 nm was used f o r d e t e c t i n g n a t i v e and a l t e r e d HCG. U n a l t e r e d HCG was e l u t e d a t 44 minutes u s i n g one TSK sw 2000 and two sw 3000 columns w i t h 0.1 M ammonium a c e t i c a c i d b u f f e r (pH 7.0) and a f l o w r a t e of 1 mL/min. Under the same c o n d i t i o n s , a l t e r a t i o n s i n the a l p h a - s u b u n i t and the b e t a - s u b u n i t caused the e l u t i o n time t o i n c r e a s e . One TSK-G 2000 sw and two TSK-G 3000 sw HPLC columns connected i n s e r i e s ( S h i m o h i g a s h i 1983) gave a r e t e n t i o n time of 40 t o 60 minutes f o r HCG when a 0.1 M ammonium a c e t a t e - a c e t i c a c i d b u f f e r (pH 7.0) and a f l o w r a t e of 1.0 mL/min were used. On the b a s i s o f a s e r i e s of r e f e r e n c e g l o b u l a r p r o t e i n s , t h e e s t i m a t e d v a l u e s of m o l e c u l a r w e i g h t s of HCG and d e g l y c o s y l a t e d HCG d e v i a t e d from t h e i r a c t u a l m o l e c u l a r w e i g h t s . S h i m o h i g a s h i (1983) found the e x t e n t of d e v i a t i o n depended on the c a r b o h y d r a t e c o n t e n t and p o s s i b l y the l o c a t i o n of the c a r b o h y d r a t e c h a i n s . Recombinant s t u d i e s (Shimogashi 1983) su g g e s t e d t h a t the b e t a - s u b u n i t i s the dominant d e t e r m i n a n t f o r t h e expanded m o l e c u l a r s i z e and thus the apparent m o l e c u l a r weight by the above methods. Reversed-phase HPLC s e p a r a t i o n of HCG and i t s s u b u n i t s (Putterman 1982) u s i n g a l i n e a r s o l v e n t g r a d i e n t from 0% B t o 60% B (A: t r i f l u o r o a c e t i c a c i d / w a t e r 0.5/1000, B: -59-t r i f l u o r o a c e t i c a c i d / w a t e r / a c e t o n i t r i l e 0.425/3.825/995.75) oc c u r e d w i t h i n 60 m i n u t e s . M o n i t o r i n g 206 nm, HCG and i t s s u b u n i t s were found t o e l u t e a t 40 t o 50 m i n u t e s , s i m i l a r t o t h a t observed w i t h the e x c l u s i o n columns. W i t h a l i n e a r g r a d i e n t of sodium phosphate (pH 6.8) t o 50% a c e t o n i t r i l e : 5 0 % sodium phosphate (pH 6.8) and a f l o w r a t e o f 2 mL/min, th e s u b u n i t s o f HCG were s e p a r a t e d u s i n g a Vydac C^- column (Parsons 1984). Reversed-phase HPLC a n a l y s i s o f HCG and i t s s u b u n i t s w i t h a pH g r a d i e n t from 7.0 t o 2.0 on an a l k y l p h e n y l column has a l s o been r e p o r t e d (Grego 1984). The a l p h a - and b e t a - s u b u n i t s of HCG found t o be homogeneous by a m i n o - a c i d a n a l y s i s gave two peaks when e v a l u a t e d by t h i s method (Grego 1984) . Moudgal (1982) was a b l e t o s e p a r a t e i n t a c t HCG from i t s s u b u n i t s u s i n g a C Q o column (4.5 x 250 mm) and a l i n e a r g r a d i e n t of n-propanol (10% t o 40%) . The e f f e c t of r e v e r s e d - p h a s e HPLC on the b i o l o g i c a l a c t i v i t y o f HCG has r e c e n t l y been r e p o r t e d by W i l k s and B u t l e r ( W i l k s 1984). F o l l o w i n g methods r e p o r t e d i n the l i t e r a t u r e (Moudgal 1982, Putterman 1982), C g and C 1 8 columns were used w i t h m obile phases p r e v i o u s l y d e s c r i b e d , as w e l l as newly d e v e l o p e d s o l v e n t s y s t e m s . Only 10% t o 60% of the b i o l o g i c a l a c t i v i t y , as d e t e r m i n e d by o v a r i a n a d e n y l a t e c y c l a s e a c t i v i t y , was r e t a i n e d f o l l o w i n g r e v e r s e d - p h a s e HPLC ( W i l k s 1984). Some of the reduced -60-b i o l o g i c a l a c t i v i t y was a t t r i b u t e d t o d i s s o c i a t i o n of the s u b u n i t s of HCG a t the low pH of the mobile phases ( W i l k s 1984). The r e m a i n i n g l o s s i n b i o l o g i c a l a c t i v i t y a f t e r n e u t r a l i z a t i o n of the pH p r i o r t o l y o p h i l i z a t i o n and b i o a s s a y i s presumably due t o o r g a n i c s o l v e n t d e n a t u r a t i o n ( W i l k s 19 84). To d a t e , o n l y p u r i f i e d i n t a c t HCG and/or i t s i n d i v i d u a l s u b u n i t s ( F o r a s t i e r i 1982, Grego 1984, Moudgal 1982, Parsons 1984, S h i m o h i g a s h i 1982; 1983, W i l k s 1984) and crude HCG i s o l a t e d from the u r i n e of pregnant women (Putterman 1982) have been e v a l u a t e d by HPLC a n a l y s i s . Amino-acid a n a l y s i s (Grego 1984, Parsons 1984, Putterman 1982, S h i m o h i g a s h i 1982), e l e c t r o p h o r e s i s (Parsons 1984, Putterman 1982), b i n d i n g a c t i v i t y ( S h i m o h i g a s h i 1982) and immunoassay methods (Putterman 1982) have been used t o v e r i f y peak i d e n t i t y , however, peak homogeneity has not been c l e a r l y e s t a b l i s h e d . A l s o , none of the s e HPLC methods of a n a l y s i s have attempted t o q u a n t i t a t e HCG ( F o r a s t i e r i 1982, Grego 1984, Moudgal 1982, P a r s o n s 1984, Putterman 1982, S h i m o h i g a s h i 1982; 1983, W i l k s 1984) . F o r a s t i e r i (1982) had problems s e p a r a t i n g i n t a c t HCG from the b e t a - s u b u n i t w i t h s i z e e x c l u s i o n HPLC. Reversed-phase HPLC a n a l y s i s of HCG w i t h b o t h C g (Moudgal 1982, W i l k s 1984) and C 1 D (Parsons 1984, Putterman 1982, W i l k s 1984) HPLC columns r e q u i r e d g r a d i e n t e l u t i o n and 40% -61-t o 90% o f the b i o l o g i c a l a c t i v i t y was l o s t d u r i n g chromatography ( W i l k s 1984). Only one peak was obser v e d f o r p u r i f i e d i n t a c t HCG by s i z e - e x c l u s i o n ( F o r a s t i e r i 1982, S h i m o h i g a s h i 1982; 1983) and r e v e r s e d - p h a s e (Moudgal 1982) HPLC, however, more r e c e n t e v a l u a t i o n of i o d i n e - 1 2 5 l a b e l l e d p u r i f i e d HCG ( W i l k s 1984) i n d i c a t e d many components were p r e s e n t . The o f f i c i a l r a t u t e r i n e w e i ght b i o a s s a y r e q u i r e d f o r q u a n t i t a t i o n of a l l p r e p a r a t i o n s of HCG i s time consuming, e x p e n s i v e , r e q u i r e s the s a c r i f i c e of a l a r g e number of a n i m a l s , and i s not s p e c i f i c f o r HCG. A c h r o m a t o g r a p h i c assay p r o c e d u r e s u i t a b l e f o r r e p l a c i n g t h i s o f f i c i a l a s say f o r q u a n t i t a t i o n of HCG would p r e s e n t many advantages. S p e c i f i c , s e n s i t i v e , r a p i d and r e l a t i v e l y i n e x p e n s i v e HPLC assay p r o c e d u r e s have been dev e l o p e d t o r e p l a c e l e s s r e l i a b l e b i o a s s a y s p r e v i o u s l y used by the USP. C o n t i n u i n g t h i s t r e n d , a t t e m p t s were made t o d e v e l o p an HPLC assay p r o c e d u r e s u i t a b l e f o r r e p l a c i n g the USP b i o a s s a y f o r HCG. Commercial p r e p a r a t i o n s of HCG, USP Refer e n c e S t a n d a r d HCG, p u r i f i e d i n t a c t HCG and p u r i f i e d s u b u n i t s of HCG were e v a l u a t e d by v a r i o u s HPLC a s s a y p r o c e d u r e s . E l e c t r o p h o r e s i s t e c h n i q u e s and a g g l u t i n a t i o n i n h i b i t i o n immunoassays were used f o r a n a l y s i s of c h r o m a t o g r a p h i c peak e l u a t e t o v e r i f y peak i d e n t i t y . -62-2. EXPERIMENTAL 2.1 S u p p l i e s 2.1.1 E l e c t r o p h o r e s i s T r i c h l o r o a c e t i c a c i d - Baker and Adamson, M o r r i s t o w n , NJ, U.S.A. Bromophenol b l u e , G l y c e r i n - B r i t i s h Drug Houses (Canada) L t d . , T o r o n t o , Ont., Canada. H y d r o c h l o r i c a c i d , G l a c i a l a c e t i c a c i d , I s o p r o p a n o l , V o r t e x - G e n i e - F i s h e r S c i e n t i f i c Co., Vancouver, b.c., Canada. B l a c k I n d i a n i n k - Hunt M a n u f a c t u r i n g Co., S t a t e s v i l l e , NC, U.S.A. Ammonium p e r o x y d i s u l f a t e , Coomassie b r i l l i a n t b l u e R-250, Dodecyl sodium s u l f a t e - Eastman Kodak Co., Kodak L a b o r a t o r y and S p e c i a l t y C h e m i c a l s , R o c h e s t e r , NY, U.S.A. A c r y l a m i d e , G l y c i n e , 2 - M e r c a p t o e t h a n o l , N,N'-Methylene-bis- a c r y l a m i d e , D a l t o n Mark VI SDS M o l e c u l a r Weight M a r k e r s , R i b o f l a v i n , Sucrose grade 1, N,N,N',N'-Tetramethy1- E t h y l e n e d i a m i n e (TEMED), Tris(hydroxymethy1)aminomethane - Sigma Chemical Co., S t . L o u i s , MO, U.S.A. Urea- M a l l i n c k r o d t Chemical Works, S t . L o u i s , MO, U.S.A. L y o p h i l i z e r - V i r t i s Co., D i v i s i o n of Cenco M e d i c a l / H e a l t h Supply c o . , G a r d i n e r , NY, U.S.A. 2.1.2 High Performance L i q u i d Chromatography -63-G e l F i l t r a t i o n S t a n d a r d - Bio - R a d L a b o r a t o r i e s (Canada) L t d . , M i s s a s s a u g a , Ont., Canada. M e t h a n o l , P r o p a n - 2 - o l , Water Omnisolv HPLC grade -B r i t i s h Drug Houses (Canada) L t d . , Vancouver, B.C., Canada Three way PTFE v a l v e ( P a r t # 1102) - O m n i f i t L t d . , Cambridge Engl a n d . T r i f l u o r o a c e t i c a c i d - Eastman Kodak Co., Kodak L a b o r a t o r i e s and S p e c i a l t y C h e m i c a l s , R o c h e s t e r , NY, U.S.A. A c e t o n i t r i l e HPLC grade - F i s h e r S c i e n t i f i c Co., Vancouver, B.C., Canada. 2.1.3 Human C h o r i o n i c G o n a d o t r o p i n A.P.L. HCG f o r I n j e c t i o n U.S.P. - A y e r s t L a b o r a t o r i e s , M o n t r e a l , Que., Canada. Human C h o r i o n i c G o n a d o t r o p i n f o r I n j e c t i o n - K-Line P h a r m a c e u t i c a l s L t d . , Downsview, Ont., Canada. USP Human C h o r i o n i c G o n a d o t r o p i n R e f e r e n c e S t a n d a r d -US Ph a r m a c o p i a l C o n v e n t i o n I n c . , R o c k v i l l e , MD, U.S.A. P u r i f i e d Human C h o r i o g o n a d o t r o p i n , P u r i f i e d A l p h a - S u b u n i t Human C h o r i o g o n a d o t r o p i n , P u r i f i e d B e t a - S u b u n i t C h o r i o g o n a d o t r o p i n - B o e h r i n g e r Mannheim Canada L t d . , D o r v a l , Que., Canada. 2.1.4 M i s c e l l a n e o u s T r i e t h y l a m i n e - A l d r i c h Chemical Co., Milwaukee, WI, U.S.A. Sodium Hy d r o x i d e - American S c i e n t i f i c and C h e m i c a l , P o r t l a n d , OR, U.S.A. B e n z y l a l c o h o l , E t h y l e n e d i a m i n e t e t r a a c e t a t e t e t r a s o d i u m - B r i t i s h Drug Houses L t d . , L a b o r a t o r y Chemicals -64-D i v . , P o o l e , E n g l a n d . Sodium a c e t a t e anhydrous ( a n a l y t i c a l r e a g e n t g r a d e ) , Sodium c h l o r i d e , Sodium phosphate monobasic monohydrate, Sodium s u l f a t e (AnalaR grade) - B r i t i s h Drug Houses (Canada) L t d . , T o r o n t o , Ont., Canada. Sodium b i c a r b o n a t e , Ammonium h y d r o x i d e , Sodium c a r b o n a t e , Phenol - Baker and Adamson, A l l i e d C h e m i c a l , S p e c i a l t y Chemicals D i v i s i o n , M o r r i s t o w n , NJ, U.S.A. C a l c i u m c h l o r i d e , P o t a s s i u m c h l o r i d e , Sodium a z i d e , Acetone - F i s h e r S c i e n t i f i c Co., Vancouver, B.C., Canada. Ammonium c h l o r i d e , Ammonium phosphate monobasic -M a i l i n c k r o d t C h e m i c a l Works, M o n t r e a l , Que., Canada. Ammonium b i c a r b o n a t e - Matheson, Coleman and B e l l , Norwood, OH, U.S.A. L a t e x Pregnancy S l i d e T e s t K i t ( B e t a - s u b u n i t ) - N.C.S. D i a g n o s t i c s I n c . , M i s s i s s a u g a , Ont., Canada. P r e g n o s i s L a t e x A g g l u t i n a t i o n I n h i b i t i o n S l i d e T e s t f o r Pregnancy, Fluram - Roche D i a g n o s t i c s D i v . , Hoffman-LaRoche I n c . , N u t l e y , NJ, U.S.A. S p e c t r a / P o r 3 d i a l y s i s t u b i n g - Spectrum M e d i c a l I n d u s t r i e s I n c . , Los A n g e l e s , CA, U.S.A. 2 . 2 Equipment 2.2 .1 E l e c t r o p h o r e s i s E-C G e l Column E l e c t r o p h o r e s i s Apparatus - E-C Apparatus Corp., S t . P e t e r s b u r g , FL, U.S.A. PS 1200 Power Supply - Hoefer S c i e n t i f i c I n s t r u m e n t s , San F r a n c i s c o , CA, U.S.A. -65-2.2.2 High Performance L i q u i d Chromatography Beckman Model 100 A S o l v e n t M e t e r i n g Pump w i t h A l t e x C RIA Chromatopac Data P r o c e s s o r - Beckman ( A l t e x ) I n s t r u m e n t s I n c . , F u l l e r t o n , CA, U.S.A. H e w l e t t P a c k a r d Model 1080 L i q u i d Chromatograph -H e w l e t t Packard L t d . , A n a l y t i c a l P r o d u c t M a n u f a c t u r i n g D i v i s i o n , A v o n d a l e , PA, U.S.A. Model U6K HPLC I n j e c t o r - Waters A s s o c i a t e s , M i l f o r d , MA, U.S.A. Rheodyne Model 7125 F i x e d Loop HPLC I n j e c t o r -Rheodyne, B e r k e l e y , CA, U.S.A. P r o t e i n Pak 125 column (7.8 mm x 30 cm), P r o t e i n Pak 300 sw columns (7.5 mm x 30 cm), P r o t e i n Pak DEAE 5pw (7.5 mm x 7.5 cm) - Waters A s s o c i a t e s , M i l f o r d , MA, U.S.A. U l t r a s p h e r e ODS column (4.6 mm x 25 cm), S p e c t r o p h o t o m e t e r 155-10 - Beckman I n s t r u m e n t s I n c . , B e r k e l e y , CA, U.S.A. S p e c t r o f l u o r o m e t e r FS970 w i t h 470 nm Emmission F i l t e r -S c h o e f f e l Instrument Corp., Westwood, NJ, U.S.A. S o l v e n t C l a r i f i c a t i o n K i t , Membrane F i l t e r s (0.45 m i c r o m e t e r ) , N o r g a n i c C a r t r i d g e s - L a b o r a t o r y P r o d u c t s D i v i s i o n , M i l l i p o r e C o r p o r a t i o n , B e d f o r d , MA, U.S.A. 2.3 Fluorescamine D e r i v a t i z a t i o n of HCG F l u o r e s c a m i n e (1.4 mg) was weighed a c c u r a t e l y and d i s s o l v e d i n HPLC grade a c e t o n i t r i l e . T h i s f l u o r e s c a m i n e s o l u t i o n (0.20 mL) was added r a p i d l y t o 0.10 mL (200 IU) -66-a l i q u o t s o f HCG w h i l e p l a c e d on a v o r t e x m i x e r . The f l u o r e s c a m i n e s o l u t i o n was s t o r e d a t room t e m p e r a t u r e , p r o t e c t e d from l i g h t , and the d e r i v a t i v e of HCG was p r e p a r e d as r e q u i r e d . 2.4 Recombination of HCG Subunits Commercial HCG samples were i n c u b a t e d w i t h 0.02 M sodium phosphate b u f f e r (pH 6.8) f o r 15 t o 20 hours a t 37°C f o l l o w e d by HPLC a n a l y s i s . 2.5 D i s s o c i a t i o n of I n t a c t HCG Samples of commercial HCG were i n c u b a t e d w i t h 8 M u r e a and sodium phosphate b u f f e r (0.02 M, pH 7.5) a t 37°C f o r 60 minutes f o l l o w e d by HPLC a n a l y s i s . 2.6 P r e p a r a t i o n of Reagents and Stock S o l u t i o n s  2.6.1 H y d r o c h l o r i c A c i d (1.0 M) C o n c e n t r a t e d h y d r o c h l o r i c a c i d (83 mL) was mixed w i t h -67-d i s t i l l e d , d e i o n i z e d water and t a k e n t o a f i n a l volume of 1 L w i t h d i s t i l l e d , d e i o n i z e d w a t e r . 2.6.2 Sodium H y d r o x i d e (1.0 M) Sodium h y d r o x i d e (4.0 g) was weighed, d i s s o l v e d i n HPLC grade water and t a k e n t o 100 mL. 2.6.3 G l a c i a l A c e t i c A c i d (0.1 M) G l a c i a l a c e t i c a c i d (0.57 mL) was added t o HPLC grade water and t a k e n t o 100 mL w i t h HPLC grade w a t e r . 2.6.4 Urea S o l u t i o n (10 M) Urea (6.0 g) was weighed, d i s s o l v e d i n d i s t i l l e d , d e i o n i z e d water and t a k e n t o a f i n a l volume o f 10 mL. 2.6.5 Sodium Phosphate B u f f e r (0.02 M)  W i t h Urea (8 M) Sodium phosphate d i b a s i c anhydrous (1.42 g) and sodium phosphate monobasic monohydrate (1138 g) were weighed and s e p a r a t e l y d i s s o l v e d i n HPLC grade water t o a f i n a l volume of 500 mL. S u f f i c i e n t sodium phosphate monobasic s o l u t i o n was added t o the d i b a s i c s o l u t i o n t o r e a c h a f i n a l pH of 7.5. Urea (48.0 g) was weighed and added t o 100 mL of the -68-pH 7.5 b u f f e r . 2.6.6 Sodium Phosphate B u f f e r (0.02 M) Sodium phosphate d i b a s i c anhydrous (1.42 g) and sodium phosphate monobasic monohydrate (1.38 g) were weighed and s e p a r a t e l y d i s s o l v e d i n HPLC grade water t o a f i n a l volume of 500 mL. S u f f i c i e n t sodium phosphate monobasic s o l u t i o n was added t o the d i b a s i c s o l u t i o n t o r e a c h a f i n a l pH of 7.5. 2.6.7 Sodium Dodecyl S u l f a t e - P o l y a c r y l a m i d e G e l  E l e c t r o p h o r e s i s 2.6.7.1 A c r y l a m i d e Stock S o l u t i o n A c r y l a m i d e (22.2 g) and b i s - a c r y l a m i d e (0.6 g) were weighed and d i s s o l v e d i n d i s t i l l e d d e i o n i z e d water t o make 100 mL of s o l u t i o n . T h i s s o l u t i o n was s t o r e d a t 4°C and pr e p a r e d f r e s h monthly. 2.6.7.2 Ammonium P e r s u l f a t e S o l u t i o n Ammonium p e r s u l f a t e (150 mg) was weighed a c c u r a t e l y and d i s s o l v e d i n d i s t i l l e d d e i o n i z e d water t o make 10 mL of s o l u t i o n as r e q u i r e d . -69-2.6.7.3 Gel B u f f e r S o l u t i o n Sodium monobasic phosphate monohydrate (7.8 g ) , sodium d i b a s i c phosphate anhydrous (38.6 g) and sodium d o d e c y l s u l f a t e (2.0 g) were d i s s o l v e d i n d i s t i l l e d , d e i o n i z e d water t o make 1 L o f b u f f e r a t pH 7.2. T h i s s o l u t i o n was p r e p a r e d f r e s h as r e q u i r e d . 2.6.7.4 R e s e r v o i r B u f f e r G e l b u f f e r was d i l u t e d 1 t o 1 w i t h d i s t i l l e d , d e i o n i z e d water t o make the r e s e r v o i r b u f f e r . 2.6.7.5 SDS-PAGE Working S o l u t i o n  (10% A c r y l a m i d e ) The 10% a c r y l a m i d e w o r k i n g s o l u t i o n was p r e p a r e d by m i x i n g 13.5 mL a c r y l a m i d e s o l u t i o n , 15.0 mL b u f f e r s o l u t i o n , 1.5 mL ammonium p e r s u l f a t e s o l u t i o n and 0.045 mL TEMED. T h i s made s u f f i c i e n t w o r k i n g s o l u t i o n f o r 12 tubes (2.5 mL s o l u t i o n / t u b e ) . 2.6.8 D i s c o n t i n u o u s P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s 2.6.8.1 S e p a r a t i n g G e l B u f f e r S o l u t i o n T r i s base (36.3 g) was weighed a c c u r a t e l y and d i s s o l v e d -70-i n a p p r o x i m a t e l y 40 mL of d i s t i l l e d , d e i o n i z e d w a t e r . TEMED (0.23 mL) was added and the s o l u t i o n was t i t r a t e d t o pH 8.9 w i t h about 48 mL of 1 M h y d r o c h l o r i c a c i d . The f i n a l s o l u t i o n was made up t o 100 mL w i t h d i s t i l l e d , d e i o n i z e d w a t e r , s t o r e d a t 4 °C and p r e p a r e d f r e s h monthly. 2.6.8.2 S e p a r a t i n g Gel A c r y l a m i d e S o l u t i o n A c r y l a m i d e (26.8 g) and b i s - a c r y l a m i d e (0.268 g) were weighed a c c u r a t e l y and d i s s o l v e d i n d i s t i l l e d , d e i o n i z e d water t o make 100 mL of s o l u t i o n which was s t o r e d a t 4°C and p r e p a r e d f r e s h monthly. 2.6.8.3 S e p a r a t i n g Gel Ammonium P e r s u l f a t e  S o l u t i o n Ammonium p e r s u l f a t e (28 mg) was weighed a c c u r a t e l y and d i s s o l v e d i n d i s t i l l e d , d e i o n i z e d water t o make 20 mL of s o l u t i o n . T h i s s o l u t i o n was p r e p a r e d o n l y as r e q u i r e d . 2.6.8.4 S e p a r a t i n g G e l Working S o l u t i o n  (7.5% w/v and 10.0% w/v A c r y l a m i d e ) U s i n g s e p a r a t i n g g e l s o l u t i o n s , the a c r y l a m i d e w o r k i n g s o l u t i o n was p r e p a r e d by m i x i n g 4 mL b u f f e r , 9 mL (7.5%) or 12 mL (10%) a c r y l a m i d e , 16 mL ammonium p e r s u l f a t e and 3 mL -71-(7.5% o n l y ) d i s t i l l e d , d e i o n i z e d w a t e r . T h i s produced s u f f i c i e n t s o l u t i o n f o r 16 g e l s (2 mL s o l u t i o n / g e l ) . 2.6.8.5 S e p a r a t i n g G e l P r e l i m i n a r y E l e c t r o p h o r e s i s  B u f f e r T r i s base (36.3 g) was weighed, d i s s o l v e d i n d i s t i l l e d , d e i o n i z e d w a t e r , t a k e n t o pH 8.9 w i t h a p p r o x i m a t e l y 48 mL of 1 M h y d r o c h l o r i c a c i d and made up t o a f i n a l volume of 1 L. 2.6.8.6 S t a c k i n g Gel B u f f e r T r i s base (5.98 g) was weighed a c c u r a t e l y and d i s s o l v e d i n d i s t i l l e d , d e i o n i z e d w a t e r . TEMED (0.46 mL) was added a l o n g w i t h s u f f i c i e n t 1 M h y d r o c h l o r i c a c i d ( a p p r o x i m a t e l y 48 mL) t o produce a pH of 6.7. The f i n a l s o l u t i o n was made up t o 100 mL w i t h d i s t i l l e d , d e i o n i z e d w a t e r , s t o r e d a t 4°C and p r e p a r e d f r e s h monthly. 2.6.8.7 S t a c k i n g G e l A c r y l a m i d e S o l u t i o n A c r y l a m i d e (10 g) and b i s - a c r y l a m i d e (2.5 g) were weighed a c c u r a t e l y , d i s s o l v e d i n d i s t i l l e d , d e i o n i z e d water t o a f i n a l volume of 100 mL, s t o r e d a t 4°C and p r e p a r e d f r e s h monthly. -72-2.6.8.8 S t a c k i n g G el R i b o f l a v i n S o l u t i o n R i b o f l a v i n (4.0 mg) was weighed a c c u r a t e l y , d i s s o l v e d i n d i s t i l l e d , d e i o n i z e d water and made up t o 100 mL. T h i s s o l u t i o n was s t o r e d a t 4°C and p r e p a r e d f r e s h monthly. 2.6.8.9 S t a c k i n g G el Sucrose S o l u t i o n Sucrose (40 g) was weighed, d i s s o l v e d i n d i s t i l l e d , d e i o n i z e d water and made up t o 100 mL. T h i s s o l u t i o n was s t o r e d a t 4°c and p r e p a r e d f r e s h monthly. 2.6.8.10 S t a c k i n g G el Working S o l u t i o n  (2.5% A c r y l a m i d e ) U s i n g the s t a c k i n g g e l s o l u t i o n s , the 2.5% a c r y l a m i d e w o r k i n g s o l u t i o n was p r e p a r e d by m i x i n g 1 mL b u f f e r , 2 mL a c r y l a m d e , 1 mL r i b o f l a v i n and 4 mL s u c r o s e s o l u t i o n s . T h i s produced s u f f i c i e n t s o l u t i o n f o r 16 g e l s (0.5 mL s o l u t i o n / g e l ) . 2.6.8.11 DISC-PAGE R e s e r v o i r B u f f e r T r i s base (3.0 g) was weighed and d i s s o l v e d i n d i s t i l l e d , d e i o n i z e d w a t e r . G l y c i n e (14.4 g) was added, the pH a d j u s t e d t o 8.3 and the f i n a l s o l u t i o n was t a k e n t o 1 L -73-w i t h d i s t i l l e d , d e i o n i z e d w a t e r . 2.6.9 S t a i n For E l e c t r o p h o r e s i s T r i c h l o r o a c e t i c a c i d (50 g) and Coomassie b r i l l i a n t b l u e R-250 (0.2 g) were weighed, d i s s o l v e d i n d i s t i l l e d , d e i o n i z e d water and made up t o 100 mL. 2.6.10 D e s t a i n For E l e c t r o p h o r e s i s I s o p r o p a n o l (100 mL r e a g e n t grade) and g l a c i a l a c e t i c a c i d (100 mL) were added t o d i s t i l l e d , d e i o n i z e d water and made up t o 1 L. 2.7 HPLC Mobile Phase P r e p a r a t i o n o The m o b i l e phase b u f f e r s were p r e p a r e d as d e s c r i b e d below f o l l o w e d by f i l t e r i n g and d e a e r a t i n g . O r g a n i c a d d i t i v e s were added a f t e r f i l t e r i n g and the f i n a l s o l u t i o n was s t i r r e d r a p i d l y f o r 30 minut e s . 2.7.1 Sodium S u l f a t e Sodium Phosphate Monobasic  (0.1 M / 0 . 0 2 M) Sodium s u l f a t e anhydrous (7.1 g) and sodium phosphate -74-monobasic monohydrate (1.38 g) were weighed a c c u r a t e l y , d i s s o l v e d i n HPLC grade w a t e r , t a k e n t o pH 6.9 w i t h 1.0 M sodium h y d r o x i d e i n HPLC grade water and made up t o 500 mL w i t h HPLC w a t e r . 2.7.2 Sodium Phosphate B u f f e r (0.01 M) Sodium phosphate d i b a s i c anhydrous (0.71 g) and sodium phosphate monobasic monohydrate (0.69 g) were weighed and s e p a r a t e l y d i s s o l v e d i n HPLC grade water t o 500 mL. S u f f i c i e n t sodium phosphate monobasic s o l u t i o n was added t o the sodium phosphate d i b a s i c s o l u t i o n t o make the f i n a l pH 7.0. 2.7.3 Sodium Phosphate B u f f e r (0.075 M) Sodium phosphate d i b a s i c anhydrous (5.33 g) and sodium phosphate monobasic monohydrate (5.18 g) were weighed a c c u r a t e l y and s e p a r a t e l y d i s s o l v e d i n HPLC grade water t o a f i n a l volume of 500 mL. The sodium phosphate d i b a s i c s o l u t i o n was t i t r a t e d t o pH 7.0 w i t h the monobasic s o l u t i o n . 2.7.4 Sodium Phosphate B u f f e r (0.1 M) Sodium phosphate d i b a s i c anhydrous (7.1 g) and sodium phosphate monobasic monohydrate (6.9 g) were weighed and -75-s e p a r a t e l y d i s s o l v e d i n HPLC grade water t o a f i n a l volume of 500 mL each. P o r t i o n s of the sodium phosphate monobasic s o l u t i o n were t i t r a t e d t o pH v a l u e s of 6.2, 6.5, 6.6, 6.8 and 7.0 w i t h the sodium phosphate d i b a s i c s o l u t i o n . 2.7.5 Sodium Phosphate B u f f e r (0.15 M) Sodium phosphate d i b a s i c anhydrous (10.65 g) and sodium phosphate monobasic monohydrate (10.35 g) were weighed a c c u r a t e l y and i n d i v i d u a l l y d i s s o l v e d i n HPLC grade water t o a f i n a l volume of 500 mL. Sodium monobasic s o l u t i o n was t i t r a t e d t o pH v a l u e s of 5.9, 6.1, 6.4, 6.5, 6.9, 7.0 and 7.8 w i t h the sodium phosphate d i b a s i c s o l u t i o n . 2.7.6 Sodium Phosphate B u f f e r (0.15 M) W i t h Sodium  C h l o r i d e Sodium phosphate d i b a s i c anhydrous (6.29 g) , sodium phosphate monobasic monohydrate (4.14 g) and sodium c h l o r i d e (1.46 g f o r 0.05 M; 2.92 g f o r 0.1 M; 5.84 f o r 0.2 M) were weighed a c c u r a t e l y , d i s s o l v e d i n HPLC grade water and t a k e n t o a f i n a l volume of 500 mL (pH 7.0). 2.7.7 Sodium Phosphate B u f f e r (0.15 M)  With EDTA (0.001 M) -76-Sodium phosphate d i b a s i c anhydrous (6.29 g ) , sodium phosphate monobasic monohydrate (4.14 g) and t e t r a s o d i u m EDTA (1.46 g) were weighed a c c u r a t e l y , d i s s o l v e d i n HPLC grade water and t a k e n t o a f i n a l volume of 500 mL (pH 7.0). 2.7.8 Sodium Phosphate B u f f e r (0.15 M) W i t h Sodium  C h l o r i d e (0.1 M) And EDTA (0.001 M) Sodium phosphate d i b a s i c anhydrous (6.29 g) , sodium phosphate monobasic monohydrate (4.14 g ) , sodium c h l o r i d e (2.92 g) and t e t r a s o d i u m EDTA (1.46 g) were weighed a c c u r a t e l y , d i s s o l v e d i n HPLC grade water and t a k e n t o a f i n a l volume of 500 mL (pH 7.0). 2.7.9 Sodium Phosphate B u f f e r (0.15 M) W i t h Ammonium  C h l o r i d e Sodium phosphate d i b a s i c anhydrous (6.29 g) , sodium phosphate monobasic monohydrate (4.14 g) and ammonium c h l o r i d e (2.67 g) were weighed a c c u r a t e l y , d i s s o l v e d i n HPLC grade water and ta k e n t o 500 mL w i t h HPLC water (pH 7.0). 2.7.10 Sodium Phosphate B u f f e r (0.15 M) w i t h P h o t assium C h l o r i d e Sodium phosphate d i b a s i c anhydrous (6.29 g) , sodium -77-phosphate monobasic monohydrate (4.14 g) and p o t a s s i u m c h l o r i d e (3.73 g) were weighed a c c u r a t e l y , d i s s o l v e d i n HPLC grade water and t a k e n t o 500 mL w i t h HPLC water (pH 7.0). 2.7.11 Sodium Phosphate B u f f e r (0.15 M)  Wi t h T r i e t h y l a m i n e (0.05 M) Sodium phosphate d i b a s i c anhydrous (10.65 g) and sodium phosphate monobasic monohydrate (10.35 g) were weighed a c c u r a t e l y and i n d i v i d u a l l y d i s s o l v e d i n HPLC grade w a t e r . T r i e t h y l a m i n e (2.4 mL) was added t o b o t h s o l u t i o n s which were t a k e n t o a f i n a l volume of 500 mL w i t h HPLC grade w a t e r . Sodium monobasic s o l u t i o n was t i t r a t e d t o pH 7.0 w i t h the sodium phosphate d i b a s i c s o l u t i o n . 2.7.12 Sodium Phosphate B u f f e r (0.15 M)  With A c e t o n i t r i l e Sodium phosphate d i b a s i c anhydrous (6.29 g) and sodium phosphate monobasic monohydrate (4.14 g) were weighed a c c u r a t e l y and d i s s o l v e d i n HPLC grade w a t e r . A c e t o n i t r i l e (HPLC grade) (5 mL f o r 1%; 10 mL f o r 2%; 25 mL f o r 5%; 50 mL f o r 10%) was added and the f i n a l s o l u t i o n was t a k e n t o 500 mL (pH 7.0) w i t h HPLC w a t e r . -78-2.7.13 Sodium Phosphate B u f f e r (0.15 M)  Wi t h I s o p r o p a n o l Sodium phosphate d i b a s i c anhydrous (6.29 g) and sodium phosphate monobasic monohydrate (4.14 g) were weighed a c c u r a t e l y and d i s s o l v e d i n HPLC grade w a t e r . I s o p r o p a n o l (HPLC grade) (5 mL f o r 1%; 10 mL f o r 2%) was added and the f i n a l s o l u t i o n was t a k e n t o 500 mL w i t h HPLC water (pH 7.0). 2.7.14 Sodium P h o s p h a t e B u f f e r (0.15 M) W i t h M e t h a n o l Sodium phosphate d i b a s i c anhydrous (6.29 g) and sodium phosphate monobasic monohydrate (4.14 g) were weighed a c c u r a t e l y and d i s s o l v e d i n HPLC grade w a t e r . Methanol (HPLC grade) (5 mL f o r 1%; 10 mL f o r 2%; 25 mL f o r 5%; 30 mL f o r 6%; 40 mL f o r 8%; 50 mL f o r 10%; 60 mL f o r 12%) was added and the f i n a l s o l u t i o n was ta k e n t o 500 mL w i t h HPLC water (pH 7.0). 2.7.15 Sodium Phosphate B u f f e r (0.2 M) Sodium phosphate d i b a s i c anhydrous (14.2 g) and sodium phosphate monobasic monohydrate (13.8 g) were weighed and i n d i v i d u a l l y d i s s o l v e d i n HPLC grade water t o a f i n a l volume o f 500 mL. The sodium phosphate monobasic s o l u t i o n was t h e n t i t r a t e d t o a pH v a l u e of 7.0 w i t h the d i b a s i c s o l u t i o n . _ 7 9 " 2.7.16 Sodium Phosphate B u f f e r (0.3 M) Sodium phosphate d i b a s i c anhydrous (21.3 g) and sodium phosphate monobasic monohydrate (20.7 g) were weighed and i n d i v i d u a l l y d i s s o l v e d i n HPLC grade w a t e r . S u f f i c i e n t sodium phosphate monobasic s o l u t i o n was then added t o the d i b a s i c s o l u t i o n t o make the f i n a l pH 7.0. 2.7.17 Sodium Phosphate B u f f e r (0.4 M) Sodium phosphate d i b a s i c anhydrous (28.4 g) and sodium phosphate monobasic monohydrate (27.6 g) were s e p a r a t e l y weighed and d i s s o l v e d i n HPLC grade water t o a f i n a l volume of 500 mL. The sodium phosphate d i b a s i c s o l u t i o n was then t i t r a t e d t o a pH of 7.0 w i t h the monobasic s o l u t i o n . 2.7.18 Sodium A c e t a t e B u f f e r (0.1 M) Sodium a c e t a t e anhydrous (4.1 g) was weighed a c c u r a t e l y and d i s s o l v e d i n HPLC grade w a t e r . T h i s s o l u t i o n was t i t r a t e d t o pH 5.6 or 7.0 w i t h 0.1 M g l a c i a l a c e t i c a c i d i n HPLC grade water and t a k e n t o a f i n a l volume of 500 mL. 2.7.19 T r i s A c e t a t e B u f f e r (0.1 M) T r i s base (2.42 g) was weighed a c c u r a t e l y and d i s s o l v e d i n HPLC grade w a t e r . T h i s was t i t r a t e d t o pH 6.6 w i t h 0.1 M -80-g l a c i a l a c e t i c a c i d i n HPLC grade water and t a k e n t o a f i n a l volume of 200 mL. 2.7.20 T r i s C h l o r i d e B u f f e r (0.02 M) T r i s base (1.21 g) was weighed a c c u r a t e l y , d i s s o l v e d i n HPLC grade w a t e r , t a k e n t o pH 8.5 w i t h c o n c e n t r a t e d h y d r o c h l o r i c a c i d and made up t o 500 mL w i t h HPLC grade w a t e r . 2.7.21 Ammonium B i c a r b o n a t e B u f f e r (0.05 M) Ammonium b i c a r b o n a t e (0.79 g) was weighed a c c u r a t e l y and d i s s o l v e d i n HPLC grade w a t e r . The pH was a d j u s t e d t o 8.5 w i t h ammonium h y d r o x i d e and the f i n a l s o l u t i o n was made up t o 200 mL w i t h HPLC grade w a t e r . 2.7.22 Ammonium B i c a r b o n a t e B u f f e r (0.1 M) Ammonium b i c a r b o n a t e (1.58 g) was weighed a c c u r a t e l y and d i s s o l v e d i n HPLC grade w a t e r . The pH was a d j u s t e d t o 8.5 w i t h ammonium h y d r o x i d e and the f i n a l s o l u t i o n was made up t o 200 mL w i t h HPLC grade w a t e r . 2.7.23 Ammonium Phosphate Monobasic B u f f e r (0.05 M) Ammonium phosphate monobasic (1.44 g) was weighed -81-a c c u r a t e l y and d i s s o l v e d i n HPLC grade w a t e r . The pH was a d j u s t e d t o 8.5 w i t h ammonium h y d r o x i d e and the f i n a l s o l u t i o n was made up t o 250 mL w i t h HPLC grade w a t e r . 2.7.24 T r i f l u o r o a c e t i c A c i d : A c e t o n i t r i l e : W a t e r A s t o c k s o l u t i o n was p r e p a r e d by a d d i n g t r i f l u o r o a c e t i c a c i d (10.0 mL) t o HPLC grade water and t a k i n g t h i s t o a f i n a l volume of 100 mL. S o l v e n t A (1 mL s t o c k s o l u t i o n d i l u t e d t o 200 mL w i t h HPLC grade water) and S o l v e n t B (0.85 mL s t o c k s o l u t i o n d i l u t e d t o 200 mL w i t h HPLC grade a c e t o n i t r i l e ) were mixed and used t o make a f i n a l s o l u t i o n of 0.05% t r i f l u o r o a c e t i c a c i d and 30% a c e t o n i t r i l e i n water (70% A:30% B ) . T h i s m i x t u r e was then degassed by s t i r r i n g r a p i d l y f o r 30 minutes p r i o r t o use. 2.8 Sodium Dodecyl S u l f a t e - P o l y a c r y l a m i d e Gel  E l e c t r o p h o r e s i s The development of a sodium d o d e c y l s u l f a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (SDS-PAGE) method f o r t h e a n a l y s i s of HCG was based on p r o c e d u r e s found i n the l i t e r a t u r e ( P a l a 1973, Q a z i 1974, Weber 1969). T h i s e l e c t r o p h o r e s i s method was used f o r e v a l u a t i o n of commercial -82-HCG p r e p a r a t i o n s , the USP R e f e r e n c e S t a n d a r d HCG and c h r o m a t o g r a p h i c peak e l u a t e c o m p o s i t i o n . 2.8.1 G e l P r e p a r a t i o n U s i n g a 10% w/v aqueous a c r y l a m i d e s o l u t i o n , 2.5 mL was poured i n t o g l a s s tubes and a l l o w e d t o s o l i d i f y i n t o g e l s . B e f o r e the g e l s were t o t a l l y s o l i d i f i e d , a drop of d i s t i l l e d , d e i o n i z e d water was p l a c e d on top t o a l l o w a smooth s u r f a c e t o d e v e l o p . The tubes were then p l a c e d i n the E-C G e l Column E l e c t r o p h o r e s i s Apparatus and the r e s e r v o i r b u f f e r was added. 2.8.2 Sample P r e p a r a t i o n Samples were p r e p a r e d f o r e l e c t r o p h o r e s i s by a d d i n g 0.020 mL SDS sample b u f f e r , 0.005 mL bromophenol b l u e s o l u t i o n as t r a c k i n g dye and one drop of g l y c e r i n t o the p r o t e i n s o l u t i o n . 2-Mercaptoethanol (0.005 mL) was added and the samples were v o r t e x e d . Urea (0.050 mL of a 10 M s o l u t i o n ) was sometimes added t o the samples. M a i n t a i n i n g the samples a t 37°C f o r 45 minutes a l l o w e d r e d u c t i o n of the d i s u l f i d e bonds. U s i n g 0.030 mL of the SDS M o l e c u l a r Weight Marker s o l u t i o n , known m o l e c u l a r w e i g h t samples were pr e p a r e d i n a s i m i l a r manner t o a l l o w c a l c u l a t i o n of m o l e c u l a r w e i g h t s . A f t e r i n c u b a t i o n , the samples were - 8 3 -p l a c e d on top of the g e l s i n the b u f f e r r e s e r v o i r w i t h a p a s t e u r p i p e t t e and sank t o the s o l i d i f i e d g e l s u r f a c e due t o the g l y c e r i n added. 2.8.3 E l e c t r o p h o r e s i s C o n d i t i o n s A f t e r sample a p p l i c a t i o n , the g e l s were s u b j e c t e d t o a c u r r e n t of 10 mA/tube f o r about 5.5 h o u r s , or u n t i l the bromophenol b l u e had m i g r a t e d t h r o u g h a t l e a s t t h r e e - q u a r t e r s of the g e l (Weber 1969, 1972). 2.8.4 S t a i n i n g Procedure V i s u a l i z a t i o n of the p r o t e i n was i n i t i a l l y s t u d i e d u s i n g Coomassie B r i l l i a n t B l u e R-250. S o l u t i o n s of 0.05% w/v Coomassie i n an a c e t i c a c i d s o l u t i o n (Weber 1969) and 0.05% w/v Coomassie i n 12.5% t r i c h l o r o a c e t i c a c i d (TCA) ( P a l a 1973, Q a z i 1974) f a i l e d t o s t a i n the q u a n t i t y (70 t o 100 IU) of HCG used when a p p l i e d t o the g e l s f o r one hour. W i t h 0.2% w/v Coomassie i n 50% w/v TCA, i t r e q u i r e d from 45 minutes t o 1 hour t o s t a i n 70 IU of HCG. T h i s s t a i n i n g p r o c e d u r e was used f o r b o t h subsequent SDS and d i s c o n t i n u o u s (DISC) e l e c t r o p h o r e s i s systems. 2.8.5 P r e p a r a t i o n of Chromatographic Peak E l u a t e For SDS-PAGE -84-A f t e r c o l l e c t i o n of the peak e l u a t e s , the samples were then p u r i f i e d by d i a l y s i s t o remove the c h r o m a t o g r a p h i c m o b i l e phase e l e c t r o l y t e s . The samples were p l a c e d i n S p e c t r a / P o r 3 d i a l y s i s t u b i n g which was t h e n immersed i n about 2 L of d i s t i l l e d , d e i o n i z e d w a t e r . The water was changed t h r e e t i m e s i n a 24 hour p e r i o d a f t e r w h i c h the samples were removed from the d i a l y s i s t u b i n g , f r o z e n w i t h a c e t o n e - d r y i c e and d r i e d f o r 24 t o 48 hours i n a l y o p h i l i z e r . F o l l o w i n g r e c o n s t i t u t i o n w i t h a s u i t a b l e q u a n t i t y o f d i s t i l l e d , d e i o n i z e d w a t e r , t h e samples were pr e p a r e d f o r and e v a l u a t e d by SDS-PAGE. 2.9 Discontinuous P o l y a c r y l a m i d e Gel E l e c t r o p h o r e s i s DISC-PAGE was c a r r i e d out as d e s c r i b e d by G a b r i e l (1971) u s i n g a system d e s i g n e d t o r e s o l v e p r o t e i n s from 4 6 10 t o 10 Da. Only the commercial p r e p a r a t i o n s of HCG and t h e USP R e f e r e n c e S t a n d a r d HCG were e v a l u a t e d u s i n g t h i s e l e c t r o p h o r e s i s method. 2.9.1 Gel P r e p a r a t i o n U s i n g e i t h e r the 7.5% or 10% aqueous a c r y l a m i d e s e p a r a t i n g g e l s o l u t i o n , 2.0 mL was poured i n t o the g l a s s -85-t u b e s . One d r o p o f d i s t i l l e d , d e i o n i z e d w a t e r was p l a c e d on top of the g e l once s o l i d i f i c a t i o n s t a r t e d . P r e l i m i n a r y e l e c t r o p h o r e s i s of the s e p a r a t i n g g e l was done u s i n g t r i s ( h y d r o x y m e t h y 1 ) a m i n o m e t h a n e ( T r i s ) h y d r o c h l o r i c a c i d b u f f e r (pH 9.5) f o r 2 hours a t 3 mA/tube. The tubes were then removed from the b u f f e r and p r e p a r e d f o r a d d i t i o n of the s t a c k i n g g e l by c a r e f u l l y d r y i n g the i n s i d e o f the tube (above the s e p a r a t i n g g e l ) . S t a c k i n g g e l (0.5 mL of 2.5% w/v a c r y l a m i d e s o l u t i o n ) was l a y e r e d on top of the s e p a r a t i n g g e l and p o l y m e r i z e d w i t h a f l u o r e s c e n t lamp. In p r e p a r a t i o n f o r use, the tubes were p l a c e d i n the E-C G e l Column E l e c t r o p h o r e s i s A p p a r a t u s and the DISC-PAGE r e s e r v o i r b u f f e r was added. 2.9.2 Sample P r e p a r a t i o n Samples were p r e p a r e d f o r e l e c t r o p h o r e s i s by a d d i n g 0.010 mL bromophenol b l u e s o l u t i o n f o r t r a c k i n g dye and 1 drop o f g l y c e r i n t o the p r o t e i n s o l u t i o n . A f t e r v o r t e x i n g the samples were p l a c e d on top of the g e l s i n the b u f f e r r e s e r v o i r w i t h a p a s t e u r p i p e t t e . 2.9.3 E l e c t r o p h o r e s i s C o n d i t i o n s The g e l s were s u b j e c t e d t o a c u r r e n t of 2 mA/tube f o r 2 t o 2.5 h o u r s , o r u n t i l the bromophenol b l u e had m i g r a t e d - 8 6 -t h r o u g h a t l e a s t t h r e e - q u a r t e r s of the g e l . 2.10 L a t e x A g g l u t i n a t i o n I n h i b i t i o n S l i d e T e s t s F o r HCG The c o m p o s i t i o n of c h r o m a t o g r a p h i c peak e l u a t e s were e v a l u a t e d u s i n g two l a t e x a g g l u t i n a t i o n s l i d e t e s t s . The main d i f f e r e n c e between th e s e two s l i d e t e s t s i s the a n t i s e r u m used. 2.10.1 P r e g n o s i s S l i d e T est The q u a l i t a t i v e p r o c e d u r e f o r the P r e g n o s i s s l i d e t e s t was used t o assay the peak e l u a t e c o l l e c t e d w i t h o u t f u r t h e r p r e p a r a t i o n . The a n t i s e r u m r e a g e n t s u p p l i e d w i t h t h i s k i t was a r a b b i t anti-HCG serum and the s e n s i t i v i t y r e p o r t e d by the m a n u f a c t u r e r was 1.5 t o 2.5 IU HCG/mL. 2.10.2 Pregnancy L a t e x S l i d e T e s t (Beta Subunit) The Pregnancy L a t e x S l i d e T est was used t o q u a l i t a t i v e l y assay c h r o m a t o g r a p h i c peak e l u a t e . The c r o s s - r e a c t i v i t y of the a n t i - H C G - b e t a serum ( r a b b i t ) used i n t h i s k i t w i t h the a l p h a s u b u n i t was low as i n d i c a t e d by a n e g a t i v e r e s u l t f o r p u r i f i e d HCG-alpha (0.5 mg/mL). T h e r e f o r e , a p o s i t i v e r e s u l t i n d i c a t e d the presence of -87-i n t a c t HCG and/or HCG-beta. The s e n s i t i v i t y r e p o r t e d by the m a n u f a c t u r e r was 2.0 IU HCG/mL. 2.11 High Performance L i q u i d Chromatography H i g h performance l i q u i d chromatography was p e r formed on a H e w l e t t P a c k a r d 1080 l i q u i d chromatograph e q u i p e d w i t h an LC t e r m i n a l or the Beckman 100 A s o l v e n t m e t e r i n g pump w i t h the C RIA d a t a p r o c e s s o r . A U6K Waters i n j e c t o r or a Rheodyne 7125 i n j e c t o r were used w i t h the Beckman HPLC system. A v a r i a b l e w a v e l e n g t h s p e c t r o p h o t o m e t e r (155-10) was used f o r a l l u l t r a v i o l e t absorbance d e t e c t i o n w h i l e the s p e c t r o f l u o r o m e t e r (FS970) w i t h a 470 nm e m i s s i o n f i l t e r was f o r f l u o r e s c e n c e d e t e c t i o n . A P r o t e i n Pak 125 sw column, two P r o t e i n Pak 300 sw columns and v a r i o u s c o m b i n a t i o n s of t h e s e columns were used f o r s i z e e x c l u s i o n c h r o m a t o g r a p h y . C o n n e c t i o n of columns i n s e r i e s was a c h i e v e d by u s i n g t h e s h o r t f i t t i n g s u p p l i e d w i t h t h e s e columns. Column t o d e t e c t o r c o n n e c t i o n s were made w i t h PTFE t u b i n g . The v a r i o u s b u f f e r s used w i t h t h e P r o t e i n Pak 300 sw column w h i l e o p t i m i z i n g the c o n d i t i o n s are shown i n T able I of the R e s u l t s and D i s c u s s i o n s e c t i o n . R e c y c l e chromatography was done u s i n g the Beckman -88-s y s t e m w i t h one P r o t e i n Pak 300 sw column. A t h r e e way PTFE v a l v e was i n s e r t e d between the d e t e c t o r and t h e r e c y c l e p o r t on the pump t o a l l o w removal or c o l l e c t i o n of peak e l u a t e s . The P r o t e i n Pak DEAE 5 pw column used f o r a n i o n exchange HPLC was e v a l u a t e d u s i n g the v a r i o u s b u f f e r s summarized i n Table I I of the R e s u l t s and D i s c u s s i o n s e c t i o n . F o l l o w i n g Putterman (1982), a mobile phase of t r i f l u o r o a c e t i c a c i d - a c e t o n i t r i l e - w a t e r was used w i t h the U l t r a s p h e r e ODS HPLC column t o determine i f r e v e r s e d - p h a s e HPLC c o u l d f u r t h e r s e p a r a t e the p r o t e i n components o f commercial HCG p r e p a r a t i o n s . -89-3. RESULTS AND DISCUSSION 3.1 E l e c t r o p h o r e s i s of Commercial HCG P r e p a r a t i o n s 3.1.1 Sodium Dodecyl S u l f a t e -P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s W i t h urea added t o the sample p r e p a r a t i o n , two bands were obse r v e d f o r HCG. A comparison of the m o b i l i t y of the M o l e c u l a r Weight Markers w i t h t h a t f o r HCG i n d i c a t e d t h a t the s l o w e s t moving band had a m o l e c u l a r w e i g h t of 33000 +_ 700 Da w h i l e the f a s t e r moving band had a m o l e c u l a r w e i g h t o f 21000 _+ 600 Da (based on 6 s a m p l e s ) . These r e s u l t s a r e s i m i l a r t o t h o s e o b t a i n e d by P a l a (1973) (35000 Da and 27000 D a ) . W i t h o u t u r e a , two bands a t 21000 +_ 1000 Da and 34000 _+ 800 Da were observed (5 samples) . Samples e v a l u a t e d by the above methods, i r r e s p e c t i v e of the commercial source of HCG ( A y e r s t o r K-Line) , y i e l d e d i d e n t i c a l r e s u l t s . S i n c e no s i g n i f i c a n t d i f f e r e n c e was produced w i t h the a d d i t i o n of urea t o thes e samples, f u r t h e r SDS-PAGE was done i n the absence of u r e a . When 200 IU samples of HCG were a s s a y e d , f a i n t bands a t 43000 +_ 1800 Da and 18000 + 1300 Da were observed a l o n g w i t h those a t 21000 Da and 34000 Da. The -90-a d d i t i o n a l bands o b s e r v e d w i t h t h e s e l a r g e r HCG samples were much f a i n t e r than those a t 21000 and 34000 Da. A l s o , the m o l e c u l a r w e i g h t s of th e s e bands (18000 and 43000 Da) have not been r e p o r t e d i n the l i t e r a t u r e f o r HCG. T h e r e f o r e these a d d i t i o n a l bands are l i k e l y due t o i m p u r i t i e s i n the commercial HCG p r e p a r a t i o n . Samples p r e p a r e d w i t h o u t 2-mercaptoethanol gave f a i n t bands a t 47800 + 1900 Da, 27300 +_ 600 Da and 17000 + 600 Da and a d a r k e r band a t 20000 +_ 700 Da (based on two s a m p l e s ) . These r e s u l t s i n d i c a t e t h a t the m o b i l i t y of the two s l o w e s t moving f r a c t i o n s i s a f f e c t e d by r e d u c t i o n of d i s u l f i d e bonds more than the f a s t e r moving f r a c t i o n s . 3.1.2 D i s c o n t i n u b u s - P o l y a c r y l a m i d e G e l  E l e c t r o p h o r e s i s U s i n g 10% w/v a c r y l a m i d e g e l (0.1% w/v b i s - a c r y l a m i d e ) , HCG m i g r a t e d a v e r y s h o r t d i s t a n c e i n t o the s e p a r a t i n g g e l , t h e r e f o r e , a 7.5% w/v a c r y l a m i d e g e l (0.08% w/v b i s - a c r y l a m i d e ) (2.0 mL) w i t h a 2.5% w/v a c r y l a m i d e s t a c k i n g g e l (0.5 mL) was employed. With t h i s system, 70 t o 100 IU of HCG o b t a i n e d from A y e r s t L a b o r a t o r i e s and K-Line P h a r m a c e u t i c a l s appeared as one f a i n t broad band. T h i s had been p r e v i o u s l y r e p o r t e d by B a h l , 1969; G r a e s s l i n , 1972; and Q a z i , 1974. A n a l y s i s of l a r g e r samples of HCG (500 t o 1000 IU) by DISC-PAGE showed f o u r s e p a r a t e bands. The s l o w e s t -91-moving f r a c t i o n was broad and s t a i n e d d a r k e s t . Two narrow f a i n t bands s e p a r a t e d t h i s b r o a d band from t h e f a s t e r moving f a i n t broad band. The two f a i n t bands are l i k e l y - d u e t o i m p u r i t i e s s i n c e HCG and i t s s u b u n i t s g i v e broad bands w i t h t h i s e l e c t r o p h o r e s i s method. However, the two broad bands may be the i n d i v i d u a l s u b u n i t s o f HCG or i n t a c t HCG and o t h e r i m p u r i t i e s . 3.2 USP Reference Standard HCG 3.2.1 Sodium Dodecyl S u l f a t e -P o l y a c r y l a m i d e Gel E l e c t r o p h o r e s i s W i t h 2 - m e r c a p t o e t h a n o l but no u r e a , SDS-PAGE o f t h e USP Re f e r e n c e S t a n d a r d HCG gave f r a c t i o n s w i t h the f o l l o w i n g m o l e c u l a r w e i g h t s ; 12600, 21400, 25500, 30400, 34000, 40000 to 61000, 75500, 81000, 84000 t o 90000 and 93200 Da. S i n c e o n l y t h r e e of th e s e f r a c t i o n s a re l i k e l y due t o HCG (61000 Da i n t a c t HCG, 34000 Da HCG-beta, 21400 Da HCG-alpha), the o t h e r f r a c t i o n s a re l i k e l y due t o i m p u r i t i e s i n the USP Refer e n c e S t a n d a r d HCG. -92-3.2.2 D i s c o n t i n u o u s - P o l y a c r y l a m i d e G e l  E l e c t r o p h o r e s i s E v a l u a t i o n o f the USP Refer e n c e S t a n d a r d HCG w i t h t h i s DISC-PAGE method gave f o u r s e p a r a t e broad bands. S i n c e i n t a c t HCG was ex p e c t e d t o g i v e o n l y one broad band, the s e r e s u l t s i n d i c a t e t h a t the USP Refer e n c e S t a n d a r d HCG c o n t a i n s i m p u r i t i e s which i s i n agreement w i t h the SDS-PAGE r e s u l t s . 3.3 P r e l i m i n a r y S t u d i e s With S i z e E x c l u s i o n Chromatography Using The P r o t e i n Pak 300 sw  HPLC Column 3.3.1 E v a l u a t i o n of P r o t e i n Pak 300 sw  Column Performance I n i t i a l a t t e m p t s t o employ s i z e e x c l u s i o n HPLC of HCG were u n d e r t a k e n u s i n g a P r o t e i n Pak 300 sw column (7.8 mm x 30 cm) w i t h d e t e c t i o n a t 280 nm. The column was e v a l u a t e d u s i n g a G e l F i l t r a t i o n S t andard (Bio-Rad) and a s u l p h a t e : p h o s p h a t e b u f f e r f o r mobile phase. Comparison of the r e s u l t s u s i n g t h i s column, shown i n F i g u r e 4 , and those o b t a i n e d by Bio-Rad L a b o r a t o r i e s on an e q u i v a l e n t column (Bio-Rad 1981) , demonstrated t h a t the performance of t h i s -93-FIGORE 4 CHROMATOGRAM OF GEL FILTRATION STANDARD 0.15-O CO 0.10-CM o < m oc o 00 < 0.05-i 10 LEGEND 1. T h y r o g l o b u l i n 670 000 Da 2. IgG 150 000 Da 3. Ovalbumin 45 000 Da 4. Myoglobin 17 000 Da 5. Cyanocobalamin 1 355 Da 20 TIME (MIN) Chromatographic c o n d i t i o n s : column, P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium s u l f a t e / sodium phosphate monobasic b u f f e r (0.1 M/0.02 M, pH 6.9); i n j e c t i o n volume, 0.020 mL; flow r a t e , 1.0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 280 nm; ch a r t speed, 0.25 cm/min. -94-column was a c c e p t a b l e . 3.3.2 Commercial HCG P r e p a r a t i o n s I n j e c t i o n of a commercial HCG sample o b t a i n e d from A y e r s t L a b o r a t o r i e s (A.P.L.) r e v e a l e d t h r e e peaks e l u t i n g a t 11.2, 13.44 and 15.92 minutes ( F i g u r e 5 ) . The chromatogram o b t a i n e d from i n j e c t i o n of a second commercial HCG sample o b t a i n e d from K-Line P h a r m a c e u t i a l s r e v e a l e d peaks a t 4.60, 8.20, 10.8, 12.4 and 13.95 minutes ( F i g u r e 6 ) . Both p r e p a r a t i o n s were r e c o n s t i t u t e d i n the s t e r i l e d i l u e n t s u p p l i e d . For the A.P.L. sample, the d i l u e n t c o n t a i n e d 2% b e n z y l a l c o h o l and the l y o p h i l i z e d sample of HCG c o n t a i n e d not more th a n 0.2% p h e n o l . In a s i m i l a r f a s h i o n , t h e K - L i n e s t e r i l e d i l u e n t was known t o c o n t a i n b e n z y l a l c o h o l (0.9%) and m a n n i t o l (100 mg) was found i n the l y o p h i l i z e d HCG sample. From i n j e c t i o n s of pure p h e n o l , b e n z y l a l c o h o l and m a n n i t o l i t was found t h a t the peak a t 15.92 minutes i n the A.P.L. sample was phenol and the peaks a t 13.44 and 13.95 minutes i n the A.P.L. and K-Line samples r e s p e c t i v e l y were due t o b e n z y l a l c o h o l . The m a n n i t o l p r e s e n t i n the l y o p h i l i z e d K-Line HCG sample e l u t e d j u s t a f t e r b e n z y l a l c o h o l a t a p p r o x i m a t e l y 17 minutes but d i d not have a s t r o n g enough absorbance t o be d e t e c t e d under th e s e c o n d i t i o n s ( F i g u r e 6 ) . F u r t h e r r e c o n s t i t u t i o n of -95-FIGORE 5 CHROMATOGRAM OF HCG (AYERST LABORATORIES) ON PROTEIN PAK 300 sw COLUMN WITH DETECTION AT 280 nm E C o CO CM Ul o z < cc o CO CQ < CM Oi in 0.05-O ^ CM * I I I O 10 2 0 TIME (MIN) Chromatographic c o n d i t i o n s : column, P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium s u l f a t e / sodium phosphate monobasic b u f f e r (0.1 M/0.02 M, pH 6.9); i n j e c t i o n volume, 0.02 mL (0.8 IU); flow r a t e , 1.0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 280 nm; ch a r t speed, 0.25 cm/min. -96-FIGORE 6 CHROMATOGRAM OF HCG (K-LINE PHARMACEUTICALS) ON PROTEIN PAK 300 sw COLUMN WITH DETECTION AT 280 nm E 0 .15-o CO CM ^ o.ioH Ul o z < 0.05 03 cc o CO it) O) CO o (O o CM o o CO * • • O CM V rrrfrrfrr*"^ 10 2 0 TIME (MIN) Chromatographic c o n d i t i o n s : column, P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium s u l f a t e / sodium phosphate monobasic b u f f e r (0.1 M/0.02 M, pH 6.9); i n j e c t i o n volume, 0.05 mL (50 IU); flow r a t e , 1.0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 280 nm; ch a r t speed, 0.25 cm/min. -97-l y o p h i l i z e d HCG samples was t h e r e f o r e done w i t h HPLC grade water a l o n e . As we were not c e r t a i n i f t h e HCG was e l u t i n g as one of the minor peaks i n the chromatogram or was, i n f a c t , c o - e l u t i n g w i t h one o f the p r e s e r v a t i v e s , the s u b s t a n c e ( s ) e l u t i n g a t 15.92 and 13.95 minutes were c o l l e c t e d from the o u t l e t of the d e t e c t o r and s u b s e q u e n t l y a n a l y s e d by SDS-PAGE. The r e s u l t s were n e g a t i v e , t h e r e b y i n d i c a t i n g t h a t the HCG was l i k e l y t o be e l u t i n g i n the e a r l i e r p a r t of thes e chromatograms. The maximum absorbance f o r HCG has been r e p o r t e d t o be 278 nm (Bahl 1973, Got 1960), however, the a b s o r p t i v i t y i s v e r y low. T h e r e f o r e a wavelength of 208 nm was s e l e c t e d i n subsequent e x p e r i m e n t s t o ta k e advantage o f h i g h energy end a b s o r p t i o n of the t y r o s i n e r e s i d u e s i n HCG and t o o b t a i n i n c r e a s e d s e n s i t i v i t y . A t a w a v e l e n g t h of 208 nm t h e A.P.L. sample e x h i b i t e d a m u l t i p l e t of p o o r l y r e s o l v e d peaks between 7 and 14.5 minutes ( F i g u r e 7 ) . An a t t e m p t , t h e r e f o r e , was made t o a d j u s t the b u f f e r pH, c o n c e n t r a t i o n and type i n o r d e r t o r e s o l v e t h e s e components. 3.3.3 O p t i m i z a t i o n of M o b i l e Phase B u f f e r The b u f f e r c o m p o s i t i o n s i n v e s t i g a t e d a re shown i n Table -98-FIGURE 7 CHROMATOGRAM OF HCG (AYERST LABORATORIES) ON A PROTEIN PAK 300 sw COLUMN WITH DETECTION AT 208 nm to in 0 10 2 0 TIME (MIN) Chromatographic c o n d i t i o n s : column, P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium s u l f a t e / sodium phosphate monobasic b u f f e r (0.1 M/0.02 M, pH 6.9); i n j e c t i o n volume, 0.015 mL (15 IU); flow r a t e , 1.0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 208 nm; c h a r t speed, 0.25 cm/min. -99-I . Both the sodium s u l f a t e / s o d i u m phosphate monobasic ( F i g u r e 7) and the sodium phosphate (0.01 M) b u f f e r s r e s u l t e d i n poor r e s o l u t i o n of the n o n - p r e s e r v a t i v e components of commercial HCG. A l t e r a t i o n of a 0.1 M b u f f e r a t pH v a l u e s from 6.2 t o 7.0 i n f i v e s t e p s produced a g r a d u a l i n c r e a s e i n r e s o l u t i o n w h i c h r e a c h e d an optimum a t pH 7.0 ( F i g u r e 8) where f o u r (or more) components, as w e l l as phenol a t 15.89 m i n u t e s , were v i s i b l e i n t h e A.P.L. sample. When the b u f f e r c o n c e n t r a t i o n was a l t e r e d from 0.01 t o 0.4 M i n s i x s t e p s a t a pH v a l u e of 7.0, the A.P.L. sample e x h i b i t e d f i v e or more components ( F i g u r e 9) which were s t i l l u n r e s o l v e d . C o l l e c t i o n s o f the e l u a t e from the u l t r a v i o l e t d e t e c t o r from 9 t o 10, 10.4 t o 12 and 14 t o 15 minutes r e v e a l e d t h a t HCG was p r e s e n t i n the peak w i t h a r e t e n t i o n t ime of 9.20 m i n u t e s . The p r e s e n c e of HCG i n peak e l u a t e s was d e t e r m i n e d by SDS-PAGE as w e l l as by a l a t e x a g g l u t i n a t i o n i n h i b i t i o n t e s t ( P r e g n o s i s L a t e x A g g l u t i n a t i o n I n h i b i t i o n S l i d e T e s t For P r e g n a n c y ) . E l e c t r o p h o r e s i s i n d i c a t e d HCG was p r e s e n t i n the 9.20 peak e l u a t e o n l y , w h i l e the P r e g n o s i s t e s t showed HCG was always p r e s e n t i n the 9.20 minute and sometimes p r e s e n t i n the 11.0 minute peak e l u a t e s . W ith a second l a t e x a g g l u t i n a t i o n i n h i b i t i o n s l i d e t e s t s p e c i f i c f o r the b e t a - s u b u n i t (Latex Pregnancy S l i d e T e s t K i t B e t a - S u b u n i t N.C.S.), o n l y the i n i t i a l peak (9.20 minutes) gave a p o s i t i v e r e s u l t -100-TABLE I VARIOUS BUFFERS USED AS MOBILE PHASE WITH THE PROTEIN PAK 300 sw COLUMN BUFFER CONCENTRATION pH (M) ADDITIVES Sodium S u l f a t e / S o d i u m Phosphate Monobasic Sodium Phosphate Sodium Phosphate Sodium Phosphate Sodium A c e t a t e T r i s A c e t a t e Sodium Phosphate Sodium Phosphate Sodium Phosphate Sodium Phosphate Sodium Phosphate Sodium Phosphate 0.10/0.02 0.01 0.10 0.15, 0.20 0.30, 0.40 0.10 0.10 0 .15 0.15 0.15 0 .15 0 .15 0 .15 7 . 0 7.0 6.2, 6.5, 6.6, 6.8, 7.0 7.0 5.6, 7.0 6.6 7.0 7.0 7.0 7.0 7.0 7.0 none none none none none none 0.1 M NaCl 0.1 M NaCl 0.001 M EDTA 0. 001 M EDTA 1, 2, 5, 10% a c e t o n i t r i l e 1, 2% i s o p r o p a n o l 1, 2, 5, 6, 8, 10, 12% methanol Sodium Phosphate 0.15 7.0 0.05 M t r i e t h y l a m i n e -101-FIGURE 8 CHROMATOGRAM OF HCG (AYERST LABORATORIES) ON A PROTEIN PAK 300 sw COLUMN WITH MOBILE PHASE OF OPTIMUM pH 0> CO in* i 1 i 0 10 20 TIME (MIN) Chromatographic c o n d i t i o n s : column, P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium phosphate b u f f e r (0.1 M,. pH 7.0); i n j e c t i o n volume, 0.025 mL (25 IU) ; flow r a t e , 1.0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 208 nm; c h a r t speed, 0.25 cm/min. -102-FIGORE 9 CHROMATOGRAM OF HCG (AYERST LABORATORIES) ON A PROTEIN PAK 300 sw COLUMN WITH OPTIMUM MOBILE PHASE 0 J ' l 1 l 0 10 2 0 3 0 TIME (MIN) Chromatographic c o n d i t i o n s : column, P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium phosphate b u f f e r (0.15 M, pH 7.0); i n j e c t i o n volume, 0.010 mL (20 IU); flow r a t e , 1.0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 208 nm; c h a r t speed, 0.25 cm/min. -103-i n d i c a t i n g i t c o n t a i n e d i n t a c t HCG and/or i t s b e t a - s u b u n i t . The second peak (11.0 minutes) was l i k e l y c o n t a m i n a t e d by the f i r s t peak or o t h e r c r o s s - r e a c t i n g compound(s), hence the r e s u l t s were sometimes p o s i t i v e . A s e r i e s o f e x p e r i m e n t s w i t h sodium a c e t a t e (pH 5.6 and 7.0) and t r i s a c e t a t e (pH 6.6) b u f f e r s and the sodium phosphate b u f f e r (pH 7.0) w i t h sodium c h l o r i d e , e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d and t r i e t h y l a m i n e as a d d i t i v e s f a i l e d t o improve r e s o l u t i o n of the o v e r l a p p i n g p e a k s . A d d i t i o n o f o r g a n i c s o l v e n t s t o the sodium phosphate b u f f e r r e s u l t e d i n s l i g h t changes i n peak shape. These r e s u l t s i n d i c a t e t h a t the commercial HCG samples are a complex m i x t u r e of components, r a t h e r t h a n a r e l a t i v e l y pure f r a c t i o n of HCG. The optimum mobile phase f o r the P r o t e i n Pak 300 sw column was found t o be 0.15 M sodium phosphate b u f f e r a t pH 7.0. 3.3.4 Two P r o t e i n Pak 300 sw Columns  Connected i n S e r i e s As i t appeared t h a t the P r o t e i n Pak 3 00 sw column c o u l d not r e s o l v e t h i s m i x t u r e , a second P r o t e i n Pak 300 sw column was c o n n e c t e d i n s e r i e s . The chromatogram shown i n F i g u r e 10 i n d i c a t e d t h a t t h e r e may be 12 or more c o n s t i t u e n t s i n -104-FIGURE 10 CHROMATOGRAM OF HCG (AYERST LABORATORIES) ON TWO PROTEIN PAK 300 sw COLUMNS CONNECTED IN SERIES WITH 0.01 M SODIUM PHOSPHATE BUFFER AS MOBILE PHASE 00 CO o CO TIME (MIN) Chromatographic c o n d i t i o n s : columns, two P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium phosphate b u f f e r (0.01 M, pH 7.0); i n j e c t i o n volume, 0.025 mL (25 IU); flow r a t e , 1.0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 208 nm; c h a r t speed, 0.25 cm/min. -105-t h i s A.P.L. p r e p a r a t i o n . When the K-Line sample was a n a l y s e d under the same c o n d i t i o n s (0.01 M Phosphate b u f f e r a t pH 7.0) s i m i l a r compound peaks were o b s e r v e d . The chromatogram shown i n F i g u r e 11 w i t h 0.15 M phosphate b u f f e r p r o v i d e d somewhat b e t t e r r e s o l u t i o n of the peaks of i n t e r e s t , however, the s e p a r a t i o n of the components was s t i l l i n c o m p l e t e . At 280 nm, t h e c o r r e s p o n d i n g chromatogram was s i m i l a r t o F i g u r e 11, w i t h the peak a t 23.8 minutes h a v i n g a h i g h e r absorbance w i t h r e s p e c t t o those a t 18 and 21 m i n u t e s . We were unable t o a l t e r the c o m p o s i t i o n of the b u f f e r t o i n c r e a s e s e p a r a t i o n beyond t h a t seen i n F i g u r e 11. 3.4 S i z e E x c l u s i o n Chromatography Using  A P r o t e i n Pak 125 sw Column 3.4.1 P r o t e i n Pak 125 sw Column F u r t h e r a t t e m p t s a t i n c r e a s i n g the r e s o l u t i o n i n c l u d e d u s i n g a P r o t e i n Pak 125 sw column. T h i s column has a narrower pore s i z e d i s t r i b u t i o n and i s quoted by the m a n u f a c t u r e r t o be c a p a b l e of r e s o l v i n g n a t i v e g l o b u l a r p r o t e i n s i n the range of 2000 t o 80000 d a l t o n s (the P r o t e i n Pak 300 sw column has been quoted as c a p a b l e of r e s o l v i n g n a t i v e g l o b u l a r p r o t e i n s i n the range 10000 t o 400000 Da) . -106-FIGURE 11 CHROMATOGRAM OF HCG (AYERST LABORATORIES) ON TWO PROTEIN PAK 300 sw COLUMNS CONNECTED IN SERIES WITH 0.15 M PHOSPHATE BUFFER AS MOBILE PHASE 0.8 -CO o CM Ul < CO cc o CO m < 0.4 -O J (O eo 1 0 2 O 30 40 TIME (MIN) Chromatographic conditions: columns, two Protein Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium phosphate buffer .(0.15 M, pH 7.0); i n j e c t i o n volume, 0.025 mL (25 IU); flow rate, 1.0 mL/min.; u l t r a v i o l e t absorbance detection wavelength, 208 nm; chart speed, 0.25 cm/min. -107-I t was hoped t h a t the narrow pore s i z e would l e a d t o i n c r e a s e d r e s o l u t i o n of the components of the HCG samples. As shown i n F i g u r e 12, the A.P.L. sample s t i l l e x h i b i t e d e v i d e n c e of s i x or more p o o r l y r e s o l v e d components. 3.4.2 P r o t e i n Pak 300 sw and 125 sw HPLC Columns  Connected i n S e r i e s C o n n e c t i o n of the P r o t e i n Pak 300 sw and P r o t e i n Pak 125 sw columns d i d not p r o v i d e a d d i t i o n a l r e s o l u t i o n when the phosphate b u f f e r , pH 7.0 a t 0.15 M was used. T h e r e f o r e f u r t h e r e f f o r t s u s i n g t h i s c o m b i n a t i o n were not pursued. 3.5 Ion Exchange Chromatography Anion exchange chromatography w i t h DEAE s t a t i o n a r y phases has been used f o r p u r i f i c a t i o n of HCG p r e p a r a t i o n s (Bahl 1969a; 1969b, C a n f i e l d 1971, Merz 1974, Van H a l l 1971). A s t u d y of a d i e t h y l a m i n o e t h y 1 HPLC column ( P r o t e i n Pak DEAE 5 pw) was t h e r e f o r e u n d e r t a k e n . T h i s HPLC column has been shown t o be e f f e c t i v e i n r e s o l u t i o n o f amino a c i d s and p r o t e i n s such as immunoglobulins (Kato 1983b, M i l l i p o r e 1984). The b u f f e r c o m p o s i t i o n s shown i n Table I I were i n v e s t i g a t e d . A number of the b u f f e r s used (0.02 M T r i s -108-FIGURE 12 CHROMATOGRAM OF HCG (AYERST LABORATORIES) ON A PROTEIN PAK 125 sw COLUMN WITH DETECTION AT 208 nm O) CO • 00 3.2 E c CO o CM Ul O z < CD OC O CO CO < 1.6 -10 2 0 TIME (MIN) 3 0 Chromatographic c o n d i t i o n s : column, P r o t e i n Pak 125 sw HPLC (7.8 mm x 30 cm); mobile phase, sodium phosphate b u f f e r (0.15 M, pH 7.0); i n j e c t i o n volume, 0.10 mL (100 IU); flow r a t e 1.0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 208 nm; c h a r t speed, 0.25 cm/min. -109-TABLE II VARIOUS BUFFERS USED AS MOBILE PHASE WITH THE PROTEIN PAK DEAE 5 pw COLUMN BUFFER CONCENTRATION pH ADDITIVES (M) T r i s C h l o r i d e 0.02 8.5 none Sodium S u l f a t e / S o d i u m Phosphate Monobasic 0.10/0.02 7.0 none Ammonium B i c a r b o n a t e 0.01, 0.05 8.5 none Ammonium B i c a r b o n a t e 0.10 8.9 none Ammonium Phosphate Monobasic 0.05 8.5 none Sodium Phosphate 0.075, 0.15, 7.0 none 0.30 Sodium Phosphate 0.15 5.9, 6.1, none 6.4, 6.5, 6.9, 7.8 Sodium Phosphate 0.15 7.0 0.05 M NaCl Sodium Phosphate 0.15 7.0 0.1, 0.2 M NaCl Sodium Phosphate 0.15 7.0 0.1 M KC1 Sodium Phosphate 0.15 7.0 0.1 M NH 4C1 -110-c h l o r i d e , 0.1 M ammonium b i c a r b o n a t e , and 0.1 M sodium b i c a r b o n a t e ) e x h i b i t e d v e r y h i g h b a s e l i n e absorbance a t 208 nm, t h e r e f o r e e x p e r i m e n t s w i t h t h e s e c o u l d not be pursued. W i t h a 0.01 M ammonium b i c a r b o n a t e b u f f e r (pH 8.5) a t 215 nm, u n r e s o l v e d peaks were observed between 3 and 14 minutes f o r the A.P.L. sample. I n c r e a s i n g the b u f f e r c o n c e n t r a t i o n up t o 0.05 M ( F i g u r e 13) d i d not improve r e s o l u t i o n . Under the s e c o n d i t i o n s , phenol e l u t e d as the broad peak a t 29.79 mi n u t e s . The o t h e r b u f f e r s g i v e n i n Table I I r e s u l t e d i n poor r e t e n t i o n and r e s o l u t i o n of t h e HCG samples on the DEAE column. S i n c e the r e s o l u t i o n of the p r o t e i n components of the commercial HCG samples was even l e s s than t h a t found w i t h the P r o t e i n Pak 300 sw column, f u r t h e r a t t e m p t s t o i n c r e a s e r e s o l u t i o n u s i n g the DEAE column were not pursued. 3.6 Reversed-Phase Chromatography W h i l e a i m i n g f o r b e t t e r r e s o l u t i o n of the p r o t e i n components of co m m e r c i a l HCG, an U l t r a s p h e r e ODS HPLC column was used t o e v a l u a t e the USP Re f e r e n c e S t a n d a r d HCG and p u r i f i e d i n t a c t HCG samples. W i t h a mo b i l e phase of t r i f l u o r o a c e t i c a c i d - a c e t o n i t r i l e - w a t e r (Putterman 1982) , poor r e s o l u t i o n of i n t a c t HCG from i m p u r i t i e s was o b t a i n e d f o r b o t h the USP Re f e r e n c e S t a n d a r d HCG ( F i g u r e 14) and - 1 1 1 -F IGORE 13 CHROMATOGRAM OF HCG (AYERST LABORATORIES) ON A PROTEIN PAK DEAE 5 pw COLUMN CO • 9 CM 0 J f I I I I I 0 10 20 3 0 4 0 5 0 TIME (MIN) Chromatographic c o n d i t i o n s : column, P r o t e i n Pak DEAE 5 pw HPLC ( 7 . 5 mm x 7 . 5 cm); mobile phase, ammonium bica r b o n a t e b u f f e r ( 0 . 0 5 M, pH 8 . 5 ) ; i n j e c t i o n volume, 0 . 0 5 0 mL (50 IU); flow r a t e , 1 . 0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 215 nm; c h a r t speed, 0 . 2 5 cm/min. -112-FIGURE 14 CHROMATOGRAM OF USP REFERENCE STANDARD HCG ON AN ULTRASPHERE ODS COLUMN •i i 5 10 15 T I M E ( M I N ) Chromatographic c o n d i t i o n s : column, U l t r a s p h e r e ODS 5JJ HPLC (4.6 mm x 25 cm); mobile phase, 0.05% t r i f l u o r o a c e t i c acid/30% a c e t o n i t r i l e i n water; i n j e c t i o n volume, 0.025 mL (1.66 IU); flow r a t e , 1.0 mL/min.; u l t r a v i o l e t d e t e c t i o n wavelength, 210 nm; c h a r t speed, 0.25 cm/min. -113-p u r i f i e d i n t a c t HCG ( F i g u r e 1 5 ) . S i n c e a pure s t a n d a r d i s r e q u i r e d f o r a q u a n t i t a t i v e a s s a y , f u r t h e r a t t e m p t s w i t h t h i s ODS column were not u n d e r t a k e n . 3 . 7 F l u o r e s c e n c e D e t e c t i o n Of The Fluorescamine D e r i v a t i v e Of HCG Due t o the l a c k of r e s o l u t i o n of HCG from i n t e r f e r i n g peaks i n s e p a r a t i o n modes examined thus f a r , an attempt was made t o form a f l u o r e s c e n t d e r i v a t i v e of HCG w i t h f l u o r e s c a m i n e (Fluram) and t o u t i l i z e d e t e c t i o n of the f l u o r o p h o r e formed. A c o m b i n a t i o n of columns ( P r o t e i n Pak 300 sw, P r o t e i n Pak 125 sw p l u s P r o t e i n Pak 300 sw, P r o t e i n Pak 125 sw, and two s e r i a l l y c onnected P r o t e i n Pak 300 sw columns) were e v a l u a t e d . The r e s u l t s o b t a i n e d ( F i g u r e s 16 t o 19) d i d not p r o v i d e r e s o l u t i o n o f the d e r i v a t i v e from the i n t e r f e r i n g components. 3.8 L o c a t i o n Of I n t a c t HCG And I t s Subunits S i n c e the components of commercial HCG were b e s t r e s o l v e d w i t h the P r o t e i n Pak 300 sw column and 0.15 M phosphate b u f f e r (pH 7.0) as m obile phase ( F i g u r e 9 ) , USP -114-FIGURE 15 CHROMATOGRAM OF PURIFIED INTACT HCG ON AN ULTRASPHERE ODS COLUMN 1.0 E c <M U 0 . 5 z < CQ CC o CO CD < o I I 1 0 2 0 T I M E ( M I N ) Chromatographic c o n d i t i o n s : column, U l t r a s p h e r e ODS 5\i HPLC (4.6 mm x 25 cm); mobile phase, 0.05% t r i f l u o r o a c e t i c acid/30% a c e t o n i t r i l e i n water; i n j e c t i o n volume, 0.025 mL (8.33 IU); flow r a t e , 1.0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 210 nm; c h a r t speed, 0.25 cm/min. -115-F I G U R E 1 6 CHROMATOGRAM OF FLUORESCAMINE DERIVATIVE OF HCG (AYERST LABORATORIES) ON A PROTEIN PAK 300 sw COLUMN WITH FLUORESCENCE DETECTION ' 1 1 0 10 2 0 T I M E ( M I N ) Chromatographic c o n d i t i o n s : column, P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium phosphate b u f f e r (0.15 M, pH 7.0); i n j e c t i o n volume, 0.025 mL (8.3 IU); flow r a t e , 1.0 mL/min.; f l u o r e s c e n c e d e t e c t i o n wavelength, e x c i t a t i o n / e m i s s i o n 390/470 nm; c h a r t speed, 0.25 cm/min. -116-FIGURE 17 CHROMATOGRAM OF FLUORESCAMINE DERIVATIVE OF HCG (AYERST LABORATORIES) ON TWO PROTEIN PAK 300 sw COLUMNS CONNECTED IN SERIES WITH FLUORESCENCE DETECTION 0.00*54 -O co in CM t 1 (fi Z UJ H Ul U O E UJ O o CO • X UJ CO o CM 0.0032 -r 10 2 0 T I M E ( M I N ) 3 0 Chromatographic c o n d i t i o n s : columns, two P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium phosphate b u f f e r (0.15 M, pH 7.0); i n j e c t i o n volume, 0.05 mL (16.7 IU); flow r a t e , 1.0 mL/min.; f l u o r e s c e n c e d e t e c t i o n wavelength, e x c i t a t i o n / e m i s s i o n 390/470 nm; c h a r t speed, 0.25 cm/min. FIGURE 18 CHROMATOGRAM OF FLUORESCAMINE DERIVATIVE OF HCG (AYERST LABORATORIES) ON PROTEIN PAK 125 sw AND 300 sw COLUMNS CONNECTED IN SERIES Chromatographic c o n d i t i o n s : columns, P r o t e i n Pak 125 sw HPLC (7.8 mm x 30 cm) and P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium phosphate b u f f e r (0.15 M, pH 7.0); i n j e c t i o n volume, 0.025 mL (8.3 IU); flow r a t e , 1.0 mL/min.; f l u o r e s c e n c e d e t e c t i o n wavelength, e x c i t a t i o n / e m i s s i o n 390/470 nm; c h a r t speed, 0.25 cm/min. -118-FIGURE 19 CHROMATOGRAM OF FLUORESCAMINE DERIVATIVE OF HCG (AYERST LABORATORIES) ON A PROTEIN PAK 125 sw COLUMN J- E Ul o E Ul Ul o z Ul o CO Ul cc o E c O 0> CO • X Ul 0.0 04-0.0 0 2 0 J m o> oo o> ^ co' -o J 1 0 2 0 TIME (MIN) 3 0 Chromatographic c o n d i t i o n s : column, P r o t e i n Pak 125 sw HPLC (7.8 mm x 30 cm); mobile phase, sodium phosphate b u f f e r (0.15 M, pH 7.0); i n j e c t i o n volume, 0.025 mL (8.3 IU); flow r a t e , 1.0 mL/min.; f l u o r e s c e n c e d e t e c t i o n wavelength, e x c i t a t i o n / e m i s s i o n 390/470 nm; c h a r t speed, 0.25 cm/min. -119-R e f e r e n c e S t a n d a r d HCG, p u r i f i e d i n t a c t HCG and p u r i f i e d i n d i v i d u a l s u b u n i t s were i n j e c t e d onto a newly a c q u i r e d P r o t e i n Pak 300 sw column under th e s e c o n d i t i o n s i n an attempt t o l o c a t e where i n t a c t HCG and the i n d i v i d u a l s u b u n i t s e l u t e d . 3.8.1 USP R e f e r e n c e Standard HCG With d e t e c t i o n a t 280 nm ( F i g u r e 20) the USP R e f e r e n c e S t a n d a r d r e v e a l e d one major peak a t 8.09 minutes and minor peaks a t 4.97, 6.31, 6.99 and 14.97 m i n u t e s . The chromatogram o b t a i n e d from the USP R e f e r e n c e S t a n d a r d shown i n F i g u r e 21 a t 208 nm i s s i m i l a r t o t h a t o b t a i n e d a t 280 nm ( F i g u r e 2 0 ) . S l i g h t d i f f e r e n c e s i n the o b s e r v e d r e t e n t i o n t imes between F i g u r e s 20 and 21 are due t o a g i n g of the column. C o l l e c t i o n of t h i s major peak e l u a t e (8.21, F i g u r e 21) and assay w i t h the b e t a - s u b u n i t L a t e x Pregnancy T e s t gave a p o s i t i v e r e s u l t i n d i c a t i n g t h a t i t c o n t a i n e d i n t a c t HCG and/or the b e t a s u b u n i t . 3.8.2 P u r i f i e d S u b u n i t s P u r i f i e d beta-HCG e l u t e d as one major peak a t 8.48 minutes ( F i g u r e 22). Assay of t h i s peak e l u a t e w i t h the b e t a - s u b u n i t L a t e x Pregnancy T e s t (N.C.S.) gave a p o s i t i v e -120-FIGORE 20 CHROMATOGRAM OF USP REFERENCE STANDARD HCG ON A PROTEIN PAK 300 sw COLUMN WITH DETECTION AT 280 nm Chromatographic c o n d i t i o n s : column, P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium phosphate b u f f e r (0.15 M, pH 7.0); i n j e c t i o n volume, 0.01 mL (6.65 IU); flow r a t e , 1.0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 280 nm; c h a r t speed, 0.25 cm/min. FIGURE 21 CHROMATOGRAM OF USP REFERENCE STANDARD HCG ON A PROTEIN PAK 300 sw COLUMN WITH DETECTION AT 208 nm 0 J 0 10 2 0 T I M E ( M I N ) Chromatographic c o n d i t i o n s : column, P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium phosphate b u f f e r (0.15 M, pH 7.0); i n j e c t i o n volume, 0.0025 mL (1.7 IU); flow r a t e , 1.0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 208 nm; c h a r t speed, 0.25 cm/min. -122-FIGURE 2 2 CHROMATOGRAM OF PURIFIED HCG-BETA ON A PROTEIN PAK 300 sw COLUMN WITH DETECTION AT 280 nm 0.0 2 -O CO CM OJ 0.01 o z < CD OC O Ui CD 0 J 00 00 If) o o CO0? r>- CO 1 0 2 0 T I M E ( M I N ) Chromatographic c o n d i t i o n s : column, P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium phosphate b u f f e r (0.15 M, pH 7.0); i n j e c t i o n volume, 0.020 mL (0.010 mg) ; flow r a t e , 1.0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 280 nm; c h a r t speed, 0.25 cm/min. -123-r e s u l t i n d i c a t i n g t h a t t h i s major peak c o n t a i n e d i n t a c t HCG and/or the b e t a s u b u n i t . S i n c e p u r i f i e d beta-HCG c o n t a i n s l e s s than 0.02% of t h e a l p h a s u b u n i t and.the s l i d e t e s t from N.C.S. has v e r y low c r o s s - r e a c t i v i t y , t h i s major peak a t 8.48 minutes c o n t a i n s the b e t a s u b u n i t . The chromatogram o b t a i n e d from i n j e c t i o n of the a l p h a - s u b u n i t ( F i g u r e 23) r e v e a l e d one major peak a t 9.81 mi n u t e s and two o t h e r peaks a t 4.94 and 8.54 m i n u t e s . Assay of peak e l u a t e s w i t h the b e t a - s u b u n i t L a t e x Pregnancy T e s t (N.C.S.) gave n e g a t i v e r e s u l t s c o n f i r m i n g t h a t n e i t h e r i n t a c t HCG nor i t s b e t a - s u b u n i t were p r e s e n t i n s i g n i f i c a n t q u a n t i t y . The a l p h a - s u b u n i t i s s m a l l e r than the b e t a - s u b u n i t and t h e r e f o r e was ex p e c t e d t o e l u t e a f t e r the b e t a s u b u n i t , a f t e r 8.48 minute s . T h e r e f o r e the a l p h a s u b u n i t may be p r e s e n t i n e i t h e r the 8.54 or 9.81 minute peaks. The major peak a t 9.81 minutes i s l i k e l y t he a l p h a - s u b u n i t s i n c e the p u r i f i e d p r e p a r a t i o n c o n t a i n s l i t t l e c o n t a m i n a t i o n . 3.8.3 P u r i f i e d I n t a c t HCG Assay of peak e l u a t e s from i n j e c t i o n of p u r i f i e d HCG ( F i g u r e 24) w i t h the N.C.S. S l i d e T e s t gave n e g a t i v e r e s u l t s f o r b o t h t h e major peak a t 4.85 minutes and t h e peak a t 8.20 minutes . S i n c e the USP Refer e n c e S t a n d a r d i n d i c a t e d i n t a c t -124-FIGURE 23 CHROMATOGRAM OF PURIFIED HCG-ALPHA ON A PROTEIN PAK 300 sw COLUMN WITH DETECTION AT 280 nm to oi 0.0 4 -O CO CM S 0 0 2 z < CD CC o CO CD < to 10 2 0 TIME (MIN) 3 0 Chromatographic c o n d i t i o n s : column, P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium phosphate b u f f e r (0.15 M, pH 7.0); i n j e c t i o n volume, 0.020 mL (0.010 mg); flow r a t e , 1.0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 280 nm; c h a r t speed, 0.25 cm/min. -125-FIGURE 24 CHROMATOGRAM OF PURIFIED HCG ON A PROTEIN PAK 300 sw COLUMN WITH DETECTION AT 280 nm 0.0 2 E c o 00 CM Uj 0.01 O z < 03 £C O V) CO < in co 0 10 20 T I M E ( M I N ) Chromatographic c o n d i t i o n s : column, P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium phosphate b u f f e r (0.15 M, pH 7.0); i n j e c t i o n volume, 0.020 mL (0.005 mg, 67.5 IU); flow r a t e , 1.0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 280 nm; c h a r t speed, 0.25 cm/min. -126-HCG e l u t e d a t 8.09 m i n u t e s , t h e 8.20 peak e l u a t e may c o n t a i n i n t a c t HCG below the s e n s i t i v i t y l e v e l of t h i s Pregnancy T e s t (2000 I U / L ) . 3.9 Comparison Of P u r i f i e d And Commercial P r e p a r a t i o n s The i n d i v i d u a l s u b u n i t s of HCG showed almost b a s e l i n e r e s o l u t i o n as shown i n F i g u r e 25, however, i n t a c t HCG c o u l d not be r e s o l v e d from the b e t a - s u b u n i t ( F i g u r e 2 6 ) . Two P r o t e i n Pak 300 sw columns c o u p l e d i n s e r i e s were used t o e v a l u a t e a sample of commercial HCG and USP R e f e r e n c e S t a n d a r d HCG. Some r e s o l u t i o n was o b s e r v e d between the 19.60 minute peak, the f i r s t major peak from A.P.L. and the 16.03 minute peak of i n t a c t HCG (USP R e f e r e n c e Standard) ( F i g u r e 27) i n d i c a t i n g t h a t the 19.60 minute peak from c o m m e r c i a l HCG c o n t a i n s components o t h e r than i n t a c t HCG and HCG-beta. A l s o , s i n c e f a r from b a s e l i n e r e s o l u t i o n was o b s e r v e d f o r the f i r s t and second major peaks from c o m m e r c i a l HCG, b o t h peaks c o n t a i n components o t h e r t h a n the i n d i v i d u a l s u b u n i t s . 3.10 Recycle S i z e E x c l u s i o n HPLC -127-FIGURE 25 CHROMATOGRAM OF PURIFIED ALPHA- AND BETA-SUBUNITS OF HCG Chromatographic c o n d i t i o n s : column, P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium phosphate b u f f e r (0.15 M, pH 7.0); i n j e c t i o n volume, 0.020 mL (0.0025 mg alpha-HCG, 0.0075 mg beta-HCG); flow r a t e , 1.0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 280 nm; c h a r t speed, 0.25 cm/min. -128-FIGURE 26 CHROMATOGRAM OF USP REFERENCE STANDARD HCG AND THE PURIFIED ALPHA- AND BETA-SUBUNITS 0.04 Chromatographic c o n d i t i o n s : column, P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium phosphate b u f f e r (0.15 M, pH 7.0); i n j e c t i o n volume, 0.020 mL (0.665 IU USP Reference Standard HCG, 0.0045 mg alpha-HCG, 0.005 mg beta-HCG); flow r a t e , 1.0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 280 nm; c h a r t speed, 0.25 cm/min. FIGURE 27 CHROMATOGRAM OF AYERST LABORATORIES AND USP REFERENCE STANDARD HCG ON TWO PROTEIN PAK 300 sw COLUMNS CONNECTED IN SERIES Chromatographic c o n d i t i o n s : columns, two P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium phosphate b u f f e r (0.15 M, pH 7.0); i n j e c t i o n volume, 0.020 mL (19.5 IU A y e r s t L a b o r a t o r i e s HCG, 0.333 IU USP Reference Standard HCG); flow r a t e , 1.0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 208 nm; c h a r t speed, 0.25 cm/min. -130-A s e r i e s o f e x p e r i m e n t s u s i n g r e c y c l e HPLC w i t h t h e new P r o t e i n Pak 300 sw column were conducted i n o r d e r t o p r o v i d e b e t t e r r e s o l u t i o n of the components of commercial HCG. A t h r e e way PTFE v a l v e was i n s t a l l e d a f t e r the d e t e c t o r t o a l l o w s e l e c t i o n of components t o be r e c y c l e d . For A.P.L., the l a r g e p r e s e r v a t i v e peak i n t e r f e r e d w i t h r e s o l u t i o n of the p r o t e i n peaks when r e c y c l e d so t h i s f r a c t i o n was j removed. A f t e r s i x c y c l e s t h r o u g h the column, t h e . r e c y c l e d peaks c o - e l u t e d ( F i g u r e 2 8 ) . R e c y c l i n g the p r o t e i n components of commercial HCG has e f f e c t i v e l y i n c r e a s e d the s e p a r a t i o n however the band b r o a d e n i n g has i n c r e a s e d . W ith subsequent i n j e c t i o n s , the components e l u t i n g a t 9.75 and 11.48 minutes were i n d i v i d u a l l y r e c y c l e d . No i n c r e a s e i n the r e s o l u t i o n of t h e components of the i n d i v i d u a l peaks was found and b o t h peak e l u a t e s c o l l e c t e d a f t e r 3 t o 4 c y c l e s t h r o u g h the column gave p o s i t i v e r e s u l t s w i t h the N.C.S. s l i d e t e s t . No f u r t h e r a t t e m p t s a t r e c y c l i n g c o m m e r c i a l HCG p r e p a r a t i o n s were done s i n c e t h e band b r o a d e n i n g had g r e a t l y i n c r e a s e d , the r e s o l u t i o n was not improved and the time i n v o l v e d would be u n p r a c t i c a l f o r an HPLC a s s a y . 3.11 R e c o m b i n a t i o n o f S u b u n i t s and D i s s o c i a t i o n Of I n t a c t HCG CHROMATOGRAM RECYCLING SIX FIGURE 28 OF HCG (AYERST LABORATORIES) AFTER TIMES ON A PROTEIN PAK 300 sw COLUMN Chromatographic c o n d i t i o n s : column, P r o t e i n Pak 300 sw HPLC (7.5 mm x 30 cm); mobile phase, sodium phosphate b u f f e r (0.15 M, pH 7.0); i n j e c t i o n volume, 0.50 mL (500 IU); flow r a t e , 1.0 mL/min.; u l t r a v i o l e t absorbance d e t e c t i o n wavelength, 208 nm; c h a r t speed, 0.25 cm/min. -132-Both the c h r o m a t o g r a p h i c d a t a and the immunoassay r e s u l t s suggest t h a t the main n o n - p r e s e r v a t i v e peaks of absorbance f o r the commercial HCG samples examined c o n t a i n i n t a c t HCG and/or HCG-beta ( e l u t i n g as t h e f i r s t major peak) and a number of p r o t e i n s and p o s s i b l y HCG-alpha e l u t i n g as the second major p e a k ) . Methods r e p o r t e d i n the l i t e r a t u r e f o r r e c o m b i n a t i o n of the s u b u n i t s and s e p a r a t i o n of i n t a c t HCG (Swaminathan 1970) were f o l l o w e d i n an e f f o r t t o c l a r i f y t h e s e r e s u l t s . N e i t h e r of t h e s e p r o c e d u r e s produced a s i g n i f i c a n t change as e v i d e n c e d by HPLC a n a l y s i s o f the samples. -133-4 . SUMMARY AND CONCLUSION The r e s u l t s o b t a i n e d f o r the USP R e f e r e n c e S t a n d a r d HCG samples i n d i c a t e t h a t i t c o n t a i n s t e n s e p a r a t e p r o t e i n s or s u b u n i t s . Three of t h e s e f r a c t i o n s have a m o l e c u l a r w e i g h t c o r r e s p o n d i n g t o t h a t r e p o r t e d i n t h e l i t e r a t u r e ( P a l a 1973) f o r HCG-alpha (27000 Da), HCG-beta (35000 Da) and i n t a c t HCG (63000 Da) by e l e c t r o p h o r e s i s . DISC-PAGE a l s o shows t h a t t h i s R e f e r e n c e S t a n d a r d sample c o n t a i n s more th a n one p r o t e i n ; one of which may be i n t a c t HCG. By HPLC, the USP R e f e r e n c e S t a n d a r d HCG a l s o appears t o c o n t a i n more t h a n one component. S i n c e a p o s i t i v e r e s u l t was o b s e r v e d w i t h the b e t a - s u b u n i t L a t e x Pregnancy Test f o r t h i s major peak, e i t h e r i n t a c t HCG, i t s b e t a - s u b u n i t or o t h e r c r o s s - r e a c t i n g compounds were p r e s e n t i n the peak e l u a t e . P u r i f i e d HCG-beta has one major peak i n u l t r a v i o l e t absorbance (at 8.48 minutes) which i s l i k e l y the b e t a - s u b u n i t . S i n c e HCG has a h i g h e r m o l e c u l a r w e i g h t than HCG-beta, i t s h o u l d e l u t e e a r l i e r than HCG-beta w i t h t h i s column. T h e r e f o r e , the peak i n u l t r a v i o l e t absorbance a t 8.21 minutes ( F i g u r e 18) f o r USP R e f e r e n c e S t a n d a r d HCG i s l i k e l y i n t a c t HCG. However, the p o s s i b i l i t y of c o - e l u t i o n of s m a l l amounts of HCG-beta or o t h e r compounds t h a t absorb u l t r a v i o l e t l i g h t , c r o s s - r e a c t w i t h the b e t a - s u b u n i t L a t e x Pregnancy T e s t and have a s i m i l a r m o l e c u l a r s i z e i n s o l u t i o n -134-can not be e x c l u d e d . P u r i f i e d HCG-alpha had one major peak i n u l t r a v i o l e t a b s o r p t i o n a t 9.81 minutes which i s l i k e l y the a l p h a - s u b u n i t . T h i s p u r i f i e d HCG-alpha peak may, however, c o n t a i n c o - e l u t i n g compounds t h a t absorb u l t r a v i o l e t l i g h t i n t h i s r e g i o n and are about the same s i z e . Chromatography of p u r i f i e d HCG shows t h a t i m p u r i t i e s are p r e s e n t a l o n g w i t h the i n t a c t HCG e l u t i n g a t 8.20 m i n u t e s . S i z e e x c l u s i o n HPLC of commercial HCG samples show two major n o n - p r e s e r v a t i v e peaks i n u l t r a v i o l e t a b s o r b a n c e . Use of the secondary methods of a n a l y s i s f o r e v a l u a t i o n of peak e l u a t e s i n d i c a t e d t h a t i n t a c t HCG, HCG-beta and/or o t h e r c r o s s - r e a c t i n g compounds e l u t e i n the i n i t i a l peak found w i t h t h e P r o t e i n Pak 300 sw column. A l s o , t h i s peak e l u a t e c o n t a i n s some HCG-alpha ( f r e e o r from i n t a c t HCG) s i n c e the e l e c t r o p h o r e s i s r e s u l t s i n d i c a t e d b o t h s u b u n i t s were p r e s e n t . The second major peak appears t o c o n t a i n a number of p r o t e i n s , one of which may be HCG-alpha. Attempts t o d i s s o c i a t e the i n t a c t HCG i n the USP R e f e r e n c e S t a n d a r d HCG were not s u c c e s s f u l due t o problems w i t h the d i s s o c i a t i o n method or the USP R e f e r e n c e S t a n d a r d . N e i t h e r of t h e s e p o s s i b i l i t i e s can be d i s r e g a r d e d a t the p r e s e n t t i m e . D i f f i c u l t y was a l s o found w i t h the r e c o m b i n a t i o n of the i n d i v i d u a l s u b u n i t s of HCG. Reasons f o r t h e l a c k of r e c o m b i n a t i o n of s u b u n i t s f o r c o m m e r c i a l HCG - 1 3 5 -samples i n c l u d e e r r o r s w i t h the method, changes i n e i t h e r the a l p h a - or b e t a - s u b u n i t such t h a t a s s o c i a t i o n c o u l d not occ u r or i n s u f f i c i e n t c o n c e n t r a t i o n of one or b o t h s u b u n i t s t o a l l o w r e c o m b i n a t i o n . These r e s u l t s i n d i c a t e commercial HCG samples ( A y e r s t L a b o r a t o r i e s and K-Line P h a r m a c e u t i c a l s ) c o n t a i n i n d i v i d u a l s u b u n i t s of HCG and p o s s i b l y i n t a c t HCG a l o n g w i t h the i m p u r i t i e s observed by e l e c t r o p h o r e s i s and HPLC. W i t h s i z e e x c l u s i o n c h r o m a t o g r a p h i c a n a l y s i s of the s e samples on a P r o t e i n Pak 300 sw column, the f i r s t major peak of u l t r a v i o l e t absorbance i s due t o HCG-beta and/or i n t a c t HCG and/or o t h e r u l t r a v i o l e t a b s o r b i n g compounds. The second major peak c o n t a i n s a number of p r o t e i n s and p o s s i b l y c o n t a i n s HCG-alpha. The USP Refe r e n c e S t a n d a r d HCG samples assayed may c o n t a i n i n t a c t HCG but a l s o has o t h e r u l t r a v i o l e t a b s o r b i n g components t h a t can be p a r t i a l l y s e p a r a t e d w i t h s i z e e x c l u s i o n chromatography. SDS-PAGE and DISC-PAGE b o t h i n d i c a t e t h a t the USP Re f e r e n c e S t a n d a r d HCG i s not pure. The s i z e e x c l u s i o n HPLC assay d e v e l o p e d u s i n g one P r o t e i n Pak 300 sw column was not a b l e t o r e s o l v e the b e t a - s u b u n i t from i n t a c t HCG. T h e r e f o r e i t would not be s u i t a b l e f o r development of a q u a n t i t a t i v e HPLC assay f o r HCG. Two c r i t e r i a o f a q u a n t i t a t i v e HPLC assay were met s i n c e t h i s a ssay was a b l e t o d e t e c t v e r y low l e v e l s of HCG -136-(0.21 IU of USP R e f e r e n c e Standard HCG) and r e q u i r e d under t e n minutes t o a n a l y s e each sample. 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