UBC Theses and Dissertations
Isolation of immunoglobulins from cheese whey and milk using ultrafiltration and immobilized metal affinity chromatography Fukumoto, Lanna Rumiko
Cheddar cheese whey was concentrated using an ultrafiltration (OF) membrane with a 50 kDa molecular weight cut-off (MWCO). The concentrated whey was applied to an immobilized metal affinity chromatography (IMAC) column to isolate IgG. With a 50 ml IMAC column loaded with 17.3 ml of 50 mM CuCl₂ to get 2/3 Cu saturation, 1240 mg of IgG could be isolated with 56% purity. About 152 mg of lactoferrin and 90 mg of lactoperoxidase could be isolated along with the IgG. By using concentrated whey instead of unconcentrated whey, the time required to apply the whey was reduced proportionally to the concentration factor. Whey could be concentrated up to 30 times without any decrease in recovery or purity of IgG obtained by IMAC. UF of whey with a 50 kDa or 100 kDa MWCO membrane at 4°C or 45°C did not affect IgG recovery or purity obtained by IMAC. Diafiltration also had no effect. By using 50 mM glycine instead of 1.0 M NH₄Cl in the eluting buffer, elution was more efficient. The type of whey used could affect the recovery and purity of IgG obtained by IMAC. IMAC was run at room temperature or 7°C without any loss in recovery or purity of IgG, but the elution profile was altered. IgG could be recovered from a whey concentrated commercially by UF as well. A method was also developed to isolate IgG from raw skim milk using UF and IMAC. Ion exchange chromatography was compared to IMAC as an alternative method to isolate IgG from concentrated whey. Ion exchange chromatography gel was about 31 times less expensive than IMAC gel. The capacity of an ion exchange chromatography column for IgG was 16 times lower than for an IMAC column, and the purity of IgG recovered was lower. Taking the capacity into account, the overall cost of IMAC was about 2 times more than ion exchange chromatography. Considering the high purity of IgG obtainable and the advantages in working with smaller columns, IMAC appeared to be a better method of isolating IgG. A small-scale shelf life study of IgG in UHT milk was also done. A thermal resistance curve for IgG in UHT milk was constructed assuming first-order destruction of IgG at 62°, 66°, 70°, 74°, 78° and 80°C. D values were obtained from the first-order destruction curves. The D values were used to plot a thermal resistance curve, and D values were extrapolated for temperatures used for the shelf life study. D values extrapolated were 1.77 x 10¹⁴ yr at 4°C, 3.85 x 10⁷ yr at 25°C, and 1.94 x 10⁵ yr at 35°C. IgG isolated by IMAC was membrane sterilized using prefiltration and final filtration with a 0.2 Jim membrane. Membrane sterilization did not affect IgG activity. Membrane sterilized IgG was aseptically injected into 250 ml Tetra Brik cartons containing 2% white UHT milk. The cartons were stored at 4°, 25° and 35°C. Over 5 months of the shelf life study, no change was observed in IgG concentration as predicted.
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