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UBC Theses and Dissertations

Enzymatic harvesting of glycosyl phosphatidylinositol anchored recombinant proteins from mammalian cells Sunderji, Rumina


Controlled release of recombinant proteins from mammalian cells enables protein product harvesting at increased concentrations and purity by separating protein expression from the protein recovery. The chinese hamster ovary (CHO) cell line investigated was genetically engineered to express glycosyl phosphatidylinositol (GPI) anchored human melanoma antigen p97 on the outer surface of the cell membrane. At intervals the cells were treated with a phosphatidylinositol phospholipase C (PT-PLC) harvest solution to selectively cleave the GPI anchor and recover the protein at high concentration and purity (cyclic harvesting). The growth of the recombinant CHO cells was investigated in the serum-free media CHO-S-SFM I, CHO-S-SFM II, HBCHO, DMEMJF12 and Ham’s F12. CHO-S-SFM II supplemented with DNase and CHO-S-SFM I achieved single suspension cells at high densities (approximately 6x10⁶ cells/mL). CHO-S-SFM I was selected for all further investigations. A repeated harvesting technique which involved re-using the PT-PLC enzyme solution to harvest separate batches of cells was investigated. This approach further increased the concentration of the desired protein product after each harvest. Preliminary repeated harvesting experiments recovered 140 ɥ/mL p97 from 7 harvests of 10⁸ cells each. The first harvest recovered approximately 60 jig/mL p97, therefore theoretically 7 harvests should have recovered 420 ɥ/mL. Since this process did not achieve the expected concentrations, the stability of p97 and PT-PLC were investigated. However, p97 was found to be stable at 37 °C in the harvesting medium (PBS with I mg/mL BSA) for 24 h. Since a suitable assay for P1-PLC was not available, a new PT-PLC assay was developed. PT-PLC was stable at 37 °C for a period of 14 days. PT-PLC and p97 were also stable in the pH range of 6.0 - 7.9. Instability of the proteins involved in the harvesting process was not the cause for the low product concentrations recovered from repeated harvesting. Loss of P1-PLC due to adsorption to the cells was studied. An equilibrium was established between P1-PLC in solution and P1-PLC adsorbed to the cell surface within 3 minutes. Since PT-PLC was adsorbed to each batch of cells in the repeated harvesting process, enzyme was removed with the cells after each harvest. Therefore loss of PT-PLC by adsorption was considered the cause for the reduced protein recovery. The repeated harvesting process was repeated with PT-PLC replenishment after each harvest. Addition of 30 or 300 mU/mL PT-PLC recovered respectively 294 or 343 ig/mL p97 from 5 consecutive harvests. The estimated purity of p9’7, based on total protein, was approximately 30 %. A continuous harvesting process was also investigated. This approach involved addition of PT-PLC to the growth medium resulting in the continuous release of p97 into the medium. The continuous harvesting process was carried out simultaneously with 0, 3 and 30 mU/niL PT-PLC. The 0 mU/niL PT-PLC control produced approximately 3.8 ɥ/mL p97 in a batch culture of 11 days. During the same period of time cultures with 3 and 30 mU/niL P1-PLC yielded 11.6 and 15.3 ɥ/mL p97 respectively. For the continuous harvesting process at 30 mU/niL PT-PLC the p97 productivity was 5.75x10⁻⁷ fig/cell-day or approximately 2-fold higher than that achieved by the cyclic and repeated harvesting processes. However, the repeated harvesting process used approximately 10 times less PT-PLC and recovered 20-fold higher p97 concentrations.

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