Duk Jae Oh

Effects of Supplementation of Various Medium Components on Chinese Hamster Ovary Cell Cultures Producing Recombinant Antibody

Thirteen vitamins, twenty amino acids, hormones, inorganic salts, and other chemical agents, which constitute typical serum-free media, were evaluated for the development of fortified medium to enhance cell growth and productivity of recombinant antibody in the cultures of the recombinant Chinese hamster ovary (rCHO) cells. Two different rCHO cell lines, rCHO-A producing recombinant antibodies against the human platelet and rCHO-B secreting recombinant antibodies against the S surface antigen of Hepatitis B, respectively, were cultivated in batch suspension mode. Concentration of interested component in the tested medium was doubled to examine the fortification effect. Growth of rCHO-A cell and its antibody production were slightly improved with addition of either choline chloride, folic acid, thiamine⋅HCl, or LongTMR3IGF-I. On the other hand, in the cultivation of rCHO-B cell which was more sensitive to its environmental changes, hormones such as LongTMR3IGF-I and triiodothyronine (T3) as well as various vitamins involving choline chloride, i-inositol, niacinamide, pyridoxine HCl, and thiamine⋅HCl enhanced the cell growth and antibody production. Particularly, when concentration of consuming amino acid was doubled, remarkable increase in specific productivity was served, resulting in high final antibody concentration. These results were believed to provide a fundamental strategy of medium fortification useful for improvement of recombinant antibody production in serum-free medium.

Application of a Cell-Once-Through Perfusion Strategy for Production of Recombinant Antibody from rCHO Cells in a Centritech Lab II Centrifuge System

Based upon the results of scale‐down intermittent perfusion processes, a cell‐once‐through (COT) perfusion concept was applied to a dual bioreactor system coupled to a Centritech Lab II centrifuge for culture of recombinant Chinese hamster ovary (rCHO) cells for monoclonal antibody production. In this new culture mode, i.e., the COT perfusion process, total spent medium was transferred to the centrifuge and a fixed percentage was removed. Approximately 99% of the viable cells are transferred to another bioreactor filled with fresh medium by single operation of the Centritech Lab II centrifuge system for about 30 min. Accordingly, a significant reduction of the cell‐passage frequency to the centrifuge led to minimization of cell damage caused by mechanical shear stress, oxygen limitation, nutrient limitation, and low temperature outside the bioreactor. The effects of culture temperature shift and fortified medium on cell growth and recombinant antibody production in the COT perfusion process were investigated. Although the suppressive effects of low culture temperature on cell growth led to a loss of stability in a long‐term COT perfusion culture system, the average antibody concentration at 33 °C was 157.8 mg/L, approximately 2.4‐fold higher than that at 37 °C. By the use of a fortified medium at 37 °C, rCHO cells were maintained at high density above 1.2 × 107 cells/mL, and antibody was produced continuously in a range of 260–280 mg/L in a stable long‐term COT perfusion culture. The proposed new culture mode, the COT perfusion approach, guarantees the recovery of rCHO cells damaged by lowered temperature or high lactate and ammonium concentration. It will be an attractive choice for minimization of cell damage and stable long‐term antibody production with high cell density.

Use of soybean protein hydrolysates for promoting proliferation of human keratinocytes in serum-free medium

Human keratinocytes are generally cultured in media containing bovine pituitary extract (BPE), an animal product that can be a source of infectious contaminants. We investigated whether a safer plant product could replace BPE in the culture medium. Medium containing both BPE and soy protein hydrolysates (Bacto Soytone and Soy Hydrolysate) produced the largest number of viable cells, followed in descending order by medium supplemented only with BPE, only with the hydrolysates, and without supplementation (basal medium only). Soybean protein is thus an excellent source of nutrients for the growth of adherent keratinocytes, although they do not fully substitute for BPE.

Effect of Environmental Parameters on Glycosylation of Recombinant Immunoglobulin G Produced from Recombinant CHO Cells

Site specific glycosylation of immunoglobulin G (IgG) occurs at Asn297 in the Fc region. The heterogeneous ensemble of glycoform occurs due to the degree of terminal galactosylation and sialylation, and these differences in glycosylation affect both the pharmacokinetic behavior and effector functions of the IgG, such as complementdependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). In this study, the differential glycosylation of IgG was compared and environmental physical and chemical parameters were evaluated in an attempt to promote glycosylation of recombinant antibodies, thereby creating more humanized glycoform antibodies and increasing their in vivo efficacy as therapeutic drugs. It was shown that cells at late stationary growth phase in batch cultures, cells with increased passage number, and the culture conditions of lowered temperature and pH promoted galactosylation and sialylation of antibodies. Galactose, fructose and mannose were found to elicit galactosylation and sialylation when they were used alone as a substitute of glucose. Mannose showed synergistic effects on glycosylation when used with other sugars, such as glucose and galactose. However when fructose was used with other sugars, the degree of galactosylation mechanism appeared to be decreased. These results support understandings of the glycosylation mechanisms in glycoprotein, particularly recombinant antibodies for therapeutics.

Production of recombinant human growth hormone by rCHO cells in a depth filter perfusion system

Human growth hormone is a single-chain polypeptide produced commercially from recombinant animal cells as well as recombinant microorganisms. Its increased applications have requested development of highly efficient production systems using particularly animal cells. Depth filter perfusion system (DFPS) has been developed and successfully used for production of recombinant proteins such as antibodies from recombinant Chinese hamster ovary (rCHO) cells. In this study, rCHO cells expressing recombinant human growth hormone (rhGH) were successfully cultivated in the DFPS for 2,200 h. Parameters affecting the performance of the DFPS for rhGH production were investgated. The depth filter with 40 µm pores was selected for stable operation. The shifts of culture temperature and pH were tested at 37 and 33℃, and 7.2 and 7.0, respectively. In the begining of the culture, more than 80% of the seeded cells were immobilized in 200 min by medium/cell circulation. When the culture temperature was lowered from 37 to 33℃, about 50% increase of the volumetric productivity (VP) at a perfusion rate of 2.0/day was achieved, and the VP reached 168 mg/L/d at perfusion rate of 3.0/day, showing the benefit of cultures in low temperature. In contrast, when pH was shifted from 7.2 to 7.0, maintaining rhGH concentration at about 60 mg/L and decreased perfusion rate from 2.0 to 1.0/day, the VP dropped to 50%. As the DFPS showed stable and efficient production of rhGH for long-term periods from this study, it can be an attractive choice for production of rhGH from recombinant animal cells.