Effect of Culture Conditions on IgM Antibody Structure, Pharmacokinetics and Activity

Abstract

Culture conditions affect the binding activity, charge heterogeneity, conformational stability, glycosylation, and pharmacokinetics of human monoclonal IgM HMAB-10058. The 10058 human/human/murine trioma was grown in serum-free airlift suspension culture, hollow fiber perfusion culture, or in nude mouse ascites. The ascites-produced antibody showed reduced conformational stability, greater charge and glycoform heterogeneity, and a lower average degree of sialylation than the in vitro culture-produced material. Mean residence time after IV injection in rats was approximately 80-fold greater for the ascites culture-produced material, but specific binding activity was less than 5% of that for the airlift-produced material. In vitro culture in serum-supplemented media (in a hollow fiber perfusion reactor or in shake-flasks) resulted in antibody with pharmacokinetics intermediate between the serum-free airlift and ascites-produced materials. Incubation of airlift-produced antibody in ascites fluid also resulted in material with intermediate pharmacokinetics. Conclusions regarding the effect of culture conditions on antibody product cannot be generalized, as in vitro-produced antibody derived from two related cell lines (HMAB-10233 and HMAB-10390) had long mean residence times similar to that of ascites-produced HMAB-10058.

Related effects of cell adaptation to serum-free conditions on murine EPO production and glycosylation by CHO cells

“…Therefore, except for the sialylation degree, EPO glycosylation was not significantly modified by the serum removal from the culture medium, which suggests that the intracellular process of EPO glycosylation involving numerous glycosyltransferases and glycan precursors seems to be not compromised. This phenomenon was already reported in the literature as serum was shown to affect the sugar content of the glycans of glycoproteins, particularly by decreasing the sialylation degree which is known to play a critical role in their biological activity (Patel et al 1992;Megaw and Johnson 1979;Maiorella et al 1993;Lamotte 1997;Gawlitzek et al 1995). However, these results seem to be closely dependent on the molecule investigated and of the culture conditions used (Gawlitzek et al 1995) as other papers reported a minor effect of the serum on the glycosylation pattern of recombinant proteins (Lifely et al 1995;Moellering et al 1990;Kopp et al 1996).…”

Apoptosis-resistant E1B-19K-expressing NS/0 myeloma cells exhibit increased viability and chimeric antibody productivity under perfusion culture conditions

“…Furthermore, increased levels of cellular-derived substances such as DNA are prone to complicate cell/medium separations (Maiorella et al, 1991). The release of proteases and other intracellular enzymes can result in oxidation, deamination and oligosaccharide hydrolysis of the proteins of interest (Karl et al, 1990;Mohan et al, 1993;Van Erp et al, 1991) with deleterious consequences such as enhanced clearance rates of injected products in therapeutic applications (Goochee and Monica, 1990;Maiorella et al, 1993;Prior et al, 1989).…”

Effect of bench-scale culture conditions on murine IgG heterogeneity

“…Although there is one consensus sequence on each IgG Fc H chain for an asparagine-linked sugar chain, the variable region of L and H chain may also be glycosylated (Abel et al, 1968;Spielberg et al, 1970). The carbohydrate on the Fc portion of immunoglobulin has been shown to be involved in different events like complement activation through the binding to Clq (Duncan and Winter, 1988), Fc receptor binding, antibody-dependent cellular cytotoxicity, and feedback immunosuppression, in many cases without affecting antigen binding (Maiorella et al, 1993;Robinson et al, 1994).…”

“…Only a limited number of papers have been published on the effect of culture conditions on antibody structural and functional homogeneity (Maiorella et al, 1993;Patel et al, 1992). Investigations on murine IgM production indicated that antibody structure, activity, and pharmacokinetics were dependent on culture conditions, resulting in wide differences in binding activity and clearance rate according to the production method used (Maiorella et al, 1993).…”

“…Investigations on murine IgM production indicated that antibody structure, activity, and pharmacokinetics were dependent on culture conditions, resulting in wide differences in binding activity and clearance rate according to the production method used (Maiorella et al, 1993). In a different study, a transfectant clonal derivative of the murine myeloma cell line, NSO, expressing the heavy and light chain genes of a recombinant human anti-HIV gp120 IgG1, was cultivated in a fed-batch mode, and MAb characterization from different time points in the process suggested the presence of differential antibody glycosylation (Robinson et al, 1994).…”