Aggregation and Chemical Modification of Monoclonal Antibodies under Upstream Processing Conditions
- 1.9k Downloads
To investigate antibody stability and formation of modified species under upstream processing conditions.
The stability of 11 purified monoclonal human IgG1 and IgG4 antibodies, including an IgG1-based bispecific CrossMab, was compared in downscale mixing stress models. One of these molecules was further evaluated in realistic bioreactor stress models and in cell culture fermentations. Analytical techniques include size exclusion chromatography (SEC), turbidity measurements, cation exchange chromatography (cIEX), dynamic light scattering (DLS) and differential scanning calorimetry (DSC).
Sensitivity in downscale stress models varies among antibodies and results in formation of high molecular weight (HMW) aggregates. Stability is increased in cell culture medium and in bioreactors. Media components stabilizing the proteins were identified. Extensive chemical modifications were detected both in stress models as well as during production of antibodies in cell culture fermentations.
Protective compounds must be present in chemically defined fermentation media in order to stabilize antibodies against the formation of HMW aggregates. An increase in chemical modifications is detectable in bioreactor stress models and over the course of cell culture fermentations; this increase is dependent on the expression rate, pH, temperature and fermentation time. Consequently, product heterogeneity increases during upstream processing, and this compromises the product quality.
KEY WORDSaggregation antibodies deamidation fermentation stress models
cation exchange chromatography
differential scanning calorimetry
dynamic light scattering
high molecular weight
melting point temperature
nephelometric turbidity units
size exclusion chromatography
chinese Hamster Ovary
Acknowledgments and Disclosures
We would like to thank Carolin Lucia and Katharina Didzus from Roche Penzberg for their support with bioreactor stress experiments, Sabrina Mahlack for her support with MAb4.2 purification, and Hubert Kettenberger, Holger Kley and Xaver Reiser for their support with DLS and DSC measurements. We would also like to thank Alexandra Schindl for carrying out the shaking stress experiments and Robert Puskeiler for support with calculations of physical parameters of the stirring stress models.
We are also grateful to Jörg Hörnschemeyer and Jonas Fast of Roche Basel for sharing information about the identity of cIEX peaks.
S.D, F.H. F.L. O.P. and K.L are all employees of Roche at the time of publication. The remaining authors declare no competing financial interests.
- 2.Schaefer W, Regula JT, Bähner M, Schanzer J, Croasdale R, Dürr H, Gassner C, Georges G, Kettenberger H, Imhof-Jung S, Schwaiger M, Stubenrauch KG, Sustmann C, Thomas M, Scheuer W, Klein C. Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies. Proc Natl Acad Sci USA. 2011;108(27):11187–92.PubMedCrossRefGoogle Scholar
- 14.Gomez N, Subramanian J, Ouyang J, Nguyen MD, Hutchinson M, Sharma VK, Lin AA, Yuk IH. Culture temperature modulates aggregation of recombinant antibody in cho cells. Biotechnol Bioeng (2011).Google Scholar
- 33.Bahrami A, Shojaosadati SA, Khalilzadeh R, Mohammadian J, Farahani EV, Masoumian MR. Prevention of human granulocyte colony-stimulating factor protein aggregation in recombinant Pichia pastoris fed-batch fermentation using additives. Biotechnol Appl Biochem. 2009;52:141–8.PubMedCrossRefGoogle Scholar
- 44.Cartaya OA. Serum-free cell culture media. United States Patent 4,205,126, 1980.Google Scholar