Mechanism of antibody reduction in cell culture production processes

Abstract

We recently observed a significant disulfide reduction problem during the scale-up of a manufacturing process for a therapeutic antibody using a CHO expression system. Under certain conditions, extensive reduction of inter-chain disulfide bonds of an antibody produced by CHO cell culture may occur during the harvest operations and/or the protein A chromatography step, resulting in the observation of antibody fragments (light chain, heavy chain, and various combination of both) in the protein A pools. Although all conditions leading to disulfide reduction have not been completely identified, an excessive amount of mechanical cell lysis generated at the harvest step appears to be an important requirement for antibody reduction (Trexler-Schmidt et al., 2010). We have been able to determine the mechanism by which the antibody is reduced despite the fact that not all requirements for antibody reduction were identified. Here we present data strongly suggesting that the antibody reduction was caused by a thioredoxin system or other reducing enzymes with thioredoxin-like activity. The intracellular reducing enzymes and their substrates/cofactors apparently were released into the harvest cell culture fluid (HCCF) when cells were exposed to mechanical cell shear during harvest operations. Surprisingly, the reducing activity in the HCCF can last for a long period of time, causing the reduction of inter-chain disulfide bonds in an antibody. Our findings provide a basis for designing methods to prevent the antibody reduction during the manufacturing process.

Tailoring recombinant protein quality by rational media design

“…Future studies, without Protein A purification, shall also show whether other fragmentation mechanisms exist. The intracellular thioredoxin system plays an important role in the protein disulfide bond reduction 186,258 . The mammalian thioredoxin system comprising thioredoxin reductase (TrxR), the thioredoxin (Trx) and NADPH generate reduced thioredoxin that catalyzes disulfide bond reduction in a great number of proteins 259 .…”

“…Selenium, a culture medium component, may favor this pathway as a result of the higher availability of the trace element during the formation of the selenocysteine residue of TrxR. On the other hand, trace elements including Hg 2+ , Cu 2+ , Zn 2+ , Co 2+ and Mn 2+ , have been reported to form complexes with thiols and selenols and thus inhibit TrxR or Trx 186 . Hence, it is suggested to further ascertain the role of Cu 2+ , Zn 2+ by extending the concentration ranges of future experiments.…”

“…Counter to chemical degradation, the thioredoxin (Trx) activity diminishes in the presence of 50-100 µM copper sulfate (CuSO 4 ), which consequently minimizes free thiols and in this way fragmentation 185,186 . Three enzyme systems including the Trx system-consisting of Trx, thioredoxin reductase (TrxR) and NADPH-, glucose-6-phosphate dehydrogenase (G6PD), and hexokinase have been identified to be responsible for antibody reduction and may be targeted to inhibit fragmentation.…”

“…On the other hand, gold complexes (ATG, ATM) are the most effective and selective inhibitors of TrxR. Dehydroepiandrosterone, epiandrosterone, pyridoxal 5'-phosphate, 1-fluoro-2,4-dinitrobenzene have effectively reduced G6PD activity and the addition of chelating agents (EDTA & EGTA), citrate, distinct types of phosphates, 6-deoxy-6-fluoroglucose, 2-C-hydroxy-methylglucose, xylose, or lyxose inhibit hexokinase 186 . L-cystine exhibited chemical inhibitory capacities for a recombinant monoclonal antibody due to its function as competitive inhibitor for reducing enzymes 55 .…”

“…Increasing concentration of cupric ions in solution resulted in greater fragmentation rates and inhibited by chelating agents 179 . Moreover, three enzymatic mechanisms including the thioredoxin system (TrxR), glucose-6-phosphate dehydrogenase (G6PD), and hexokinase have been identified to be responsible for disulfide bond reduction 185,186 .…”