See also Oxidation and Reduction in Biochemistry

Disulfide Bond Variants 

Disulfide reduced variants have one more disulfide bonding cystein(s) chemically reduced to the free thiol form. This variant can be monitored by HIC or by sizing methodology such as Capillary Electrophoresis with Sodium Dodecyl Sulfate (Harris, US2009/0202546).

Half-antibodies: Occasionally the disulfide bonds between the heavy chain polypeptides of antibodies are not formed, resulting in the formation of an antibody with no interchain disulfide bonds between two pairs of heavy and light chains. These antibodies have been called “half antibodies” (or “Hab”). Certain antibody classes and types are more susceptible to half antibody formation such as IgG4. In both natural and recombinant antibody production, a significant proportion of IgG4 antibodies at least as high as 35% are produced as half antibodies (Brower US 14/668820, published as US 2016/0108127).

Causes of Antibody Reduction in Cell Culture Production Processes:

Reducing enzymes: A very important structural feature of an antibody is the disulfide bonds that link its light and hiavy chains (inter-chain disulfides) together to form a quaternary complex. Under specific conditions, extensive reduction of the antibody’s inter-chain disulfides bonds are observed after harvest operations (centrifugation and filtration) and/or the first purificaiton step (i.e., protein A chromatography). On one such instance, it was estimated that as little as 10% of the antibody remained intact after the protein A step. This antibody reduction is not a unique issue to a specific antibody or a particular cell line. The reduction is reportedly caused by an active thioredoxin (Trx) system or other reducing enzymes with thioredoxin-like activity in the harvested cell culture fluid. The Trx system (i.e., Trx and TrxR) is one of the two intracellular enzymatic systems (the other is the glutaredoxin (Grx) system) that regualtes the cellular redox status and maintains a reducing environment in the cytosol. *Kao “Mechanism of antibody reduction in cell culture production processes” Biotechnology and Bioengineering” (2010). 

During purificaiton of IgG2, high level of fragment in the prcoess intermedaites was detected by HP-SEC. Fruther investigations traced the casue to be antibdoy reduction in the harvested cell culture fluid (HCCF). In addition, when the HCCF was redued, larger increase in aggregate content was observed druing low pH treatment. When purificaiton was initiated immediately after harvest, antibody reduction was not observed in the HCCF and aggregate formation during low pH treatment was minimized. This can be attributed to the shorter exposure time of the antibody to reducing enzymes in teh HCCF. Increasing cystine concentraitons also resulted in lower free thiol levels when the HCF was held. (Chun “Effects of antibody disulfide bond reduction on purificaiton process performance and final drug substance stability” Tiotechn Bioeng, 114(6) (2017). 

Methods to Decrease Reduction of Disulfide bonds

Reduction of the disulfide bonds that internally anchor disulfide bond-containing proteins can occur during manufactering. Reduction of the inter-chain disulfide bonds results in loss of functional antibodies and requires more coplex purification. During mAb process development, fragments and aggregates have to be removed to adequate levels due to their associated risks with increased immunogenicity and unknown effects on drug efficacy (Ahuja, US 2019/0112357)

Using Charged Depth Filter

Hoang (US 15/751,231, published as US 2018/0230180) discloses a method of reducing the perentage of reduced antigen-binding protein molecules using a charged depth filter.

Control of Disulfide bonds and Half-antibody formation using reduction/oxidation (redox) agents:

–-Addition of 2-MEA, reduced gluathione, oxidized glutathione, 2-mercaptoethanol, DTT, cystein, cystein, dithiobutylaine or sodium sulfite or Oxidizing metal ions (Cu2+, F32+, Fe3+, Zn2+)

—-During Cell Harvest

Brower (US 14/668820, published as US 2016/0108127) discloses a process to control the formation of disulfide bonds between polypeptides by contacting the polypeptides with a conditioned solution that includes predetermined solution parameters such as a redox reagent like 2-mercaptoethylamine (2-MEA), reduced glutahione, oxidized glutathione, 2-mercaptoethanol, dithiothreitol (DTT), cystine, dithiobutylamine or sodium sulfite. at a specific time point during the bioprocess. In some embodiments, the specific time point occurs during the step of viral inactivation, adjustment, chromatography, filtration, dilution, concentration or any bioprocess step that is cell free. In other embodimetns, the specific time point occurs during chromatogaphy.  In one embodiment, Brower discloses that the level of half antibody decreased in the presence of 1 mM 2-MEA but that higher levels (25 and 50 mM) results in increassed level of half-antibody and also generated H and L fragments.The level of half antibody also decreased within about 30 minutes in the presence of 2-MEA at 1-3 mM at pH 4.8. 

Chaderjian (“Effect of Copper Sulfate on performance of a serum-free CHO cell culture process and the level of free thiol in the recombinant antibody expressed” Biotechnol. Prog. 21: 550-3 (2005) discloses that cooper sulfate can at as an oxidizing agent, thereby facilitating disulfide bond formation when added to cell culture production medium. Purified antibody showed that either 50 or 100 uM copper sulfate additions reduce the level of free thiol by more than 10-fold. 

Dillon (WO 2006/047340) discloses a method of producing a IgG antibody by contacting the antibody that has been recombinantly produced by mammalian cells with a reduction/oxidation coupling reagent at a pH of about 5-11, optionally with a chaotropic agent. The method reduces the heterogeneity of the various antibody isotypes. For example, the method can be used to refold IgG4 molecules to decrease the present of IgG4 half molecuels. The redox coupling reagent may be any redox coupling reagents such a reduced glutatione and oxidized glutathione. 

Schotte (WO2010/125187) discloses a method for producing a domain antibody in a host other than E. coli, preferably yeast, by applying conditions that promote the formation of disulfide bridges in the domain antibody, specifically the addition of oxidizing agent, preferably oidizing metal ions such as Cu2+, Fe2+, Fe3+, Zn2+, or removing domain antibodies lacking at least one disulfide bridge or a combination of both. The inventor found that despite the high yield and funcitonality of domain antibodies produced in non-E coli hosts, there is a quantiatively significant fraction of product that represents a structural variant, specifically a product that lacks at least one disulfide bond. 

–—–Addition of Proxides to prevent antibody disulfide bond reduction:

During the manufacturing process, extensive reduction of antibodies has been observed after harvest operation or Protien A chromatography and multiple process parameters correlate to the extent of the reduction. For example, maintaining high levels of dissolved oxygen (DO) during harvest is vital to keep antibody molecuels intact. Mechanical shear forces, which cause cell lysis and cellular components to leak into harvest fluids also significantly contribues to the reduction. Other process parameters such as harvest hold time, media componetents (such as copper ions, cystein/cystine and pH) and termpature also have an effect on the extent of disulfide reduction. The reduciton of antibody products due to enzyme systems may be complete or partial Human IgG contain two H and 2 L chains that are held together by non-covalent interactions as well as inter chain disulfide bonds. There are a total of 12 intra chain disulfide bonds and one disulfide bond between L and H chains in all four IgG subclasses. The nubmer of inter-chain disulfide bonds between the two H chains is 2 for IgG1 and IgG4, 4 for IgG2 and 11 for IgG3. Additionally, the interchain disulfide bonds are more solvent acccessible than intra-chain bonds and the exposed cysteine residues are considered more reactive than non-exposed cysteine residues. Hydrogen proxide can oxidize reducing agents and prevent the reduction of disulfide bonds and prevent the generation of LMW species. There are many peroxide containing compounds, inorganic or oragnic that can be substituted for hydrogen peroxide. Two inorganic forms, sodium percabonate and sodium perborate, can also effectively suppress the reduction of disulfide bonds in a concentraiton dependent manner. (Du, “Using hydrogen peroxide to prevent antibody disulfide bond reduction during manufacturing process” MABS, 10(3), pp. 500-510 (2018). 

——-Addition of Peroxides + Red Light:

Color variation due to antibody process related impurities can be a major concern as a problem of lack of process control. One of the main sources of pink/red color in final drug substance has been identified as the vitamin B12-mAb complex.  Cyanocoblamin is the vitamin B12 form typically included in cell culture media as a cofactor required for DNA synthesis. Under cell culture conditions, however, cyanocobalamin is readily converted to hydroxo-cobalamin due to light exposure, with up to 80% converted after 10 d in culture. The red color intensity can be minimized by reducing the medium concentration of vitamin B12. Since disulfide reduction mediated mAb dissocaition can be prevented at the manufacturing scale( with putblished methods, the risk of red color can also be minimimized with these published methods. (Derfus “Red colored IgG4 casued by vitamin B12 form cell culture media combined with disulfide reduction at harvest” mAbs 2014).

The intensity of pink color depends on concentraitons of both free sulfhydryl groups on reduced mAb and hydroxocoalamin, the active form of vitamin B12. Both reactants are necessary to generate pink colr. Thus process control strategy can consider limiting either one or both factors. (Du, “Vitamin B12 association with mAbs: mechanism and potential mitigation strategeis” Biotechn. and Bioeng. 2018, 115: 900-909). 

Tan (US 16/612,175, published as US 20230103511) discloses preventing the reduction of antibodies by adding peroxides (e.g., hydrogen peroxide, sodium percarbonate or sodium perborate) to the harvest solution and inhibiting the conversion of vitamine B 12 (cyanocoalamin (CN-Cbl)) to hydroxocobalamin (HO-Cbl) by culturing cells under red light conditions during manufacture or cell harvest. Tan showed that the generation of pink colr during antibody manufacturing is dependent on concetnrations of both free sulfhydryl groups on reduced antibodies and hydroxocobalamin, the active form of vitamin B12. Both reactants are necessary and neither one alone is sufficient to generate pink color. The active form of vitamin B12 attaches to the free sulfhydryl group of the cystein located at H chain 134, LC 214, HC 321, 367 and 425. The five cysteins residues are distributed among both the Fab and the Fc regions. Cynocobalamin convertion to hydroxocobalamin is inhibited or reduced by replacing white light with red colored light (wavelenghts >600 nm) in the areas where the cell culture media is prepared and sotred and optionally in cell culture manufacturing and harvesting areas. 

–During Chromatography Process

Godavarti (US 7,825,223) discloses purifying an AB binding protein having an Fc region such as an anti-AB antibody by absorbing the antibody to an Fc binding agent such as Protein A, followed by a wash with a divalent cation such as copper, nickel, manganese, to remove impurities. 

Zou (WO 2006/060083) discloses subjecting column isolated preparations of polypetpides with a reduction/oxidation reagent such as cysteins and cystine, reduced and oxidized gluathione, dithiothreitol (DTT), 2-mercaptoethanol, hydrogen peroxide (oxidizer) and dithionitrobenzoate and a chatropic agent and isolating the refolded active protein produced from said contacting. In one embodiment, the redox reagent is cystein/cystine. The chaotropic agent can be urea, SDS or guanidine hydrochloride. In other embodimetns, refolding of the protein can be performed using other redox reagents such as copper. The refolding can occur on a column such as an affinity resin (e.g., Protein A) of a Fc domain containing polypeptide.

Isolation of domain antibodies with oxidizing agent(s)

Schotte (US13/266503 and US2012/0157664) discloses a method of producing a domain antibody in yeast by applying conditions such as addition of oxidizing agents, preferably oxidizing metal ions such as Cu2+, Fe2+, Fe3+ and Zn2+ that promote the formation of disulfide bridges in the domain antibodies and/or removing domain antibodies lacking at least one disulfide bride. The method results in the production of domain antibodies wherein the quality of the domain antibodies is improved with a reduced level of free thiol or its absence. 

Oxidation of Amino Acids:

Oxidation is a common degradation pathway that may occur during the life cycle of prtoeins and peptides. The resulting chemical modificaitons may affect in vitro stability and in vivo biologcial functions. In order to assess the potentail susceptibility of protein products to oxidation, different oxdiation stress conditions have been studies such as hydrogen peroxide (H2O2). , transition metal ions or UV exposure. Scuh treatment often result in a myriad of modificaitons on several amino acid residues, such as histidine, phenylalanine and tyrosine. However, methionine and tryptophan are the residues that are oxidized most cmmonly  and to the highest extent. (Folzer, “Selective oxidation of methionine and tryptophan residues in a therapeutic IgG1 molcules” J of Pharmaceutical Sciences, 2015). 

Recombinant mAbs are constantly exposed to oxidizing environments, including dissolved oxygen, oxygen in teh air and free radicals such as those generated by reactions with metals and impurities form raw materials (e.g., peroxides). Forced oxidation is used to probe residues taht are susceptible to oxdiation and to udnerstand if there is a practical impact on this degradation pathway (e.g., oxidation of a residue in the CDR may affect binding and potency). The most commonly used approaches are incubation of recombinant mAbs with hydrogen peroxide or tert-butyl hydrogen peroxide for methionine residues. (Nowark “Forced degradation of recombinant monocloanl antibodies: A practical guide” mAbs (2017)). 

Oxidation of methionine: 

Although protein oxidation can occur at cysteine, tryptophan, lysine and other amino acids, methionine is often the most susceptible residue to oxidation. The most common product of methionine oxidation is methionine sulfoxide, which is more polar and less hydrophobic than methionine. Susceptible methionines are typically located on the surface of the protein and exposed to the solvent. Met oxidaiton can have adverse effects on proteins, including decreased stability and decreased biological activity. (Pan “Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn” Protein Science, 2008, 18, 424-433).

Folzer, “Selective oxidation of methionine and tryptophan residues in a therapeutic IgG1 molcules” J of Pharmaceutical Sciences, 2015) disclsoes that oxidation levels of Fc methionine was first quantified by prtoein A chromatogrpahy. the mAb species eluted at 23 min. The oxidized species formed upon inucation with oxidant eluted earlier (before 19.8 min) because of the lower affintiy of oxidized Fc methionine to protein A. The incubation of the mAb with 1% H2O2 for 24 hr at 5C as well as the incubation with 2% t-BHP for 120 h at 25C allowed for teh nearly complete oxidation of the methionine residues form teh Fc region.