Identification of multiple sources of the acidic charge variants in an IgG1 monoclonal antibody

Abstract

Charge variants, especially acidic charge variants, of recombinant monoclonal antibodies are the major critical quality attributes in the biotechnology industry due to their potential influence on stability and biological activity. The chemical properties of the acidic charge variants have been challenging to fully characterize, and it is critical for process development and optimization. To completely understand the multiple sources of acidic charge variants, the major charge forms of an IgG1 monoclonal antibody were firstly isolated and then analyzed by a battery of characterization tools. It was found that various degrees of disulfide bond reduction, the deamination of HC-T8 Asn84 and HC-T35 Asn388 and aggregation account for the majority of acidic charge heterogeneity and the terminal galactosylation content was in relation to the acidic charge heterogeneity. The correlation between acidic charge heterogeneity and galactosylation content was further explored by weak cation exchange chromatography with the use of β1-4 galactosidase digestion. The results showed that galactosylation was not the source of the acidic charge variants per se. Meanwhile, to gain insights into the impact on binding affinity of monoclonal antibody to IgE and FcRn, charge variants were also analyzed by competitive ELISA and surface plasmon resonance, respectively. All isolated charge variants had similar affinity binding to IgE and FcRn binding relative to the starting material.

Macro- and Micro-Heterogeneity of Natural and Recombinant IgG Antibodies

“…These free cysteines have been shown to lower thermal stability [67] and increase the formation of reducible covalent aggregates [72,73,74]. The second scenario is the detection of relatively high levels of free Cys often due to the incomplete formation of a particular disulfide bond, mainly in the heavy chain variable domain [4,37,44,75,76] or the disulfide bond between the light chain and heavy chain [13,77]. The incomplete variable domain disulfide bond reduces the potency of one mAb [44], but has no impact on a different mAb [75].…”

“…MAb variants with the incomplete variable domain disulfide bond are more hydrophobic [15,37,44]. MAb variants without the disulfide bond between the light chain and heavy chain are enriched in the acidic species [13,77]. The incomplete heavy chain variable domain disulfide bond can be reformed in vivo [4].…”

“…However, galactosylation has not been reported to cause mAb heterogeneity in charge or hydrophobicity. The slight separation of mAb variants with different levels of galactosylation is probably caused by conformational differences since galactose should not change the charge properties [77]. Galactosylation can cause subtle conformational changes around the glycosylation site [54,112,113,114,115].…”

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis

“…A strategy for the direct coupling of MS with IEX involved the application of a pH gradient using volatile salts. 23,24,[29][30][31] Online weak cation exchange (WCX)-MS has been reported for analyzing IgG2 mAbs by using an ammonium hydroxide-based pH gradient. 32 Characterization of digested mAbs and proteins with molecular weights below~30 kDa has also been described by utilizing an ammonium formate and ammonium acetate-based pH and salt gradient.…”

Modulating cell culture oxidative stress reduces protein glycation and acidic charge variant formation

“…29 These control strategies modulated the charge profile typically by reducing the deamidation, glycation, or oxidation levels, based on information from in-house peptide mapping and literature reports. 7,[30][31][32] However, a clear and direct link is still missing on how exactly these factors influence these modifications. These three reactions are all chemically oxidative reactions that result in the loss of one electron and formation of ammonia (deamidation) or water (oxidation and glycation).…”