Characterization of monoclonal antibody size variants containing extra light chains

Lü, Chan; Liu, Dandan; Liu, Hongbin; Motchnik, Paul A.

doi:10.4161/mabs.22965

Cited by 54 publications

(58 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Furthermore, the following analytical methods were applied: Size-exclusion chromatography, non-gel sieving denaturing capillary electrophoresis (CE-SDS; non-reduced and reduced), capillary isoelectric focusing (CE-IEF), reversephase UPLC of released and labeled oligosaccharides, mAb1specific SPR target binding assay and mAb1-specific qualified cell-based functional assay (the latter two assays were developed in-house). [38][39][40][41][42][43][44] The five samples were comparable with regard to quality and physico-chemical properties by using the above mentioned analytical methods, e.g., very low aggregate content, unaltered charge distribution, identical oligosaccharide profiles, in vitro target binding and functionality (data not shown).…”

Section: Physico-chemical and Functional Characterizationmentioning

confidence: 99%

A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

Stracke¹,

Emrich²,

Rueger

et al. 2014

mAbs

102

View full text Add to dashboard Cite

Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.

show abstract

Section: Physico-chemical and Functional Characterizationmentioning

confidence: 99%

A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

Stracke¹,

Emrich²,

Rueger

et al. 2014

mAbs

102

View full text Add to dashboard Cite

show abstract

“…IgG2 antibodies contain 18 disulfide bonds (similar distribution but two additional disulfide bonds between the two HCs). The following disulfide‐related modifications are observed for IgG disulfide bonds: Partial cleavage of inter‐ and intrachain disulfide bonds. Disulfide scrambling . Cysteinylation /glutathionylation /addition of LCs in presence of cysteine (Cys), glutathione or free light chains (FLCs). …”

Section: Introductionmentioning

confidence: 99%

Assessment of disulfide and hinge modifications in monoclonal antibodies

Moritz

Stracke

2017

Electrophoresis

View full text Add to dashboard Cite

During the last years there was a substantial increase in the use of antibodies and related proteins as therapeutics. The emphasis of the pharmaceutical industry is on IgG1, IgG2, and IgG4 antibodies, which are therefore in the focus of this article. In order to ensure appropriate quality control of such biopharmaceuticals, deep understanding of their chemical degradation pathways and the resulting impact on potency, pharmacokinetics, and safety is required. Criticality of modifications may be specific for individual antibodies and has to be assessed for each molecule. However, some modifications of conserved structure elements occur in all or at least most IgGs. In these cases, criticality assessment may be applicable to related molecules or molecule formats. The relatively low dissociation energy of disulfide bonds and the high flexibility of the hinge region frequently lead to modifications and cleavages. Therefore, the hinge region and disulfide bonds require specific consideration during quality assessment of mAbs. In this review, available literature knowledge on underlying chemical reaction pathways of modifications, analytical methods for quantification and criticality are discussed. The hinge region is prone to cleavage and is involved in pathways that lead to thioether bond formation, cysteine racemization, and iso‐Asp (Asp, aspartic acid) formation. Disulfide or sulfhydryl groups were found to be prone to reductive cleavage, trisulfide formation, cysteinylation, glutathionylation, disulfide bridging to further light chains, and disulfide scrambling. With regard to potency, disulfide cleavage, hinge cleavage, disulfide bridging to further light chains, and cysteinylation were found to influence antigen binding and fragment crystallizable (Fc) effector functionalities. Renal clearance of small fragments may be faster, whereas clearance of larger fragments appears to depend on their neonatal Fc receptor (FcRn) functionality, which in turn may be impeded by disulfide bond cleavage. Certain modifications such as disulfide induced aggregation and heterodimers from different antibodies are generally regarded critical with respect to safety. However, the detection of some modifications in endogenous antibodies isolated from human blood and the possibility of in vivo repair mechanisms may reduce some safety concerns.

show abstract

“…Two other examples of a mAb containing a third light chain have been reported in the literature. These species are believed to be formed through a disulfide bond either to an engineered cysteine, 10,11 or the C-terminal cysteine 12 in the mAb light chain, neither is the case for the three light chain species discussed in this paper. We have shown that the third light chain is not associated through disulfide bonding, but rather is non-covalently attached to mAb-X monomer.…”

Section: Methodsmentioning

confidence: 90%

“…Genentech researchers recently reported the thorough characterization of a mAb species containing variants with one and two additional light chains present at 0.2% when produced in Chinese hamster ovary (CHO) cell culture. 12 They determined that the relative quantity of this species was related to the redox environment of the cell culture, which implicates disulfide bonding as the mode of binding for the additional light chains.…”

Section: Resultsmentioning

confidence: 99%

Analytical characterization of a monoclonal antibody therapeutic reveals a three-light chain species that is efficiently removed using hydrophobic interaction chromatography

Wollacott

Casaz

Morin

et al. 2013

mAbs

View full text Add to dashboard Cite

show abstract

Characterization of monoclonal antibody size variants containing extra light chains

Cited by 54 publications

References 16 publications

A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

Assessment of disulfide and hinge modifications in monoclonal antibodies

Analytical characterization of a monoclonal antibody therapeutic reveals a three-light chain species that is efficiently removed using hydrophobic interaction chromatography

Contact Info

Product

Resources

About