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Identification of a CE-SDS shoulder peak as disulfide-linked fragments from common CH2 cleavages in IgGs and IgG-like bispecific antibodies

ABSTRACT: ABSTRACT Fragmentation is a well-characterized degradation pathway of therapeutic antibodies and is usually monitored by capillary electrophoresis–sodium dodecyl sulfate (CE-SDS). Although fragments due to cleavage in CH2 domains linked by intrachain disulfide bonds are common and can be detected by reduced reversed-phase – liquid chromatography mass spectrometry (RP-LCMS) and reduced CE-SDS methods, their separation in nonreduced CE-SDS (nrCE-SDS) has not been reported but speculated as comigrating with intact IgG. A shoulder peak in nrCE-SDS was observed in the stability samples of an IgG-like bispecific antibody and was determined to be mainly caused by fragments from clipping at the C-terminus of leucine (L)306 or L309 (EU numbering) in the CH2 domain of both heavy chains (HCs) and, to a lesser degree, at the C-terminus of L182 in the CH1 domain of the knob HC. Subunit LCMS analysis verified that the crystallizable fragment contained variants with one or multiple mass additions of ~18 Da due to clipping. Further investigation revealed that CH2 clippings at L306 and L309 were largely due to proteolytic activity, and cleavages were present at various levels in all in-house IgG1 and IgG4 molecules studied. Our study shows that CH2 domain cleavages, with complementary fragments still linked by intrachain disulfide, can be electrophoretically resolved as a front shoulder of the main peak in nrCE-SDS. Given the high occurrence of CH2 cleavages in antibodies, these findings will have broad applicability and could help manufacturers of therapeutic antibodies in process improvement, product characterization, investigations, formulation stability, and stability comparability studies.

SUBMITTER: Cao M

PROVIDER: S-EPMC8565840 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Bispecific antibodies (bsAbs) are antibodies with two binding sites directed at different antigens, enabling therapeutic strategies not achievable with conventional monoclonal antibodies (mAbs). Since bispecific antibodies are regarded as promising therapeutic agents, many different bispecific design modalities have been evaluated, but as many of them are small recombinant fragments, their utility could be limited. For some therapeutic applications, full-size IgGs may be the optimal format. Two challenges should be met to make bispecific IgGs; one is that each heavy chain will only pair with the heavy chain of the second specificity and that homodimerization be prevented. The second is that each heavy chain will only pair with the light chain of its own specificity and not with the light chain of the second specificity. The first solution to the first criterion (knobs into holes, KIH) was presented in 1996 by Paul Carter's group from Genentech. Additional solutions were presented later on. However, until recently, out of >120 published bsAb formats, only a handful of solutions for the second criterion that make it possible to produce a bispecific IgG by a single expressing cell were suggested. We present a solution for the second challenge-correct pairing of heavy and light chains of bispecific IgGs; an engineered (artificial) disulfide bond between the antibodies' variable domains that asymmetrically replaces the natural disulfide bond between CH1 and CL. We name antibodies produced according to this design "BIClonals". Bispecific IgGs where the artificial disulfide bond is placed in the CH1-CL interface are also presented. Briefly, we found that an artificial disulfide bond between VH position 44 to VL position 100 provides for effective and correct H-L chain pairing while also preventing the formation of wrong H-L chain pairs. When the artificial disulfide bond links the CH1 with the CL domain, effective H-L chain pairing also occurs, but in some cases, wrong H-L pairing is not totally prevented. We conclude that H-L chain pairing seems to be driven by VH-VL interfacial interactions that differ between different antibodies, hence, there is no single optimal solution for effective and precise assembly of bispecific IgGs, making it necessary to carefully evaluate the optimal solution for each new antibody.

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Identification of a CE-SDS shoulder peak as disulfide-linked fragments from common CH2 cleavages in IgGs and IgG-like bispecific antibodies

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