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. 2018 Jul;29(7):1512-1523.
doi: 10.1007/s13361-018-1968-0. Epub 2018 May 7.

Investigation of Sequence Clipping and Structural Heterogeneity of an HIV Broadly Neutralizing Antibody by a Comprehensive LC-MS Analysis

Vera B Ivleva  1 , Nicole A Schneck  2 , Deepika Gollapudi  2 , Frank Arnold  2 , Jonathan W Cooper  2 , Q Paula Lei  3
Affiliations

Investigation of Sequence Clipping and Structural Heterogeneity of an HIV Broadly Neutralizing Antibody by a Comprehensive LC-MS Analysis

Vera B Ivleva et al. J Am Soc Mass Spectrom. 2018 Jul.

Abstract

CAP256 is one of the highly potent, broadly neutralizing monoclonal antibodies (bNAb) designed for HIV-1 therapy. During the process development of one of the constructs, an unexpected product-related impurity was observed via microfluidics gel electrophoresis. A panel of complementary LC-MS analyses was applied for the comprehensive characterization of CAP256 which included the analysis of the intact and reduced protein, the middle-up approach, and a set of complementary peptide mapping techniques and verification of the disulfide bonds. The designed workflow allowed to identify a clip within a protruding acidic loop in the CDR-H3 region of the heavy chain, which can lead to the decrease of bNAb potency. This characterization explained the origin of the additional species reflected by the reducing gel profile. An intra-loop disulfide bond linking the two fragments was identified, which explained why the non-reducing capillary electrophoresis (CE) profile was not affected. The extensive characterization of CAP256 post-translational modifications was performed to investigate a possible cause of CE profile complexity and to illustrate other structural details related to this molecule's biological function. Two sites of the engineered Tyr sulfation were verified in the antigen-binding loop, and pyroglutamate formation was used as a tool for monitoring the extent of antibody clipping. Overall, the comprehensive LC-MS study was crucial to (1) identify the impurity as sequence clipping, (2) pinpoint the clipping location and justify its susceptibility relative to the molecular structure, (3) lead to an upstream process optimization to mitigate product quality risk, and (4) ultimately re-engineer the sequence to be clip-resistant. Graphical Abstract ᅟ.

Keywords: Comprehensive LC-MS/MSE; HIV-1 bNAb; IdeS; Peptide mapping; Pyroglutamination; Sequence clipping; Subunit; Vaccine product quality risk.

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Figures

Figure 1.
Figure 1.
CE profiles of the reduced CAP256 bNAb: HEK cell line (top) and CHO (bottom). The CHO cell line platform resulted in two additional peaks, in addition to the expected light and heavy chains
Figure 2.
Figure 2.
TIC of the reduced CAP256 with medium clipping, showing separation of the subunits, the subunit fragments with modifications, and the light chain glycoforms. Inset: TIC of the reduced and deglycosylated CAP256 produced in HEK cell line
Figure 3.
Figure 3.
Light chain characterization: TIC of the reduced CAP256 (inset) with the individual components of the light chain numbered, corresponding to the deconvoluted spectra of the light chain glycoforms (a–d) and deconvoluted spectrum of the deglycosylated light chain (e). An additional peak on the mass spectrum (e) corresponds to incomplete cysteine alkylation, a sample preparation artifact
Figure 4.
Figure 4.
Deconvoluted mass spectra. (a) The heavy chain and its ~ 40-kDa fragment, extracted from the 6.4 ± 0.3-min time range and (b) the small (~ 14 kDa) fragment of the heavy chain (4.5–5.2-min time range)
Figure 5.
Figure 5.
Summary of the identified portion of the CAP256 primary sequence: tryptic coverage (solid underline); chymotryptic coverage (dotted underline) complementing the missing portions of the tryptic coverage; both digest types contributing to a cumulative 98% sequence coverage. PTMs with % abundance (when applicable) denoted above the modified site marked in bold
Figure 6.
Figure 6.
TIC chromatogram of the CAP256 bNAb after IdeS digestion, reduction, and deglycosylation with PNGase F, demonstrating the clipped heavy chain fragments among other IdeS digested bNAb major products
Figure 7.
Figure 7.
Identification of the clipping location based on MSE de novo sequencing of the unassigned tryptic component. Top: unassigned peak on the chromatogram. Middle: assignment of pyroQLPCAK (a) and its non-modified analog QLPCAK (b); XIC of the corresponding peptides: 699.3 Da (pyroQLPCAK) (a, inset), 716.3 Da (QLPCAK) (b, inset), (0.1 Da extracted mass window). Bottom: Fab portion of CAP256 heavy chain showing the clipping site and the identified tryptic peptide marked in bold
Figure 8.
Figure 8.
Clipping site identification. (a) A protruding loop in the CDR-H3 region (left); clipping location with a disulfide bond preventing the non-reduced bNAb to fall apart into two fragments (right). (b Disulfide bond arrangement, planar view: Cys residues are numbered, verified disulfide bonds marked in red. The sequence of the heavy chain Fab region is shown, along with the corresponding schematic view zoomed into the clipping site: the tryptic semidigested peptide (highlighted in orange) identified as a peptide dimer (tryptic component held together by a disulfide bond), covering the clipping site area
Scheme 1.
Scheme 1.
Various LC-MS workflows used to characterize the CAP256 bNAb
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