Identification and characterization of a -1 reading frameshift in the heavy chain constant region of an IgG1 recombinant monoclonal antibody produced in CHO cells

Abstract

Frameshifts lead to complete alteration of the intended amino acid sequences, and therefore may affect the biological activities of protein therapeutics and pose potential immunogenicity risks. We report here the identification and characterization of a novel -1 frameshift variant in a recombinant IgG1 therapeutic monoclonal antibody (mAb) produced in Chinese hamster ovary cells during the cell line selection studies. The variant was initially observed as an atypical post-monomer fragment peak in size exclusion chromatography. Characterization of the fragment peak using intact and reduced liquid chromatographymass spectrometry (LC-MS) analyses determined that the fragment consisted of a normal light chain disulfide-linked to an aberrant 26 kDa fragment that could not be assigned to any HC fragment even after considering common modifications. Further analysis using LC-MS/MS peptide mapping revealed that the aberrant fragment contained the expected HC amino acid sequence (1-232) followed by a 20-mer novel sequence corresponding to expression of heavy chain DNA sequence in the -1 reading frame. Examination of the DNA sequence around the frameshift initiation site revealed that a mononucleotide repeat GGGGGG located in the IgG1 HC constant region was most likely the structural root cause of the frameshift. Rapid identification of the frameshift allowed us to avoid use of a problematic cell line containing the frameshift as the production cell line. The frameshift reported here may be observed in other mAb products and the hypothesis-driven analytical approaches employed here may be valuable for rapid identification and characterization of frameshift variants in other recombinant proteins.

Expression vector-derived heterogeneity in a therapeutic IgG4 monoclonal antibody

“…Aware of other examples of mAb heterogeneity with genetic origins, [41][42][43][44][45] we sought to identify whether a similar mechanism could have led to C-terminal modification observed here. To explore this possibility, we performed in A) Annotated reverse-phase total ion chromatogram; B) deconvoluted mass spectrum for the peak eluting at 7.8 minutes corresponding to the reduced Fc (C-terminal 210 amino acids of the HC); C) deconvoluted intact mass spectrum for the peak eluting at 8.4 minutes, consistent with the Fc plus 1177 Da.…”

“…Fusions of mAb chains with other sequences, large-scale truncations, and combinations of fusions and truncations have been observed. [41][42][43][44][45][46] These types of variants typically have genetic origins, but the extent to which any such form persists through purification is likely species-and process-dependent. Here, we describe evidence for an additional contributor to mAb heterogeneity: an expression vector-derived twelve amino acid C-terminal extension on mAb heavy chains (HCs).…”

Identification and characterization of an IgG sequence variant with an 11 kDa heavy chain C-terminal extension using a combination of mass spectrometry and high-throughput sequencing analysis

“…Sequence variants produced by either genomic mutations or misincorporation during translation have been reported for therapeutic protein products. [3][4][5][6][7][8] Variants can be introduced into the encoding gene sequences during vector propagation in bacteria prior to transfection or may result from errors in DNA replication in the production cell line once the vector has been incorporated. For example, a point mutation in a single cell may lead to a sub-population of the production cell line that expresses a single nucleotide variant (SNV), resulting in a percentage of the final drug product carrying a single amino acid substitution.…”

“…3 However, data from LC-MS/MS should be carefully examined and verified by orthogonal methods due to prevalent false positive results. 14 Beyond SNVs, DNA deletions or insertions can cause a frame-shift leading to an altered amino acid sequence, 5 or a premature stop codon and the formation of a truncated product. Largerscale genomic recombination events can produce extension products, including molecules in which entire domains were added.…”

“…6,19 In addition, peptide mapping methods alone are usually not sufficient to identify low-level sequence variants (<1%) other than single amino acid substitutions. Even though low-level sequence variants can be enriched by chromatography approaches, such as size exclusion, 5,15 ion exchange, 21 or reversed-phase 20 chromatography, it is still time-consuming and resource-intensive to enrich enough material for multi-enzyme de novo sequencing. The lack of peak identification and annotation is a limiting factor for proteomics experiments that can be overcome by proteogenomics, a new field that is based on the use of high-throughput data from different sources as part of an iterative refinement process of gene models.…”