Eliminating tyrosine sequence variants in CHO cell lines producing recombinant monoclonal antibodies

Abstract

Amino acid sequence variants are defined as unintended amino acid sequence changes that contribute to product variation with potential impact to product safety, immunogenicity, and efficacy. Therefore, it is important to understand the propensity for sequence variant (SV) formation during the production of recombinant proteins for therapeutic use. During the development of clinical therapeutic products, several monoclonal antibodies (mAbs) produced from Chinese Hamster Ovary (CHO) cells exhibited SVs at low levels (≤3%) in multiple locations throughout the mAbs. In these examples, the cell culture process depleted tyrosine, and the tyrosine residues in the recombinant mAbs were replaced with phenylalanine or histidine. In this work, it is demonstrated that tyrosine supplementation eliminated the tyrosine SVs, while early tyrosine starvation significantly increased the SV level in all mAbs tested. Additionally, it was determined that phenylalanine is the amino acid preferentially misincorporated in the absence of tyrosine over histidine, with no other amino acid misincorporated in the absence of tyrosine, phenylalanine, and histidine. The data support that the tyrosine SVs are due to mistranslation and not DNA mutation, most likely due to tRNA(Tyr) mischarging due to the structural similarities between tyrosine and phenylalanine.

In vitro and in vivo modifications of recombinant and human IgG antibodies

“…147 Low levels of sequence variants have been more widely reported recently, [148][149][150][151][152][153][154] which can be attributed to the advance of modern analytical techniques offering much higher sensitivity. Sequence variants are introduced because of mutation at the DNA level, 153,155 during transfection, 147 or translation.…”

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

“…In recent years, numerous examples of such expression errors in production of recombinant proteins at various stages of the manufacturing process have been reported. [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] These reported examples are predominately single amino acid substitution caused by either DNA mutations [3][4][5][6][7][8][9][10][11] or misincorporation during translation, [12][13][14][15][16] which are commonly referred as sequence variants. Expression errors leading to alteration of longer segments of amino acid sequence have also been reported.…”

Toward an era of utilizing methionine overproducing hosts for recombinant protein production inEscherichia coli

“…Amino acid sequence variants, which can occur as a result of DNA mutations, errors during transcription, translation, and/or posttranslational processing, are one of the well-known product variants formed during manufacturing of recombinant protein therapeutics both in microbial 1,2,3 and mammalian systems. 2,4,5 Sequence variants have received considerable attention in recent years as there has been a tremendous advancement in the analytical techniques to detect these variants even at low levels. 2,3,6 Sequence variants in the final drug product can have a potential impact on biological activity and could possibly result in immunogenicity when the product is in injected into patients.…”