Evolution of a comprehensive, orthogonal approach to sequence variant analysis for biotherapeutics

Abstract

Amino acid sequence variation in protein therapeutics requires close monitoring during cell line and cell culture process development. A cross-functional team of Pfizer colleagues from the Analytical and Bioprocess Development departments worked closely together for over 6 years to formulate and communicate a practical, reliable sequence variant (SV) testing strategy with state-of-the-art techniques that did not necessitate more resources or lengthen project timelines. The final Pfizer SV screening strategy relies on next-generation sequencing (NGS) and amino acid analysis (AAA) as frontline techniques to identify mammalian cell clones with genetic mutations and recognize cell culture process media/feed conditions that induce misincorporations, respectively. Mass spectrometry (MS)-based techniques had previously been used to monitor secreted therapeutic products for SVs, but we found NGS and AAA to be equally informative, faster, less cumbersome screening approaches. MS resources could then be used for other purposes, such as the in-depth characterization of product quality in the final stages of commercial-ready cell line and culture process development. Once an industry-wide challenge, sequence variation is now routinely monitored and controlled at Pfizer (and other biopharmaceutical companies) through increased awareness, dedicated cross-line efforts, smart comprehensive strategies, and advances in instrumentation/software, resulting in even higher product quality standards for biopharmaceutical products.

Evidence for co-translational misincorporation of non-canonical amino acid hydroxyproline in recombinant antibodies produced in Chinese Hamster Ovary (CHO) cell lines

“…Wong et al [ 15 ] created a catalogue of these potential amino acid misincorporations in CHO based expression systems including those arising from amino acid starvation. Since amino acid starvation-induced misincorporations can be controlled by replenishing the limiting nutrient, Lin et al [ 18 ] have developed an effective strategy to monitor and address such misincorporations using a combination of amino acid profiles and tandem mass spectrometry.…”

A general evidence-based sequence variant control limit for recombinant therapeutic protein development

“…As a result of these collective efforts, several control strategies have been developed and proposed by different companies to best monitor and mitigate the SV issue during their product and process development. 6,8,13 As expected, these proposed strategies highlighted the importance of a multi-assay, multi-tier SV screening approach to guide process development from early cell line selection to small-scale cell culture process development to scale-up confirmation. Together, these strategies have provided a valuable and industry-wide framework and highlevel guidance toward the goal of establishing some common best practices in terms of SV control.…”

“…Among the various product quality attributes, sequence variants (SVs) resulting from unintended amino acid substitution are an increasing concern and have been discussed by both the biopharmaceutical industry and regulatory agencies. 4 – 6 Such SVs have been shown to exist in both natural and recombinant proteins, 7 and are believed to be caused by a number of mechanisms including DNA mutations during replication, and transcriptional and translational errors during the protein biosynthesis process. 5 Thanks to the high fidelity of biologic systems, which evolved to prevent the occurrence of such spontaneous errors, the SVs are usually present at a very low level (<0.1%) in natural biologic proteins.…”

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

“…25 The extra information gathered from these analyses defines a more comprehensive search space for MS/MS identification. 27 Strategies using an orthogonal approach for sequence variants detection have evolved as reported recently by Lin et al 28 Here, we report the discovery, identification, and characterization of an 11 kDa Fc C-terminal extension sequence variant of a recombinant IgG1 mAb (mAb-A) from a CHO manufacturing cell line by using a combination of MS methods and HTS. Intact mass analysis and peptide mapping were used to deduce that the 11 kDa increase in molecular mass resulted from an addition to the heavy chain Fc.…”