Advancing stability in cell line development 

Roxana McCloskey, Senior Global Marketing Manager, Protein Therapeutics, SCIEX explores how innovative analytical approaches can help improve stability for cell line development.  

Cell line development (CLD) is a critical stage in the early production of biotherapeutic molecules. During the CLD stage, a key goal is to generate, identify and advance the most stable cell lines for the robust production of high-quality therapeutic molecules in large quantities. This is achieved by screening for both product quality and productivity. Therapeutic proteins are becoming increasingly complex, expanding beyond traditional monoclonal antibodies (mAbs) to include molecules such as bi-, tri- and multi-specific antibodies; antibody-drug conjugates (ADCs); and fusion proteins. This growing diversity in biologics is accompanied by more characterization challenges that require innovative analytical approaches to support CLD groups in identifying and developing stable cell lines. 

The vast number of potential clones entering the development pipeline necessitates methods to rapidly prioritise candidates. Capillary electrophoresis (CE)- and mass spectrometry (MS)-based workflows have pivotal roles in achieving this goal. CE provides rapid assays that can assess key factors that affect stability, while MS offers precise detailed information about post-translational modification (PTM) localization and protein structure.  

Various conditions during cell processing and growth can induce changes that can impact the final product and that therefore must be comprehensively understood. Changes in titer, cell culture media, and other process conditions can induce PTMs that affect the stability and potential activity of the molecules. To understand the stability of the proteins manufactured by the cell lines, it is crucial to gather comprehensive information about their key quality attributes, including PTMs. Obtaining a complete analytical understanding of the impact of cell culture conditions on the manufactured product is critical to making process determinations within the development pipeline. This information can be used to help identify problematic areas that could be adapted, or to select for more resilient cell lines. In addition, decisions on the need for further analytical techniques and resources can be made earlier in the pipeline. 

At the intact level, monitoring PTMs can be accomplished by charge variant analysis. PTMs induce a shift in the isoelectric point (pI) of a molecule. Therefore, charge heterogeneity is a standard quality attribute that is monitored throughout the development pipeline. Changes in charge profile can offer insights into how clones respond to process changes. As molecule types become more complex, charge heterogeneity increases, and monitoring becomes more challenging.  

CE-based workflows offer a rapid and straightforward assessment of key factors affecting stability, including charge profile, charge heterogeneity, and glycan profile. Higher throughput assays enable the rapid narrowing down of potential clones, expediting the development pipeline. MS provides precise information about PTM localisation and protein structure through assays such as subunit analysis and peptide mapping. However, traditional peptide mapping can be time-consuming and is typically not appropriate to deploy during early clone selection. Modular and visually intuitive software platforms aid in the rapid identification of key PTMs, addressing some of the challenges faced by scientists utilising peptide mapping early in the development pipeline.  

Ideally, scientists would be able to obtain more detailed information about a molecule and its charge profile at both the intact and subunit levels. The direct integration of CE and MS can explain observed pI shifts without requiring extensive fractionation or peptide mapping. Technologies such as icIEF-UV/MS provide high-resolution separation and characterization of protein charge variants and aid in the identification of unexpected PTMs. By providing critical information about proteoform identification at the intact protein level, these systems contribute to risk reduction in CLD and the development pipeline overall. As a result, they help facilitate informed decisions on clonal selection and the optimisation of bioreactor conditions earlier. 

Recent virtual and conference presentations highlight the use of both icIEF-UV/MS and MS workflows within CLD groups. These strategies hold great potential for an intact-level multi-attribute monitoring platform approach, offering a streamlined and accelerated characterization of charge variants. An icIEF-UV/MS workflow on the Intabio ZT system is applied as a disruptive combination for the rapid and detailed characterization of mAb charge variants. This system aids in providing clonal identification and characterization early in the CLD process, enabling timely mitigation strategies. A middle-down MS strategy to characterize multi-specific antibodies has proved valuable in obtaining sequence coverage and insights into disulfide linkages. An orthogonal approach using icIEF-UV/MS and peptide mapping successfully investigated the discoloration of mAbs and next-generation antibodies. Discoloration is important as it might indicate impurities or other process-related changes to the antibody product, which could impact its stability, efficacy, and safety. The study identified advanced glycation end products (AGEs) as a contributor to the coloration of mAbs.  

The integrated online icIEF-UV/MS workflow is appropriate for monitoring and evaluating quality attributes in complex biologics, including mAbs, ADCs, and Fc fusion proteins. The icIEF-UV/MS workflow successfully identified glycosylation states, deamidation, glycation, and succinimide variants in the mAb sample. Similarly, it identified drug-to-antibody ratios (DARs), C-terminal amidation, C-terminal lysine, and ring-opening hydrolysis as key modifications in a cysteine-linked ADC.  

In conclusion, advancements in analytical techniques, such as CE, MS, and icIEF-UV/MS, significantly contribute to reducing risk and optimising stability in CLD. These approaches enable scientists to navigate through the complexity of therapeutic formats, identify potential issues early in development, and optimise conditions for stable cell line selection. The integration of these technologies, such as in icIEF-UV/MS and electron activated dissociation (EAD)-based workflows, gives scientists new tools to make informed decisions early in the development pipeline, ensuring the selection of the most stable and productive cell lines for therapeutic molecule production.  

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