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www.biopharminternational.com Emerging Therapies eBook September 2024 BioPharm International ® 11 Cell Line Development Recent presentations highlight the use of icIEF-UV/ MS and MS workflows within CLD teams. For instance, an icIEF-UV/MS workf low (Intabio ZT system ) has been applied as a disruptive combination for the rapid and detailed characterization of mAb charge variants (see Figure 2) (4–6). This system aids in providing clonal identification and characterization early in the CLD process, enabling timely mitigation strategies. Additionally, a middle-down MS strategy has proved valuable in characterizing multi-specific antibodies, providing insights into sequence coverage and disul- fide linkages. An orthogonal approach using icIEF-UV/ MS and peptide mapping successfully investigated the discoloration of mAbs and next-generation antibodies, identifying advanced glycation end products (AGEs) as contributors to coloration. This integration of CE and MS workflows allows for the monitoring and evaluation of key quality attributes in complex biologics, including mAbs, ADCs, and Fc fusion proteins (7–9). Case studies Enhancing clone selection with icIEF-UV/MS Integration Scientists at Johnson & Johnson Innovative Medicine have demonstrated the effectiveness of combining icIEF with MS to identify and mitigate PTM characterization issues early on. Kristen Nields, PhD, senior scientist at Johnson & Johnson Innovative Medicine, emphasized the importance of monitoring PQAs throughout mAb development. Utilizing the icIEF–UV/MS system, her team was able to identify PTMs during clone selection, thereby formulating a mitigation strategy to move promising clones forward efficiently (8–10). This sys- tem allows for high-resolution separation of protein charge variants using icIEF–UV and simultaneous mass analysis, providing real-time insights into the nature of charge variants. Characterizing charge heterogeneity is essential throughout m Ab development to understand the native state of the mAb and the impact of any PTMs, including deamidation, C-terminal lysine trunca- tion, glycation, and sialylation. Identif ying these PTMs early in the process enables the formulation of strategies to better manage them and to do so early in the process. A common phenomenon arising during m Ab bioproduction is C-terminal lysine clipping, which leads to variable amounts of process-related charge variants in the final product. C-terminal ly- sine appears as a basic proteoform, which is typically cleaved off during method validation. This can only be achieved enzymatically, which can become costly during method validation. Once the method is vali- dated, further variability can be provided by different carboxypeptidase, creating supply chain issues when producing an out-of-specification product. While icIEF can provide a high-resolution sepa- ration of protein charge variants and is commonly used throughout the development pipeline to mon- itor PQAs, it has proved challenging and time con- suming in the past to identify and characterize dif- ferent PTMs from these charge variant peaks in a single assay. Researchers have been able to employ integrated systems that allow them to correlate icIEF FIGURE 2. Components of imaged capillary isoelectric focusing ultraviolet detection with MS (icIEF–UV/MS) analysis on a microfluidic chip (4,6).