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Influences of Free Thiols on the Product Quality of Therapeutic Antibodies
kim, michael tae-jong
kim, michael tae-jong
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Abstract
Therapeutic antibodies are an important class of modern medicines. During the biomanufacturing of therapeutic antibodies, many attributes relevant to product quality must be considered to ensure an efficacious and safe end product. One of those attributes is the free thiol, or an unpaired cysteine residue, which is interesting because of its innate and diverse reactivities. In this thesis, we first review topics - including the structure and function of antibodies, antibodies as therapeutics, the biomanufacturing of therapeutic antibodies, free thiols as a post-translational modification, and basic reactions involving free thiols – and then discuss three case studies that explore free thiols as they pertain to the product quality of therapeutic antibodies. We explored the physiological outcome of free thiols on therapeutic antibodies after administration. We designed non-clinical studies where therapeutic antibodies with elevated free thiols at various sites are administered. Plasma samples were collected at various timepoints and we utilized a bioanalysis workflow involving immunopurification and mass spectrometry techniques to track site-specific free thiol levels at each timepoint. We observed that in vivo free thiol reoxidation kinetics are molecule- and disulfide site-dependent. We drew a novel connection positively associating free thiol reoxidation propensity and its solvent accessibility, providing some mechanistic basis explaining the different reoxidation rates observed. In this study, we examined the in vivo free thiol reoxidation in a more diverse panel of therapeutic antibodies than any previous study. We also determined in vivo free thiol reoxidation with far more site-specificity than any previous study. Next, we explored the possibility of enhancing a classic optical method with a novel yet simple augmentation step to significantly improve quantitation limits of free thiols. We screened commercially available fluorogenic probes and identified one that successfully transduces the UV absorbance signal of the Ellman’s method into a fluorescent signal. The augmented method, which we coin the Fluorescent Ellman’s method, exhibited superior quantitation limits for free thiol detection compared with the Ellman’s method while preserving the key advantages of the Ellman’s method. We demonstrated the usefulness of the Fluorescent Ellman’s method by quantifying the free thiol content of different antibody therapeutics, including several that would be challenging or impossible to analyze by alternative chromatography methods for free thiol determination. The Fluorescent Ellman’s method is a rapid, sensitive, inexpensive, and substrate-agnostic means of determining free thiol content. Finally, we explored the co-purification of a host cell protein which harbors a free thiol and a therapeutic antibody. This complex study involved identifying the contaminant host cell protein, expressing the recombinant host cell protein, and designing experiments to replicate the co-purification phenomena. Initial attempts to replicate the co-purification phenomena were confounded by the host cell protein’s free thiol. After alkylating the free thiol however, co-purification was successfully replicated using a scaled-down Protein A purification system. Co-purification was also successfully replicated after bioconjugating affinity handles to the host cell protein’s free thiol and performing magnetic bead pulldowns. After exhausting the more accessible analytical methods, we performed diazirine photo-crosslinking experiments coupled with mass spectrometry to confidently implicate the therapeutic antibody’s CDR loops in the host cell protein-antibody interaction.
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Date
2021-08-31
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University of Kansas
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Keywords
Analytical chemistry, antibodies, biotransformation, ellman, free thiol, host cell protein, product quality