ELUCIDATION OF THE IMPACT OF N-GLYCAN STRUCTURE ON PHYSICAL STABILITY AND LOCAL FLEXIBILITY OF WELL-DEFINED IgG1-Fc GLYCOFORMS FROM A PHARMACEUTICAL DEVELOPMENT PERSPECTIVE
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Issue Date
2017-08-31Author
More, Apurva Shirish
Publisher
University of Kansas
Format
227 pages
Type
Dissertation
Degree Level
Ph.D.
Discipline
Pharmaceutical Chemistry
Rights
Copyright held by the author.
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Therapeutic efficacies of IgG monoclonal antibodies (mAbs) depend on their physicochemical structural integrity, stability, flexibility and biological functionality. IgG-Fc glycosylation at Asn-297 is important for the structural integrity and effector function activities of IgG mAb therapeutics and is a key contributor of their heterogeneity. Molecular heterogeneities (like glycosylation) must be closely monitored and assessed during development of mAbs from drug candidate to marketed product and during biosimilar development. A variety of techniques are being developed to monitor chemical structure and glycosylation profiles of mAbs, however, novel analytical techniques are needed to better understand the effects of glycosylation on their complex higher order structures and dynamics for their effective development. In comparability and biosimilarity exercises, analytical characterization tools serve as the foundation for establishing similarity between pre change versus post change products and biosimilar versus innovator product. Hence, better tools are needed for drawing comparisons between the results. In this study, four highly purified, well-defined recombinant IgG1-Fc glycoform variants (High-mannose-Fc (HM-Fc), Man5-Fc (truncated glycoform), GlcNAc-Fc (truncated glycoform), and non-glycosylated-Fc (N297Q-Fc) were produced which served as model glycoproteins to study the effect of IgG1-Fc N-linked glycosylation on various pharmaceutical properties. This work explores the utility of yeast expression along with in vitro enzymatic digestion for obtaining homogenous glycoform profiles of IgG Fc glycans; which is extremely challenging to achieve considering the observed inherent glycan heterogeneity in expression hosts. We developed and utilized various orthogonal analytical techniques to explore the relationship between glycosylation and physical stability of these IgG1-Fc glycoforms and compared their physical stability in two different formulations to mimic follow on therapeutics or biosimilars that might be formulated differently. Hence, this study contributed towards the ongoing development of data visualization and mathematical modeling tools for comparability and biosimilarity assessments. To simulate a more practical biosimilar comparability scenario, various mixtures of the four glycoforms were made to mimic heterogeneity found in biosimilars. The physical stability of these mixtures was evaluated by combination of high-throughput biophysical techniques. The ability of various biophysical techniques to demonstrate the estimated differences between the physical stability of the mixtures was examined. Even though the biophysical techniques utilized in this study are the most commonly used tools in the industry, these are low in resolution and only show the average conformational outcome. Hence, to obtain high-resolution peptide-level information on changes in local dynamics because of changes in IgG1-Fc glycans, hydrogen exchange (HX-MS) in combination with pepsin proteolytic digestion and liquid chromatography-mass spectrometry (LC-MS) was utilized. Previous HX-MS studies on IgG mAbs in our laboratory showed an increased flexibility of a particular peptide segment in the Fc region in the presence of excipients and salts. This result was attributed to the disruption in packing of the heterogeneous glycan structures with the Fc region. Another goal of this dissertation was to explore the mechanisms of interactions between glycans and IgG1-Fc. The correlations between glycosylation and physicochemical structure, stability and flexibility of well-defined IgG1-Fc glycoforms in this dissertation, will serve as important tools to enable rational design and optimization of stable IgG formulation conditions to avoid conformational destabilization and aggregation issues during their manufacture, long-term storage and administration. The extensive analytical characterization approach utilized in this dissertation will contribute towards the ongoing development of statistical tools as required by the US Food and Drug Administration (FDA), for similarity analysis between biosimilar and innovator therapeutics.
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