| dc.description.abstract | The patents of several best-selling biologic therapeutic products are expiring soon. Consequently, the interest of developing biosimilar products is growing. A biosimilar product is developed if there are no clinically meaningful differences in terms of safety, efficacy, and purity after evaluating side-by-side with the originator. Biosimilar products are anticipated to be accessible to healthcare providers and patients at a lower cost compared to the originators. Unlike small-molecule generic drug products, which are structurally replicable and well-defined, that guarantee the safety and efficacy, biologic products are structurally complex, larger in size and often contain mixtures of various posttranslational modifications. Consequently, demonstrating the similarity of a biosimilar molecule with the reference product requires extensive characterization to ensure safety and efficay. One of the challenges in developing a biosimilar product is the lack of knowledge about the reference product’s manufacturing process, which is not accessible to the public because it is a proprietary knowledge. Therefore, the biosimilar sponsor needs to develop a process by extensive characterization of a biosimilar candidate side-by-side with a reference product. This is an iterative process aimed at developing a biosimilar molecule similar to the reference product. The first step in biosimilarity assessment is to establish structural similarity by extensive characterization using several analytical techniques. Therefore, analytical tools play a vital role in demonstrating structural similarity as well as process development of a biosimilar candidate. In addition, the level of similarity established by the analytical tools guides the type of non-clinical and clinical data packages required for regulatory approval. If a high degree of similarity is demonstrated using analytical techniques, then phase II clinical trials are not required for registration of a biosimilar candidate. Consequently, this will lower the cost of developing a biosimilar product. Hence, developing sensitive and robust analytical techniques is vital in a biosimilar development process. In this dissertation, four homogeneous glycoforms of IgG1 Fc (HM-Fc, GlcNAc-Fc, Man5-Fc, and N297Q-Fc) were produced using recombinant protein expression combined with in-vitro enzymatic reactions to be utilized as a model for biosimilar comparability analysis. These glycoforms were characterized by mass spectrometry, CIUE, SDS-PAGE, and cIEF. The main focus of the project was to produce homogeneous glycoforms of IgG1 Fc and to utilize them to develop new biolayer interferometry (BLItz) assay methods. Two biolayer interferometry methods with different immobilization techniques were developed to measure the binding affinity of IgG1 Fc glycoforms to FcRIIIa and FcRIIb. In addition, these four glycoforms were mixed in pre-defined composition to examine the characteristics of mixtures of glycoforms and to study important biological and physicochemical features of protein drugs in a biosimilar analysis. For the mixture samples, differences in binding were observed when the two immobilization formats were employed. Furthermore, these glycoforms were incubated at low and elevated temperatures for an elongated time, and a trend of decreasing binding affinity was observed with the increasing incubation period. | |
