Glycan Profiling of A Fusion Glycoprotein Product Rutwik Patel Deparment of Chemistry
Glycans are known to affect protein structure and function. Sialic acid is one such glycan located at the terminal end of the carbohydrate chain, glycosylated to the fusion glycoprotein product. The sialic acid content in the fusion glycoprotein product was analyzed from different batches. Then, optimization of the reproducibility for such sialic acid assay was done. An efficient reversed phase HPLC technique for sialic acid detection by labeling the sialic acid with o-phenylene diamine (OPD) and examining the fluorescent character of the resulting derivativized sialic acid was developed. Once the sialic acid was detected, its ratio in the entire glycoprotein was carried out by preparation of a standard curve. When the procedure was repeated again, the results demonstrated reasonable reproducibility. However, in order to address some minor problems regarding a small reproducibility discrepancy in one of the batches, the experiment will be repeated.
INTRODUCTION The primary structure of a protein is made up of long amino acid sequences connected through amide linkage. This sequence undergoes local stabilization via a variety of non-covalent interactions. These non-covalent interactions include hydrogen bonding, hydrophobic interactions, electrostatic interactions and ionic bonding. These interactions result in the formation of a secondary structure. Secondary structures primarily include alpha helices and beta sheets. Then, the protein undergoes long range interactions primarily involving disulfide bond formation between two cysteines. These interactions result in the tertiary structure of a protein. Two such proteins can interact between them and can form a quaternary structure of a protein. Glycoproteins are proteins that contain oligosaccharide chains (glycans) covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein as a result of cotranslational or posttranslational modifications. This process is known as glycosylation. Glycosylation is of two types: O-linked glycosylation and N-linked glycosylation. In O-linked glycosylation, the addition of sugar chains can happen on the hydroxyl oxygen on the side chain of serine or threonine. In case of N-linked glycosylation, the addition of sugar
chains can happen on the amide nitrogen present on the asparagine side chain. It follows a consensus sequence of Asp-[X]-Ser/Thr, where X can be any amino acid except proline. The fusion glycoprotein used in this experiment exhibits N-linked glycosylation. The fusion glycoprotein is comprised of naturally�occurring protein structures that selectively binds to complement activated cells and locally regulates the complement system through Complement Factor - H. The fusion glycoprotein is being developed as a potential treatment of inflammatory and autoimmune diseases, and some immune�related renal and hematology diseases. However, the glycan structure in the glycoprotein can vary based on different manufacturing processes. This varying glycan structure can affect protein structure and function, as well as pharmacokinetics. As a result, it is essential to analyze the glycan structure in order to ensure the process consistency. So, it is essential to study different glycans within this protein and determine a mole ratio of each glycan to the entire protein. Sialic acid is one such glycan. The two common kinds of sialic acids are N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NAGA). Sialic acids are 9-C oligosaccharides linked through glycosidic linkage with other glycans. In case of the protein in discussion, it is the terminal glycan in the carbohydrate UMLJUR 57