Large scale purification and characterization of A21 deamidated variant-most prominent post translational modification (PTM) for insulins which is also widely observed in insulins pharmaceutical manufacturing and storage.

Biopharmaceutical development demands appropriate understanding of product related variants, which are formed due to post-translational modification and during downstream processing. These variants can lead to low yield, reduced biological activity, and suboptimal product quality. In addition, these variants may undergo immune reactions, henceforth need to be appropriately controlled to ensure consistent product quality and patient safety. Deamidation of insulin is the most common post-translational modification occurring in insulin and insulin analogues. AsnA21 desamido variant is also the most prominent product variant formed during human insulin manufacturing process and/or during the storage. Often, this deamidated variant is used as an impurity standard during in-process and final product analysis in the QC system. However, purification of large quantity of purified deamidated material is always being challenging due to highly similar mass, ionic, hydrophobic properties, and high structural similarity of the variant compared to the parent product. Present work demonstrates the simplified and efficient scalable process for generation of AsnA21 deamidated variant in powder form with ~96% purity. The mixed-mode property of anion exchange resin PolyQuat was utilized to purify the deamidated impurity with high recovery. Subsequent reversed-phase high performance liquid chromatography (RP-HPLC) step was introduced for concentration of product in bind elute mode. Elution pool undergone isoelectric precipitation and lyophilisation. The lyophilized product allows users for convenient use of the deamidated impurity for intended purposes. Detailed characterization by Mass spectrometry revealed deamidation is at AsnA21 and further confirmed that, structural and functional characterization as well as the biological activity of isolated variant is equivalent to insulin.

A Mycobacterium smegmatis gyrase B specific monoclonal antibody reveals association of gyrase A and B subunits in the cell.

DNA gyrase is a unique topoisomerase, which plays important roles in macromolecular events like DNA replication, transcription and genetic recombination. In this study a high affinity monoclonal antibody to the gyrase B (GyrB) subunit of Mycobacterium smegmatis was characterized, which did not cross-react with either the Escherichia coli GyrB subunit or with GyrB subunits from other mycobacterial species. The antibody recognized an epitope in the N-terminus, novobiocin-binding domain of GyrB. Immunoprecipitation of gyrase from M. smegmatis cell lysate revealed an association, mediated by ionic interactions, of gyrase A and GyrB subunits in the cell. This antibody is a valuable tool for structure-function analysis and immunocytological studies of mycobacterial DNA gyrase.

Biochemical characterization of the intracellular domain of the human guanylyl cyclase C receptor provides evidence for a catalytically active homotrimer.

Guanylyl cyclase C (GCC) is the receptor for the family of guanylin peptides and bacterial heat-stable enterotoxins (ST). The receptor is composed of an extracellular, ligand-binding domain and an intracellular domain with a region of homology to protein kinases and a guanylyl cyclase catalytic domain. We have expressed the entire intracellular domain of GCC in insect cells and purified the recombinant protein, GCC-IDbac, to study its catalytic activity and regulation. Kinetic properties of the purified protein were similar to that of full-length GCC, and high activity was observed when MnGTP was used as the substrate. Nonionic detergents, which stimulate the guanylyl cyclase activity of membrane-associated GCC, did not appreciably increase the activity of GCC-IDbac, indicating that activation of the receptor by Lubrol involved conformational changes that required the transmembrane and/or the extracellular domain. The guanylyl cyclase activity of GCC-IDbac was inhibited by Zn(2+), at concentrations shown to inhibit adenylyl cyclase, suggesting a structural homology between the two enzymes. Covalent cross-linking of GCC-IDbac indicated that the protein could associate as a dimer, but a large fraction was present as a trimer. Gel filtration analysis also showed that the major fraction of the protein eluted at a molecular size of a trimer, suggesting that the dimer detected by cross-linking represented subtle differences in the juxtaposition of the individual polypeptide chains. We therefore provide evidence that the trimeric state of GCC is catalytically active, and sequences required to generate the trimer are present in the intracellular domain of GCC.