A novel peptide design aids in the expression and its simplified process of manufacturing of Insulin Glargine in Pichia pastoris.

Manufacturing of insulin and its analogues relied upon in vitro enzymatic cleavages of its precursor forms (single chain precursor, SCP) at both ends of a connecting peptide (C-peptide) that links the respective B-chain and A-chains to corresponding final forms. We have demonstrated a simplified approach of cleaving P. pastoris expressed SCP, distinctly at one site for conversion to insulin glargine. The design of the precursor was made in such a way that there is no C-peptide in the precursor which needs to be removed in the final product. Instead of traditional both side cleavage of the C-peptide and removing the C-peptide (by trypsin), followed by 2nd enzyme reaction (typically carboxipeptidase B), present work established only one side cleavage of the sequence by only trypsin converts the precursor to final insulin glargine product. The novel design of the precursor helped in producing insulin glargine in a single step with an application of single enzyme brought high degree of process efficiencies. Highly purified product was generated through two reversed phase high pressure chromatographic steps. Purified product was compared with the reference product Lantus®, for various physico-chemical and biological properties. Primary, secondary and tertiary structures as well as biological pharmaco-dynamic effects were found comparable. High cell density fermentation that gave a good yield of the SCP, a single step conversion to insulin glargine, enabled by a unique design of SCP and a distinct purification approach, has led to a simplified and economical manufacturing process of this important drug used to treat diabetes. KEY POINTS: • Novel concept for processing single chain precursor of insulin glargine • Simple and economic process for insulin glargine • Physicochemical characterization and animal Pharmacodynamics show similarity to Lantus.

Mass spectrometric distinction of in-source and in-solution pyroglutamate and succinimide in proteins: a case study on rhG-CSF.

Formation of cyclic intermediates involving water or ammonia loss is a common occurrence in any reaction involving terminal amines or hydroxyl group containing species. Proteins that have both these functional groups in abundance are no exception, and presence of amino acids such as asparagine, glutamines, aspartic acids, and glutamic acids aid in formation of such intermediates. In the biopharma scenario, such intermediates lead to product- or process-related impurities that might be immunogenic. Mass spectroscopy is a powerful technique that is used to decipher the presence and physicochemical characteristics of such impurities. However, such intermediates can also form in situ during mass spectrometric analysis. We present here the detection of in-source and in-solution formation of succinimide and pyroglutamate in the protein granulocyte colony stimulating factor. We also propose an approach for quick differentiation of such in-situ species from the tangible impurities. We believe that this will not only reduce the time spent in unambiguous identification of succinimide- and/or pyroglutamate-related impurity in bio-pharmaceutics but also provide a platform for similar studies on other impurities that may form due to stabilized intermediates.

PMT1 gene plays a major role in O-mannosylation of insulin precursor in Pichia pastoris.

Protein mannosyltransferases (PMTs) catalyze the O-mannosylation of serine and threonine residues of proteins in the endoplasmic reticulum. The five PMT genes coding for protein mannosyltransferases, designated as PMT1, 2, 4, 5 and 6, were identified from Pichia pastoris genome based on the homology to PMT genes in Saccharomyces cerevisiae genome, which has seven PMT genes. The homologues of S. cerevisiae PMT 3 &7 genes are absent in P. pastoris genome. Approximately 5% of the recombinant insulin precursor expressed in P. pastoris is O-mannosylated. In this study, we attempted to prevent O-mannosylation of insulin precursor in vivo, through inactivation of the Pichia PMT genes. Since multiple PMTs are found to be expressed, it was important to understand which of these are involved in O-mannosylation of the insulin precursor. The genes encoding PMT1, 4, 5 and 6 were knocked out by insertional inactivation method. Inactivation of PMT genes 4, 5 and 6 showed ∼16-28% reductions in the O-mannosylation of insulin precursor. The PMT1 gene disrupted Pichia clone showed ∼60% decrease in O-mannosylated insulin precursor, establishing its role as an important enzyme for insulin precursor O-mannosylation.