Nitesh Dave

Development of a process to manufacture PEGylated orally bioavailable insulin

To make insulin orally bioavailable, insulin was modified by covalent attachment (conjugation) of a short‐chain methoxy polyethylene glycol (mPEG) derivative to the ε‐amino group of a specific amino acid residue (LysB29). During the conjugation process, activated PEG can react with any of the free amino groups, the N‐terminal of the B chain (PheB1), the N‐terminal of the A chain (GlyA1), and the ε‐amino group of amino acid (LysB29), resulting in a heterogeneous mixture of conjugated products. The abundance of the desired product (Methoxy‐PEG3‐propionyl—insulin at LysB29:IN‐105) in the conjugation reaction can be controlled by changing the conjugation reaction conditions. Reaction conditions were optimized for maximal yield by varying the proportions of protein to mPEG molecule at various values of pH and different salt and solvent concentrations. The desired conjugated molecule (IN‐105) was purified to homogeneity using RP‐HPLC. The purified product, IN‐105, was crystallized and lyophilized into powder form. The purified product was characterized using multiple analytical methods including ESI‐TOF and peptide mapping to verify its chemical structure. In this work, we report the process development of new modified insulin prepared by covalent conjugation of short chain mPEG to the insulin molecule. The attachment of PEG to insulin resulted in a conjugated insulin derivative that was biologically active, orally bioavailable and that showed a dose‐dependent glucose lowering effect in Type 2 diabetes patients.

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.

Quantitative determination of oxytocin receptor antagonist atosiban in rat plasma by liquid chromatography-tandem mass spectrometry

A kinetic study of atosiban was conducted following repeated intravenous administration in Wistar rats. Sample analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) following full validation of an in-house method. Eptifibatide, a cyclic peptide, was used as an internal standard (IS). The analyte and internal standard were extracted using solid phase extraction (SPE) method. Chromatographic separation was carried out using an ACE C18 5 microm 50 mm x 4.6 mm column with gradient elution. Mass spectrometric detection was performed using TSQ Quantum ultra AM. The lower limit of quantification was 0.01 microg/ml when 100 microl rat plasma was used. Plasma concentrations of atosiban were measured at 0 (pre-dose), 2, 15, 30, 45, 60, 120 min at the dosage levels of 0.125 mg/kg (low dose), 0.250 mg/kg (mid dose), and 0.500 mg/kg (high dose), respectively. Atosiban plasma concentration measured at Day 1 showed mean peak atosiban concentration (C(max)) 0.40, 0.57, 1.95 microg/ml for low, mid and high dose treated animals and mean peak concentration on Day 28 was 0.41, 0.88, 1.31microg/ml on Day 28 for low, mid and high dose treated animals.

A novel one-pot de-blocking and conjugation reaction step leads to process intensification in the manufacture of PEGylated insulin IN-105

Bio-catalytic in vitro multistep reactions can be combined in a single step in one pot by optimizing multistep reactions under identical reaction condition. Using this analogy, the process of making PEGylated insulin, IN-105, was simplified. Instead of taking the purified active insulin bulk powder as the starting material for the conjugation step, an insulin process intermediate, partially purified insulin ester, was taken as starting material. Process intensification (PI) was established by performing a novel de-blocking (de-esterification) of the partially purified insulin ester and conjugation at B-29 Lys residue of B chain with a short-chain methoxy polyethylene glycol (mPEG) in a single-pot reactor. The chromatographic profile at the end of the reaction was found similar irrespective of whether both the reactions were performed sequentially or simultaneously. The conjugated product of interest, IN-105 (conjugation at LysB29), was purified from the heterogeneous mixture of conjugated products. The new manufacturing process was deduced to be more simplified and economical in making the insulin conjugates as several downstream purification steps could be circumvented. The physicochemical characteristics of IN-105 manufactured through this economic process was found to be indifferent from the product formed through the traditional process where the conjugation starting material was purified from bulk insulin.

A STUDY ON INTERACTION OF ION-PAIRING AGENT IN SEPARATION OF HUMAN INSULIN AND GLYCOSYLATED HUMAN INSULIN USING REVERSE PHASE LIQUID CHROMATOGRAPHY

Glycosylated proteins formed during expression are present as impurities in yeast based recombinant production of Human Insulin (HI). These closely related glycosylated impurities pose challenge in purification of HI by RP-HPLC. In this work, separation of glycosylated Human Insulin (gHI) and HI in presence of other HI related impurities under preparative loading conditions was studied at pH between 2.8 to 4.0. Sodium salts of acetate, citrate, formate, perchlorate, and succinate were used as ion-pairing agents in mobile phase. The study was performed by varying the concentration of ion-pairing agent, pH of mobile phase, and HI loading on the column. The effect of ion-pairing agent, in conjunction with pH, on resolution of gHI and HI was evaluated based on recovery, purity of HI, and percent reduction of gHI. Ion-pair formation of sodium perchlorate in the presence of acetonitrile as the organic modifier resulted in effective separation of gHI and HI yielding HI purity of 98.5% under preparative loading conditions (5 to 15 g/L). In order to comply with the purity and impurity specifications as per pharmacopeia, development of additional purification methods would be required, based on analysis results of the product using pharmacopeia methods. Supplemental materials are available for this article. Go to the publishers online edition of Journal of Liquid Chromatography & Related Technologies to view the free supplemental file.