Harish Iyer

Nimotuzumab, a promising therapeutic monoclonal for treatment of tumors of epithelial origin

Nimotuzumab is a humanized therapeutic monoclonal antibody against epidermal growth factor receptor (EGFR). Clinical trials are ongoing globally to evaluate nimotuzumab in different indications. Nimotuzumab has been granted approval for use in squamous cell carcinoma of head and neck (SCCHN), glioma and nasopharyngeal cancer in different countries. This review focuses on the unique functional characteristics of nimotuzumab. Also, it discusses the safety and efficacy data obtained from the Phase IIb clinical trial conducted in India in SCCHN. Post Marketing Surveillance data from Cuba for the use of nimotuzumab in pediatric and adult glioma is also discussed. Overall, nimotuzumab has immense therapeutic potential in cancers of epithelial origin.

Oral insulin - a review of current status.

Oral insulin is one of the most exciting areas of development in the treatment of diabetes because of its potential benefit in patient convenience, rapid insulinization of liver, adequate insulin delivery avoiding peripheral hyperinsulinaemia while potentially avoiding adverse effects of weight gain and hypoglycaemia. Growing evidence that earlier initiation of intensive insulin therapy produces sustained tight glycaemic control resulting in substantial delay in complications makes an effective oral insulin product even more vital for the management of patients with diabetes. Despite knowledge of this unmet medical need, oral delivery of insulin has been unsuccessful because of several barriers. For several decades, researchers have tried to develop oral insulin using various technologies without much clinical or commercial success. This review summarizes the development status of oral insulins which are publicly reported to be undergoing clinical studies. Currently, two oral insulin products are in an advanced stage of clinical development and first data from long‐term therapy are expected to be available in the second half of 2010.

European Medicines Agency workshop on biosimilar monoclonal antibodies: July 2, 2009, London, UK.

The European Medicines Agency (EMEA) workshop on biosimilar monoclonal antibodies (mAbs), held July 2, 2009 at the EMEA headquarters in London, was a harbinger with potentially far-reaching implications for all groups interested in antibody therapeutics development. These groups include not only regulators and the innovator and generic biopharmaceutical industries, but also physicians, patients and payers. The objective of the workshop was to discuss and assess the feasibility of the development and authorization of mAbs using EMEA’s biosimilar regulatory pathways. The workshop sequentially focused on questions relevant to three areas: 1) chemistry, manufacturing and controls (CMC); 2) non-clinical issues; and 3) clinical issues, including outcome measures. Proceedings of the workshop are presented in Part 1 of this report, and discussed within the context of the legal, regulatory, and business environments of the European Union, Asia, and the United States in Parts 2, 3 and 4, respectively

A dose range finding study of novel oral insulin (IN-105) under fed conditions in type 2 diabetes mellitus subjects.

Aim: The objective of the study was to establish the dose response of IN‐105 tablets and explore a possible therapeutic window in type 2 diabetes subjects poorly controlled on metformin.

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.

A tandem mass spectrometric approach to the identification of O‐glycosylated glargine glycoforms in active pharmaceutical ingredient expressed in Pichia pastoris

Glycoforms of glargine expressed in Pichia pastoris were isolated by high-performance liquid chromatography and analyzed by a series of chemical and mass spectrometric methods for the identification of various glycoforms, glycosylation position, nature and structure of glycans. Reduction and alkylation, peptide mapping techniques were used to decipher the amino acid site at which glycosylation had taken place. Chemical methods were coupled with mass spectrometry techniques such as electrospray ionization and matrix-assisted laser desorption/ionization for identification of the glycosylation site.

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 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.

Influence of cell specific productivity on product quality.

The micro heterogeneity or quality of a protein has been shown to have a significant impact on its physical, chemical and biological properties both in vitro and in vivo [1]. Micro heterogeneity is evaluated in terms of post translational modifications such as glycosylation, charge variants, aggregates and fragments profile. The biggest challenge in process development is to find a balance between increasing productivity while maintaining product quality. In our study we focus on protein glycosylation, which is a process in which oligosaccharides are added to the protein during synthesis. There are multiple possible reactions in the pathway and it takes a long time for a glycosylated protein to be fully processed. If some protein molecules have a shorter residence time in the ER and Golgi, the glycan may be only at an intermediate stage [1]. For recombinant glycoprotein, increase in cell specific productivity (amount of product produced per cell per unit time) which may result in shorter residence time in the ER and Golgi, must be weighed against possible changes in product quality attributes like glycosylation [2]. Our study concludes that it is possible to produce a protein with desired product quality profile with high specific productivity. Two different clones with the same productivity can have different product quality profiles; alternatively, the same clone with different specific productivity can be manipulated to produce the same desired product quality by altering the cell culture parameters or addition of supplements. This observation also influences the acknowledged methodology for selecting clones with higher productivity while still maintaining their product quality profile. Various process manipulations were evaluated as an attempt to improve on the product quality profiles without compromising the productivity.

Challenges in scaling up a perfusion process

Development and scale-up of a perfusion process has challenges due to the complex nature of the process and unavailability of direct scale-up of the perfusion equipment. The initial scale-up to production scale resulted in poor cell growth profile. Upon investigation, the reason for low cell concentration was attributed to poor cell retention by the perfusion device. Changes were introduced in the type of mesh used for filter construction and perfusion pumps to improve retention. These modifications helped in better cell culture profiles and yields. However, the product quality was impacted because of these changes. Further changes in the perfusion flow rates were done to address the product quality differences.