Recipharm Pharmaservices Pvt Ltd.

To develop and validate a rapid, accurate, economical, effective and greenery RP-HPLC method for the determination of Zolmitriptan in tablet dosage form.

RP-HPLC method was developed using Luna (C₁₈) (4.6×250mm, 5μm) column and Sodium phosphate buffer (pH 4.7): Methanol [75: 25, v/v] was used as mobile phase at a flow rate of 1.0mL/min. The detection was carried out at 227nm. Further, eco-friendliness, productivity and performance of the optimized analytical method were assessed by green and white tools.

The retention time of Zolmitriptan was found to be 3.25min with acceptable chromatographic parameters. The optimized RP-HPLC method was more eco-friendly, efficient, throughput and practicable than the reported methods as confirmed by AES, AGREE, GAPI and RGB tools. Further, the proposed analytical method showed all the validation parameters within the acceptance limit of ICH Q₂ R₁ guidelines. The linear regression analysis indicated a good linear response in the 10 to 120μg/mL concentration range with R² of 0.99998. The percentage content and percentage assay of Zolmitriptan in Zomig-5mg tablet was found to be 103.36±0.356% and 97.86±0.693%.

The developed and validated method has several advantages compared to the reported HPLC methods and is useful in the systematic analysis of Zolmitriptan in its dosage form.

Host cell proteins are process related impurities generated during manufacturing of biopharmaceuticals with engineered cell line.These host cell proteins are associated with a risk of generating an immune response in patients if present in higher than acceptable levels as part of regulatory guideline.Host cell proteins are removed by affinity and ion exchange chromatog. based on specificity and charge difference, resp.However there has been increasing evidence of certain class of host cell proteins which co-elutes or become associated with the target protein and therefore builds significant challenge in clearance to acceptable levels.Presence of these host cell proteins in the formulated product may cause proteolytic activity and lead to degradation of protein during stability, thus impacting shelf life.This challenge is more intensified, if a non-affinity-based purification process is followed for recombinant protein without an Fc domain.Controlling complex host cell proteins during the purification may require multiple stages with different modes of chromatog. with conventional ligand chem., based on the nature of the HCPs.Addnl., detection of these associated or co-eluting host cell proteins is difficult during routine inprocess or release testing which includes quant. ELISA based methods.Therefore, it becomes necessary to develop a combined strategy with a novel purification step which can control these host cell proteins along with other process and product related impurities.This article aims to describe the development of a membrane-based chromatog. step with a novel ligand chem. which facilitates the clearance of associated host cell proteins in a non-affinity-based purification process.This step also controls high and low mol. weight species, host cell DNA and viruses to below acceptable levels.Thus, in addition to control of impurities, the membrane-based chromatog. unit also helps in achieving a COGs efficient process.

Host cell proteins are an inevitable byproduct of fermentation-based biomanufg. and are considered as critical quality attributes (CQA).The biggest concern of not removing HCP (Host cell protein) from a therapeutic protein preparation is the potential for HCPs to elicit severe immunogenic responses in patients, particularly with high-dose drugs like recombinant therapeutic antibodies.HCP profile can be affected by numerous upstream process decisions such as cell culture duration, feeding strategies, culture temperature or by production up-scale for commercialization.Along with three routes by which HCPs can challenge purification steps: Product association through strong attractive interactions with mAbs products under Protein-A loading concentrations, variable expression during extended cell culture, or co-elution with mAbs on polishing steps.Enzyme-linked immunosorbent assays (ELISA) are still gold standard method as it provide the global HCP amount as an output without individual identification of the HCPs present and its coverage is incomplete.This limitation directly relates to immunogenic risk and antibody degradation which raises urgent need for alternative methods.Mass spectrometry has become the most promising and powerful tool to unravel individual HCP identification and provide unbiased relative quantitation.This presentation will be built on multiple case studies discussing the outcomes of the host cell proteome population and its dynamics during cell culture supernatant at different growth phases of fed-batch cell culture to purificationAlong with it, we are summarizing the correlation between proteases and their implications on antibody quality and stability.Using this as the basis for the case study, the capabilities of this novel approaches from sample preparation to data collection for HCP identification and relative quantitation were evaluated in the context of high-speed and sensitive methods for the identification of low abundant HCPs, protein-associated HCPs and host cell proteases including their localization.