scholarly journals Comparison of Three Complementary Analytical Techniques for the Evaluation of the Biosimilar Comparability of a Monoclonal Antibody and an Fc-Fusion Protein

2021 ◽  
Vol 9 ◽  
Author(s):  
Alice Demelenne ◽  
Arij Ben Yahia ◽  
Delphine Lempereur ◽  
Jacques Crommen ◽  
Anne-Catherine Servais ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Fusion Protein ◽  
Method Development ◽  
Reference Product ◽  
Degradation Products ◽  
Stress Conditions ◽  
Size Exclusion ◽  
Mild Stress ◽  
Molecular Weight Determination ◽  
Fc Fusion Protein

In this work, a monoclonal antibody, adalimumab, and an Fc-fusion protein, etanercept, were studied and compared to one of their biosimilars. Samples submitted to stress conditions (agitation and high temperature) were used for method development. The developed methods were also applied to samples reduced by beta-mercaptoethanol to evaluate their capability to distinguish the expected species. Capillary gel electrophoresis (CGE), reversed-phase liquid chromatography (RPLC), and size-exclusion chromatography (SEC) methods coupled with UV detection were used to analyze the biopharmaceuticals. Their complementarity was investigated. For further molecular weight determination, SEC-multi angle light scattering and RPLC-quadrupole time-of-flight were occasionally used. For adalimumab, a larger amount of fragments and aggregates was observed in the biosimilar compared with the reference product. For etanercept, more related species were found in the reference product. Those three separation techniques showed good complementarity. Indeed, RPLC enabled the separation of hydrophilic and hydrophobic degradation products. CGE provided good selectivity for several adalimumab fragments, and SEC was useful for the analysis of aggregates and certain fragments that cannot be separated by the other approaches. Moreover, those formulations were submitted to mild stress conditions (30°C, 300 rpm for 4 h) that mimic shipping conditions. No additional peak was found under these conditions for the two studied biopharmaceuticals.

scholarly journals Stability-Indicating RP-HPLC Method Development and Validation for Duloxetine Hydrochloride in Tablets

2010 ◽  
Vol 93 (1) ◽  
pp. 123-132 ◽  
Author(s):  
Sejal K Patel ◽  
Natavarlal J Patel ◽  
Arun M Prajapati ◽  
Dipti B Patel ◽  
Satish A Patel
Keyword(s):  
Method Development ◽  
Degradation Products ◽  
Hplc Method ◽  
Stress Conditions ◽  
Photodiode Array Detection ◽  
Heat Condition ◽  
Stability Indicating ◽  
Dry Heat ◽  
Duloxetine Hydrochloride ◽  
Rp Hplc

Abstract This paper describes the development of a stability-indicating RP-HPLC method for duloxetine hydrochloride (DLX) in the presence of its degradation products generated from forced decomposition studies. The drug substance was found to be susceptible to stress conditions of acid, base, oxidation, wet heat, dry heat, and photodegradation. The drug was found to be stable to the dry heat condition attempted. Successful separation of the drug from the degradation products formed under stress conditions was achieved on a Phenomenex C18 column (250 4.6 mm id, 5 µm particle size) using acetonitrilemethanol0.032 M ammonium acetate buffer (55 + 05 + 40, v/v/v) as the mobile phase at a flow rate of 1.0 mL/min at 40°C temperature. Quantification was achieved with photodiode array detection at 290 nm over the concentration range 0.25 µg/mL with mean recovery of 101.048 ± 0.53 for DLX by the RP-HPLC method. Statistical analysis proved the method is repeatable, specific, and accurate for estimation of DLX. Because the method could effectively separate the drug from its degradation products, it can be used as a stability-indicating method.

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR EVALUATING THE STABILITY PARAMETERS FOR THE SIMULTANEOUS ESTIMATION OF CLOPIDOGREL BISULPHATE AND OMEPRAZOLE USING RP-HPLC

INDIAN DRUGS ◽  
2016 ◽  
Vol 53 (01) ◽  
pp. 60-67
Author(s):  
J. B Nagavi ◽  
◽  
B. M. Gurupadayya
Keyword(s):  
High Performance ◽  
Method Development ◽  
Degradation Products ◽  
High Performance Liquid Chromatographic ◽  
Stress Conditions ◽  
Simultaneous Estimation ◽  
Stability Parameters ◽  
Ich Guidelines ◽  
The Stability ◽  
Neutral Conditions

A simple, sensitive, accurate and specific stability-indicating high-performance liquid chromatographic method was developed and validated for the simultaneous estimation of clopidogrel bisulphate and omeprazole in bulk. Extensive testing of clopidogrel bisulphate and omeprazole in different stress conditions were carried out as per the ICH guidelines Q1A (R2) Clopidogrel bisulphate and omeprazole was exposed to various stress conditions like oxidation, hydrolysis, photolysis and neutral decomposition. Clopidogrel bisulphate, which was found to degrade considerably in acidic and photo conditions, was found to be stable in alkaline and neutral conditions, whereas omeprazole was found to be degrading in alkaline, oxidative and photo conditions, but stable in acidic and neutral condition. Apart from the formation of minor degraded products under accelerated conditions, the drugs were reasonably stable in solid state. A good linear relationship over the concentration range of 50-500μg/mL was shown. Validation of the method was carried out as per the ICH guidelines. The method developed was found to be specific, selective, precise and accurate. Clopidogrel bisulphate showed degradation in 5M hydrochloric acid at 80oC, in 3% hydrogen peroxide for 5min the drug showed around 35% of degradation, when exposed to sunlight for 15 min, forming around 25-30% of degradation products. Omeprazole showed 15-18% degradation in alkaline and photo condition.Statistical analysis shows that the method is reproducible and selective for the estimation of clopidogrel bisulphate and omeprazole in dosage form.

Degradation products analysis of an Fc fusion protein using LC/MS methods

2009 ◽  
Vol 44 (1) ◽  
pp. 81-85 ◽  
Author(s):  
Da Ren ◽  
Gayathri Ratnaswamy ◽  
Jill Beierle ◽  
Michael J. Treuheit ◽  
David N. Brems ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Degradation Products ◽  
Fc Fusion Protein ◽  
Products Analysis

Analytical Method Development and Validation for the Analysis of Donepezil Hydrochloride and Its Related Substances Using Ultra Perfomance Liquid Chromatography

2017 ◽  
Vol 9 (5) ◽  
Author(s):  
Mahalingam V. ◽  
Kalaivani V. ◽  
Somanathan T. ◽  
Vijayabaskar S.
Keyword(s):  
Analytical Method ◽  
Method Development ◽  
Degradation Products ◽  
Gradient Elution ◽  
Stress Conditions ◽  
Base Hydrolysis ◽  
Forced Degradation ◽  
Equilibration Time ◽  
Elution Time ◽  
Donepezil Hydrochloride

A novel, economic and time-efficient reverse-phase ultra-performance liquid chromatographic (RP-UPLC) method has been developed for the analysis of Donepezil hydrochloride in the presence of both impurities and degradation products generated by forced degradation. When Donepezil hydrochloride was subjected to acid hydrolysis, oxidative, base hydrolysis, photolytic, and thermal stress, degradation was observed only in oxidative and base hydrolysis. The drug was found to be stable to other stress conditions. Successful chromatographic separation of the drug from impurities formed during synthesis and from degradation products formed under stress conditions was achieved on a Waters Acquity C18, 50 mm x 2.1mm, 1.7µ particle size column, UV detection at 286nm and a gradient elution of Trifluoroacetic acid, Acetonitrile and methanol as mobile phase. The method was validated for specificity, precision, linearity, accuracy, robustness and can be used in quality control during manufacture and for assessment of the stability samples of Donepezil hydrochloride. Total elution time was about 6 min and equilibration time of about 2 min which allowed analysis of more than 100 samples per day. The analytical method discussed in British Pharmacopeia was pH sensitive and not compatible to LC-MS analysis but the method reported in this study is more compatible to LC-MS which will be more suitable to perform LC-MS.

Evaluation of the Efficiency of Protein A Affinity Chromatography to Purify a Monoclonal Antibody for Cancer Treatment and its Purity Analysis

2020 ◽  
Vol 7 (2) ◽  
pp. 121-133
Author(s):  
Ayesha Akhtar ◽  
Shivakumar Arumugam ◽  
Shoaib Alam
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Affinity Chromatography ◽  
Protein A ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
High Yield ◽  
Staphylococcal Protein A ◽  
Purification Step ◽  
Purity Analysis

Background:: Protein A affinity chromatography is often employed as the most crucial purification step for monoclonal antibodies to achieve high yield with purity and throughput requirements. Introduction:: Protein A, also known as Staphylococcal protein A (SPA) is found in the cell wall of the bacteria staphylococcus aureus. It is one of the first discovered immunoglobulin binding molecules and has been extensively studied since the past few decades. The efficiency of Protein A affinity chromatography to purify a recombinant monoclonal antibody in a cell culture sample has been evaluated, which removes 99.0% of feed stream impurities. Materials and Method:: We have systematically evaluated the purification performance by using a battery of analytical methods SDS-PAGE (non-reduced and reduced sample), Cation Exchange Chromatography (CEX), Size-exclusion chromatography (SEC), and Reversed phased-Reduced Chromatography for a CHO-derived monoclonal antibody. Results and Discussion:: The analytical test was conducted to determine the impurity parameter, Host Cell Contaminating Proteins (HCP). It was evaluated to be 0.015ng/ml after the purification step; while initially, it was found to be 24.431ng/ml. Conclusion:: The tests showed a distinct decrease in the level of different impurities after the chromatography step. It can be concluded that Protein A chromatography is an efficient step in the purification of monoclonal antibodies.

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