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Three batches of product were supplied for Extinction Coefficient Determination, with one batch being used for the full qualification. Following qualification, a comparative assessment of the extinction coefficient for all three samples was performed side by side using the qualified method. Data from duplicate injections of 12 separate hydrolysis preparations were used for calculation of the extinction coefficient for each of the samples.
Sample aliquots were purified prior to analysis to minimize the effects of interfering buffer excipients such as amino acids, mono- and di-saccharides and Polysorbate.
The Optical Density (OD) of sample aliquots was then measured prior to each session at 280nm (against a suitable buffer blank). Sample aliquots for amino acid analysis were prepared with the addition of internal standards.
A time course hydrolysis was performed with the data obtained being used to determine the optimal hydrolysis time for the product. Once determined, all further sample aliquots were hydrolyzed for the optimal time.
Following hydrolysis, sample aliquots were derivatized and injected into BioPharmaSpec’s Agilent 1260 Infinity Binary LC system equipped with a fluorescence detector.
This method qualification includes the following experiments, which are based on the requirements within ICH Topic Q2(R1) - ICH HARMONISED TRIPARTITE GUIDELINE VALIDATION OF ANALYTICAL PROCEDURES: TEXT AND METHODOLOGY:
- HYDROLYSIS TIME OPTIMISATION
- Intermediate precision
- DETECTION LIMIT
- QUANTITATION LIMIT
Technical considerations- Extinction Coefficient
BioPharmaSpec scientists can help you understand when a determination of the extinction coefficient of your product, rather than an experimental estimation, is required.
An accurate determination of the extinction coefficient becomes essential for considerations of the dosage and concentration of your product. It is not sufficient to rely on theoretical (using software) or experimental (using amino acid analysis) estimations, which can have up to 15% error from the true value. When determining an extinction coefficient, BioPharmaSpec scientists use their working knowledge of ICH guideline ICH Q2(R1) to perform a qualification of the UV spectrophotometry and amino acid analysis for the product.
“At Intas, we are committed to characterizing our products to the highest standard and to producing quality products. BioPharmaSpec has a clear project plan in place for determining the extinction coefficient of biopharmaceuticals and we found this easy to follow and apply to our molecule.” – Senior General Manager, Intas Pharmaceuticals Ltd.