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Amino Acid Sequence and Terminal Sequence Analysis

Creative Biolabs have established a platform to analyze amino acid sequence of therapeutic drug candidates, strictly following the ICH Topic Q6b guideline. The amino acid sequence of the desired product should be evaluated utilizing approaches such as amino acid composition, terminal amino acid sequence, peptide map, sulfhydryl group(s) and disulphide bridge analysis and then compared with the amino acid sequence deduced from the gene sequence of the desired product. Terminal amino acids should be analyzed to determine the nature and homogeneity of the amino (N-) and Carboxy (C-) terminal amino acids. When the desired product is found to be heterogeneous with the respect on amino acids, the relative proportion of variant forms should be identified using an appropriate analytical procedure. The terminal amino acids should also be compared with the ones deduced from the gene sequence of the desired protein.

Physico-chemical Characterization

Degradation products exist commonly together with the desired products. Degradation products are molecular variants, generated from alterations in the desired protein product or product-related substances over time and/or by the factors of, e.g., light, pH, temperature, water, or by reaction with an excipient and/or the immediate container/closure system. Such changes may be caused during processing and/or storage by oxidation, deamidation, aggregation, or proteolysis.

For the assessment of the N-termini of the desired product, Creative Biolabs utilize automated Edman Degradation (N-terminal sequencing) for divided light chains and heavy chains of desired antibodies. This procedure includes reduction and separation of light chains and heavy chains for independent sequencing (15 cycles). To provide an orthogonal confirmation of the N-termini (to the extent possible), mass spectrometry based sequencing via MSe and MS/MS (Q-TOF) will be performed. For the assessment of the C-termini of the product, Creative Biolabs combine data from intact molecular weight analysis (superlink) and peptide mapping (superlink) to identify and sequence the C-terminal peptides. All the proteins are characterized in house in 2-3 week.

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