Creative Biolabs have established a platform to analyze amino acid sequence of therapeutic drug candidates. The primary amino acid sequence of the desired product should be confirmed and compared with the sequence of the amino acids deduced from the gene sequence of the desired product. During the protein drug production and storage, specific impurities vary from batch to batch and primary amino acid sequencing is appropriate in many cases. The most frequently encountered molecular variants of the desired product are truncated forms. During protein production procedure, cellular peptidases may catalyze the removal of amino acids or catalyze internal cleavages. Creative Biolabs offer the primary amino acid sequencing approach to confirm the amino acid sequence of the desired product.
Besides truncated form, process-related impurities occur quite often during the manufacture and are classified into three major categories: cell substrate-derived; culture-derived and downstream-derived. Cell substrate-derived impurities include protein/polypeptides derived from the host organism. Culture derived impurities include inducers and media components. Downstream-derived impurities include enzymes, carrier/ligands (e.g. monoclonal antibodies). Creative Biolabs have utilized primary amino acid sequencing approaches to analyze the protein therapeutic drugs in the previous customer cases, to validate the purity of the desired protein.
As regulatory authorities requested, confirmation of primary amino acid sequences for protein therapeutic drugs is indispensable. Creative Biolabs provide the following protein assays for sequencing analysis:
(a) On-line LC/ES-MS/MS and/or MALDI-TOF/TOF analysis, as appropriate, with MS/MS and MSe analysis of the peptides from multiple digestion of the desired protein. The analysis provides confirmation of single light chain and heavy chain sequences of the desired antibodies.
(b) As a further option, while the MS-based sequencing may not discriminate the isobaric amino acids Ile and Leu, digested peptides containing these amino acids will be purified and further subjected to Automated Edman Degradation (N-terminal sequencing) in order to identify the Ile/Leu positions unambiguously.
Besides mass spectroscopy, Creative Biolabs also utilize HPLC, capillary electrophoresis and circular dichroism to characterize the different variants of the therapeutic protein drugs. All the primary amino acid sequencing is analyzed in house in 1-2 months.