![]() ![]() Those data ultimately can be used as indications of a molecule’s intactness.īioPharmaSpec provides an N-terminal sequencing service (also known as gas- phase sequencing, Edman sequencing, and Edman degradation) using Shimadzu instrumentation for automated N-terminal sequencing. If a molecule shows posttranslational processing resulting in ragged termini, more than one terminal amino acid sequence will be detected. For successful sequencing, the chains of multichain proteins should be separated to prevent ambiguity in the data generated. Therefore, antibodies have two different N-termini and two different C-termini. Monoclonal antibodies (MAbs) comprise four protein chains: usually two identical heavy chains and two identical light chains. Not all proteins are composed of a single chain. That fact allows use of specific protein chemical procedures for sequencing proteins by means of the termini. Because these two protein termini are chemically different, they naturally will have different chemical properties. The free carboxylic acid end is called the C-terminus or carboxyl terminus. The free amine end of the chain is called the N-terminus or amino terminus. This sequential linking of amine and carboxylic acids through the length of a protein chain means that once a protein chain is completed, one end of the protein will have a free amine group, and the other end will have a free carboxylic acid group. This process is controlled by the cells’ protein translational machinery and results in the conversion of mRNA to protein. Linking the carboxylic acid group of one amino acid with the amine group of another produces this amide bond. Each amino acid has an amine functional group at one end and a carboxylic acid functional group at the other. Proteins comprise linear chains of amino acids linked to one another through amide bonds. N- and C-terminal sequencing services to assess intactness of a product and determine amino acids at the respective termini of a protein/glycoprotein product.intact molecular-weight analysis using online ultraperformance liquid chromatography (UPLC) with electrospray ionization mass spectrometry (UPLC/ES-MS) to assess intactness.The sequence of these terminal amino acids should be compared with the terminal amino acid sequence deduced from the gene sequence of the desired product.īioPharmaSpec’s protein characterization services include If the desired product is found to be heterogeneous with respect to the terminal amino acids, the relative amounts of the variant forms should be determined using an appropriate analytical procedure. Terminal amino acid analysis is performed to identify the nature and homogeneity of the amino- and carboxy-terminal amino acids. ICH Q6B guideline section 6.1.1 c states: Regulations require that biomanufacturers assess the intactness of protein and glycoprotein products as well as confirm the terminal sequences to look for existing variations. ![]()
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