Physio-Chemical Characterization Tests

PHYSIO-CHEMICAL CHARACTERIZATION TESTS Structural Characterization and Confirmation 

The amino acid sequence of the desired product should be determined to the extent possible using customary approaches and then compared with the sequence of the amino acids deduced from the gene sequence of the desired product. 

The overall amino acid composition is determined using various hydrolytic and analytical procedures and compared with the amino acid composition deduced from the gene sequence for the desired product, or the natural counterpart, if considered necessary. In many cases, amino acid composition analysis provides some useful structural information for peptides and small proteins, but such data are generally less definitive for large proteins. Quantitative amino acid analysis data can also be used to determine protein content in many cases. 

Terminal amino acid analysis is performed to identify the nature and homogeneity of the amino- and carboxy-terminal amino acids. 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. 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. 

based on the gene sequence for the desired product, cysteine residues are expected, the number and positions of any free sulfhydryl groups and/or disulfide bridges should be determined, to the extent possible. Peptide mapping (under reducing and nonreducing conditions), MS, or other appropriate techniques may be useful for this evaluation. 

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Viral selection is based on (1) relevant viruses that are actual viruses (or of the same species as actual viruses and relevant to the host cell) that have been identified as contaminants (or potential contaminants) of the process, (2) specific model viruses that are closely related to actual viruses (e.g., same genus or family) and have similar physico-chemical properties, and (3) nonspecific model viruses believed to be representative of the spectrum of different virus physio-chemical characteristics [5, 50]. Nonspecific model viruses are used to show inactivation/ removal of viruses in general and to characterize purification robustness [50]. Virus clearance studies should cover emerging viruses and viruses currently believed to be absent in raw materials. These concerns are not addressed when relying on direct testing to ensure safety, specifically consideration of future virus removal requirements in anticipation of future regulatory changes [5, 41]. 

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