An international team including 12 laboratories from 11 independent biopharmaceutical companies in the United States and Switzerland was formed to evaluate the precision and robustness of imaged capillary isoelectric focusing for the charge heterogeneity analysis of monoclonal antibodies. The different laboratories determined the apparent pI and the relative distribution of the charged isoforms for a representative monoclonal antibody sample using the same capillary isoelectric focusing assay. Statistical evaluation of the data was performed to determine within and between laboratory consistencies and outlying information. The apparent pI data generated for each charged variant peak showed very good precision between laboratories with RSD values of less than 0.8%. Similarly, the RSD for the therapeutic monoclonal antibody charged variants percent peak area values are less than 11% across different laboratories using different analyst, different lots of ampholytes and multiple instruments. These results validate the appropriate use of imaged capillary isoelectric focusing in the biopharmaceutical industry in support of process development and regulatory submissions of therapeutic antibodies.
Interlaboratory comparisons are essential to bringing emerging technologies into biopharmaceutical industry practice and regulatory acceptance. As a result, an international team including 12 laboratories from 10 independent biopharmaceutical companies in the United States and Switzerland was formed to evaluate the precision and robustness of capillary isoelectric focusing (CIEF) to assess the charge heterogeneity of monoclonal antibodies. The different laboratories determined the apparent pI and the relative distribution of the charge isoforms of a representative monoclonal antibody (rMAb) sample using the same CIEF method. Statistical evaluation of the data was performed to determine within and between-laboratory consistencies and outlying information. The apparent pI data generated for each charge variant peak showed very good precision between laboratories with percentage of RSD values of B0.5%. Similarly, the percentage of RSD for the rMAb charge variants percent peak area values are B4.4% across different laboratories with different analysts using different lots of ampholytes and multiple instruments. Taken together, these results validate the appropriate use of CIEF in the biopharmaceutical industry in support of regulatory submissions.
Hyaluronic acid is a negatively charged polysaccharide with a high degree of polydispersity that makes the separation of its oligomers extremely difficult. Through the use of columns filled with a highly viscous polyacrylamide matrix, the unit resolution of hyaluronate oligomers could be achieved, up to at least 80 kDa of mass, through capillary electrophoresis. As analytical application examples, the fractions of enzymatically or ultrasonically degraded hyaluronates were monitored through this method. Because of the very high resolving power, peaks additional to the regular oligomers can be observed that are assumed to be conformers of this regular, unbranched biopolymer.
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