ACUVRA: Anion-Exchange Chromatography UV-Ratio Analysis-A QC-Friendly Method for Monitoring Adeno-Associated Virus Empty Capsid Content To Support Process Development and GMP Release Testing.

The genome content of adeno-associated virus (AAV) vectors is critical to the safety and potency of AAV-based gene therapy products. Empty capsids are considered a product-related impurity and a critical quality attribute (CQA) of the drug product, thus requiring characterization throughout the production process to demonstrate they are controlled to acceptable levels in the final drug product. Anion exchange chromatography has been used to achieve separation between empty and full capsids, but requires method development and gradient optimization for different serotypes and formulations. Here, we describe an alternative approach to quantitation that does not rely on achieving separation between empty and full capsids, but instead uses the well-established relationship between absorbance at UV A260/A280 and relation to DNA/protein content, in combination with anion-exchange chromatography to allow one to calculate the relative proportion of empty and full capsids in AAV samples from a single peak. We call this approach ACUVRA: Anion-exchange Chromatography UV-Ratio Analysis, and show the applicability of the method through a case study with recombinant AAV2 (rAAV2) process intermediates and drug substance. Method qualification and GMP validation in a quality control (QC) laboratory results show that ACUVRA is a fit-for-purpose method for process development support and characterization, while also being a QC-friendly option for GMP release testing at all stages of clinical development. Graphical abstract.

Global intercompany assessment of ICIEF platform comparability for the characterization of therapeutic proteins.

An international team spanning 19 sites across 18 biopharmaceutical and in vitro diagnostics companies in the United States, Europe, and China, along with one regulatory agency, was formed to compare the precision and robustness of imaged CIEF (ICIEF) for the charge heterogeneity analysis of the National Institute of Standards and Technology (NIST) mAb and a rhPD-L1-Fc fusion protein on the iCE3 and the Maurice instruments. This information has been requested to help companies better understand how these instruments compare and how to transition ICIEF methods from iCE3 to the Maurice instrument. The different laboratories performed ICIEF on the NIST mAb and rhPD-L1-Fc with both the iCE3 and Maurice using analytical methods specifically developed for each of the molecules. After processing the electropherograms, statistical evaluation of the data was performed to determine consistencies within and between laboratory and outlying information. The apparent isoelectric point (pI) data generated, based on two-point calibration, for the main isoform of the NIST mAb showed high precision between laboratories, with RSD values of less than 0.3% on both instruments. The SDs for the NIST mAb and the rhPD-L1-Fc charged variants percent peak area values for both instruments are less than 1.02% across different laboratories. These results validate the appropriate use of both the iCE3 and Maurice for ICIEF in the biopharmaceutical industry in support of process development and regulatory submissions of biotherapeutic molecules. Further, the data comparability between the iCE3 and Maurice illustrates that the Maurice platform is a next-generation replacement for the iCE3 that provides comparable data.

Robustness of iCIEF methodology for the analysis of monoclonal antibodies: an interlaboratory study.

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.