Comparability assessments of process and product changes made during development of two different monoclonal antibodies.

To assess the impact of manufacturing changes on antibody structure and function during the course of product development, three comparability studies were performed for each of two different IgG1 monoclonal antibody product candidates. Comparability study #1 evaluated the effect of changing the cell line and bulk drug substance manufacturing process for cell culture and purification. Results indicated that these process changes led to differences in sialylation of N-glycans and/or C-terminal lysine levels. Comparability study #2 results confirmed that scale-up of the bulk process and transfer to the commercial site, combined with changing from a lyophilized to a liquid dosage form, did not impact the structural or functional integrity of the antibodies. Comparability study #3 examined possible differences arising when the liquid formulation filled into pre-filled syringes and vials. Results indicated nearly identical molecular structure, biological activity, and degradation profiles except for a small yet statistically significant increase in the levels of subvisible particles in pre-filled syringes. These results from comparability studies with two different monoclonal antibodies are discussed with respect to the timing of the manufacturing changes and overall comparability strategies to assure safety and efficacy during development.

Facile method of quantification for oxidized tryptophan degradants of monoclonal antibody by mixed mode ultra performance liquid chromatography.

Oxidation in therapeutic monoclonal antibody is a common occurrence and it may affect potency. Thus controlling and monitoring the amount of oxidized variant in the drug product sample is important since it may impact the purity. Here, we present the development of a fast and easy method utilizing size exclusion - ultra performance liquid chromatography (SE-UPLC) run under moderate hydrophobic conditions (mixed mode) to monitor the heterogeneity in drug product samples. The best separation was obtained using Waters Acquity BEH200 size exclusion column along with a mobile phase consisting of sodium acetate and sodium sulfate that separates IgG into aggregate, monomer, and fragment. The moderate salt concentration resulted in a second mode of separation based on hydrophobicity, resolving a monomer pre-peak from the monomer main peak. Multi-angle light scattering (MALS) determined the pre-peak has a similar mass as the IgG monomer. Characterization of the purified pre-peak fraction using mass spectrometry (MS), and bioactivity revealed this degradant to be a Trp-oxidized IgG monomer with significantly reduced bioactivity. Method qualification of the mixed mode UPLC method showed good recovery for the spiked monomer pre-peak and Fab fragment. However, the recovery of spiked dimer was low. This method is suitable for determining the relative distribution of the oxidized monomer and the native monomer species.

Method development and validation of capillary sodium dodecyl sulfate gel electrophoresis for the characterization of a monoclonal antibody.

A capillary sodium dodecyl sulfate gel electrophoresis (cSDS) method has been developed and qualified for purity and impurity analysis of monoclonal antibodies. This method was optimized and qualified for the analysis of monoclonal antibody (mAb1) under reduced and non-reduced conditions. Some of the sample preparation parameters including sample buffer pH, incubation temperature and duration, alkylation conditions with iodoacetamide (IAM), and reduction conditions with 2-mercaptoethanol (2-ME) were optimized. It was observed that under slightly acidic conditions (pH 5.5-6.5) the thermally induced fragmentation of non-reduced mAb1 was greatly decreased. As such, a citrate-phosphate buffer at pH 6.5 was used for sample preparation to replace the original Beckman sample buffer (pH 9.0). The optimal sample preparation conditions were found to be as follows: (1) incubation temperature and duration (reduced and non-reduced), 65 degrees C for 5 min; (2) alkylation condition, 10 microL of 0.25 M IAM; (3) reduction condition, 10 microL of 5-fold diluted 2-ME. The method was qualified by evaluating specificity, accuracy, precision, limit of quantitation (LOQ), and linearity. The method exhibited no interference from sample buffer matrix. The method was found to be linear, accurate, and precise in the range of 0.25-3.0mg/mL protein concentration. The LOQ of the method was determined to be 0.02 mg/mL for reduced and non-reduced mAb1. In addition, some aspects of sample stability were examined during qualification.

Evaluation of a dual-wavelength size exclusion HPLC method with improved sensitivity to detect protein aggregates and its use to better characterize degradation pathways of an IgG1 monoclonal antibody.

The evaluation of a dual wavelength size exclusion high performance liquid chromatography (DW-SE-HPLC) method with improved sensitivity to detect aggregates in a high concentration IgG1 monoclonal antibody formulation is presented. This technique utilizes ultraviolet detection at two different wavelengths to monitor the levels of monomer, aggregate, and fragments and was shown to have improved sensitivity for the detection aggregates and fragments compared to light scattering (LS) detection. After assay optimization including the use of column conditioning, the limit of quantitation for aggregates was determined to be 0.04% with essentially complete recovery of aggregates from the column (>99.5%). The DW-SE-HPLC method was used to evaluate the level of protein aggregates generated by different environmental conditions such as exposure to elevated temperatures/acidic pH or intense light. The detection and characterization of protein aggregates by DW-SE-HPLC was compared with an orthogonal biophysical technique (sedimentation velocity analytical ultracentrifugation, SV-AUC). A good overall correlation was observed for levels of monomer, aggregates (dimer and multimers), and fragments as measured by the two analytical techniques (e.g., 6.0% vs. 5.3% and 14% vs. 11% for dimeric aggregates generated by elevated temperature/acidic pH and light exposure, respectively). The stability profile of a high concentration IgG1 monoclonal antibody formulation was investigated under stressed storage conditions (40 degrees C over 3 months) using the DW-SE-HPLC method including the loss of monomeric species with the concomitant accumulation of both aggregates and fragments. The nature and composition of the aggregates (primarily noncovalent dimers) and fragments (primarily loss of Fab from an intact IgG1) formed during storage were further characterized by a combination of LS measurements and mass spectroscopy analysis of deglycosylated IgG1 samples isolated by preparative SE-HPLC. The combination of DW-SE-HPLC, SV-AUC, LS, and mass spectroscopy results provided a detailed overall understanding the monomer, aggregate, fragment degradation pathway(s) for a high concentration IgG1 monoclonal antibody formulation during storage.