CMC Regulatory Considerations for Antibody-Drug Conjugates.

Antibody-drug conjugates unite the specificity and long circulation time of an antibody with the toxicity of a chemical cytostatic or otherwise active drug using appropriate chemical linkers to reduce systemic toxicity and increase therapeutic index. This combination of a large biological molecule and a small molecule creates an increase in complexity. Multiple production processes are required to produce the native antibody, the drug and the linker, followed by conjugation of afore mentioned entities to form the final antibody-drug conjugate. The connected processes further increase the number of points of control, resulting in necessity of additional specifications and intensified analytical characterization. By combining scientific understanding of the production processes with risk-based approaches, quality can be demonstrated at those points where control is required and redundant comparability studies, specifications or product characterization are avoided. Over the product development lifecycle, this will allow process qualification to focus on those areas critical to quality and prevent redundant studies. The structure of the module 3 common technical document for an ADC needs to reflect each of the production processes and the combined overall approach to quality. Historically, regulatory authorities have provided varied expectations on its structure. This paper provides an overview of essential information to be included and shows that multiple approaches work as long as adequate cross-referencing is included.

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.

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.

Characterization of the photodegradation of a human IgG1 monoclonal antibody formulated as a high-concentration liquid dosage form.

The photodegradation of a human IgG1 monoclonal antibody has been examined in a high concentration (100 mg/mL) liquid formulation. It was observed that a yellowish color is generated when the formulation is exposed to intense and prolonged light exposure, and this discoloration occurs along with a loss in bioactivity. Extensive analytical characterization was performed to determine light induced degradation pathways that occur during exposure to intense light of ICH photodegradation conditions. It has been shown that the monoclonal antibody undergoes a combination of physical and chemical reactions under these conditions, including covalent aggregate formation, fragmentation at the hinge region, oxidation of Trp, His, and Met residues, and deamidation of Asn residues. Oxidation of Trp 94 and deamidation of Asn 93, located in the light chain CDR region, correlates with loss of bioactivity under these conditions. A series of formulation experiments were performed to elucidate the impact of the extent of light exposure, oxygen, protein concentration, and solution pH on the photostability of the formulation. Results demonstrated that photodegradation of the IgG, after intensive light exposure, can be prevented by proper secondary packaging. In addition, it is also shown that a high concentration, liquid dosage form of a human monoclonal antibody is stable upon exposure to the ambient light conditions encountered during routine manufacturing, long-term storage, and administration with proper design of formulation conditions, the primary container as well as the secondary package.