Optimization of a platform process operating space for a monoclonal antibody susceptible to reversible and irreversible aggregation using a solution stability screening approach.

Platform manufacturing processes are widely adopted to simplify and standardize the development and manufacturing of monoclonal antibodies (mAbs). However, there are mAbs that do not conform to a platform design due to instability or other protein properties leading to a negative impact on product quality or process performance (non-platform mAb). Non-platform mAbs typically require prolonged development times and significant deviations from the platform process to address these issues due to the need to sequentially optimize individual process steps. In this study, we describe an IgG2 mAb (mAb A) that is susceptible to aggregation and reversible self-association (RSA) under platform conditions. In lieu of a sequential optimization approach, we evaluated the solution stability of mAb A across the platform operating space (solution stability screen). This screening design was used to identify interacting parameters that affected the non-platform mAb stability. A subsequent response surface design was found to predict an acceptable operating space that minimized aggregate formation and RSA across the entire process. This information guided the selection of optimal parameters best suited to avoid destabilizing conditions for each process step. Substantial time savings was achieved by focusing development around these factors including protein concentration, buffer pH, salt concentration, and excipient type. In addition, this work enabled the optimization of a cation exchange chromatography step that removed aggregate without yield losses due to the presence of reversible aggregation. The final optimized process derived from this study resulted in an increase in yield of ˜30% over the original process while maintaining the same level of aggregate clearance to match product quality. Solution stability screening is readily adapted to high throughput technologies to minimize material requirements and accelerate analytical data availability. Implementation of high throughput approaches will further expedite process development and enable enhanced selection of candidate drugs by including process development objectives.

Intracellular response to process optimization and impact on productivity and product aggregates for a high-titer CHO cell process.

A key goal in process development for antibodies is to increase productivity while maintaining or improving product quality. During process development of an antibody, titers were increased from 4 to 10 g/L while simultaneously decreasing aggregates. Process development involved optimization of media and feed formulations, feed strategy, and process parameters including pH and temperature. To better understand how CHO cells respond to process changes, the changes were implemented in a stepwise manner. The first change was an optimization of the feed formulation, the second was an optimization of the medium, and the third was an optimization of process parameters. Multiple process outputs were evaluated including cell growth, osmolality, lactate production, ammonium concentration, antibody production, and aggregate levels. Additionally, detailed assessment of oxygen uptake, nutrient and amino acid consumption, extracellular and intracellular redox environment, oxidative stress, activation of the unfolded protein response (UPR) pathway, protein disulfide isomerase (PDI) expression, and heavy and light chain mRNA expression provided an in-depth understanding of the cellular response to process changes. The results demonstrate that mRNA expression and UPR activation were unaffected by process changes, and that increased PDI expression and optimized nutrient supplementation are required for higher productivity processes. Furthermore, our findings demonstrate the role of extra- and intracellular redox environment on productivity and antibody aggregation. Processes using the optimized medium, with increased concentrations of redox modifying agents, had the highest overall specific productivity, reduced aggregate levels, and helped cells better withstand the high levels of oxidative stress associated with increased productivity. Specific productivities of different processes positively correlated to average intracellular values of total glutathione. Additionally, processes with the optimized media maintained an oxidizing intracellular environment, important for correct disulfide bond pairing, which likely contributed to reduced aggregate formation. These findings shed important understanding into how cells respond to process changes and can be useful to guide future development efforts to enhance productivity and improve product quality.

Storage Conditions of Conjugated Reagents Can Impact Results of Immunogenicity Assays.

Consistent performance of anti-drug antibody (ADA) assays through all stages of clinical development is critical for the assessment of immunogenicity and interpretation of PK, PD, safety, and efficacy. The electrochemiluminescent assays commonly employed for ADA measurement use drug conjugated with ruthenium and biotin to bind ADA in samples. Here we report an association between high nonspecific ADA responses in certain drug-naïve individuals and the storage buffer of the conjugated reagents used in a monoclonal antibody ADA assay. Ruthenylated reagents stored in phosphate-buffered saline (PBS) buffer had increased levels of aggregate and produced variable and high baseline responses in some subjects. Reagents stored in a histidine-sucrose buffer (HSB) had lower aggregate levels and produced low sample responses. In contrast to PBS, conjugated reagents formulated in HSB remained low in aggregate content and in sample response variability after 5 freeze/thaw cycles. A reagent monitoring control (RMC) serum was prepared for the real-time evaluation of conjugated reagent quality. Using appropriate buffers for storage of conjugated reagents together with RMCs capable of monitoring of reagent aggregation status can help ensure consistent, long-term performance of ADA methods.

Semi-Quantitative Analysis of Inherent Visible Particles for Biopharmaceutical Products.

Visible particles must be monitored as part of the control strategy for pharmaceutical products. Extraneous (foreign) particles are not acceptable in parenteral drug products. In biopharmaceuticals, formation of protein particles is recognized as an inherent quality attribute. All protein therapeutics contain particles that vary greatly in visibility and size from invisible (sub-micron) to visible (millimeter) and, as part of the control strategy, biopharmaceutical companies are required to monitor and minimize the presence of visible and sub-visible particles in their products. There is an industry-wide unmet need for particle standards for visual inspection of protein therapeutics. A new, semi-quantitative method using particle standards for assessing the levels of small, inherent visible particles is presented. This method can be used during product development to identify a formulation that minimizes particle formation and also during release and stability testing to monitor and control inherent proteinaceous visible particles. LAY ABSTRACT: Visible particles must be monitored as part of the control strategy for parenteral biopharmaceutical drug products. In these products, formation of protein particles is a natural occurrence. All protein drugs contain particles that vary greatly in visibility and size from invisible (sub-micron) to visible (millimeter), and pharmaceutical companies are required to monitor and minimize the presence of visible and sub-visible particles in their products. There is an industry-wide unmet need for particle standards for visual inspection of protein drugs. A new, semi-quantitative method using particle standards for assessing the levels of small, naturally occurring visible particles is presented. This method can be used during drug development to identify a formulation that minimizes particle formation and also during testing of final clinical or commercial drug product to monitor and control naturally occurring proteinaceous visible particles.