Process intensification has shown great potential to increase productivity and reduce costs in biomanufacturing. This case study describes the evolution of a manufacturing process from a conventional processing scheme at 1000-L scale (Process A, n = 5) to intensified processing schemes at both 1000-L (Process B, n = 8) and 2000-L scales (Process C, n = 3) for the production of a monoclonal antibody by a Chinese hamster ovary cell line. For the upstream part of the process, we implemented an intensified seed culture scheme to enhance cell densities at the seed culture step (N-1) prior to the production bioreactor (N) by using either enriched N-1 seed culture medium for Process B or by operating the N-1 step in perfusion mode for Process C. The increased final cell densities at the N-1 step allowed for much higher inoculation densities in the production bioreactor operated in fed-batch mode and substantially increased titers by 4-fold from Process A to B and 8-fold from Process A to C, while maintaining comparable final product quality. Multiple changes were made to intensify the downstream process to accommodate the increased titers. New high-capacity resins were implemented for the Protein A and anion exchange chromatography (AEX) steps, and the cation exchange chromatography (CEX) step was changed from bind-elute to flow-through mode for the streamlined Process B. Multi-column chromatography was developed for Protein A capture, and an integrated AEX-CEX pool-less polishing steps allowed semi-continuous Process C with increased productivity as well as reductions in resin requirements, buffer consumption, and processing times. A cost-of-goods analysis on consumables showed 6.7-10.1 fold cost reduction from the conventional Process A to the intensified Process C. The hybrid-intensified process described here is easy to implement in manufacturing and lays a good foundation to develop a fully continuous manufacturing with even higher productivity in the future.
The structural characteristics of the HyperCel family of cellulosic ion-exchange materials (Pall Corporation) were assessed using methods to gauge the pore dimensions and the effect of ionic strength on intraparticle architecture. Inverse size exclusion chromatography (ISEC) was applied to the S and STAR AX HyperCel derivatives. The theoretical analysis yielded an average pore radius for each material of about 5 nm, with a particularly narrow pore-size distribution. Electron microscopy techniques were used to visualize the particle structure and relate it to macroscopic experimental data. Microscopy of Q and STAR AX HyperCel anion exchangers presented some qualitative differences in pore structure that can be attributed to the derivatization using conventional quaternary ammonium and salt-tolerant ligands, respectively. Finally, the effect of ionic strength was studied through the use of salt breakthrough experiments to determine to what extent Donnan exclusion plays a role in restricting the accessible pore volume for small ions. It was determined that Donnan effects were prevalent at total ionic strengths (TIS) less than 150 mM, suggesting the presence of a ligand-containing partitioning volume within the pore space.
Monoclonal antibodies (mAbs) and related recombinant proteins continue to gain importance in the treatment of a great variety of diseases. Despite significant advances, their manufacturing can still present challenges owing to their molecular complexity and stringent regulations with respect to product purity, stability, safety, and so forth. In this context, protein aggregates are of particular concern due to their immunogenic potential. During manufacturing, mAbs routinely undergo acidic treatment to inactivate viral contamination, which can lead to their aggregation and thereby to product loss. To better understand the underlying mechanism so as to propose strategies to mitigate the issue, we systematically investigated the denaturation and aggregation of two mAbs at low pH as well as after neutralization. We observed that at low pH and low ionic strength, mAb surface hydrophobicity increased whereas molecular size remained constant. After neutralization of acidic mAb solutions, the fraction of monomeric mAb started to decrease accompanied by an increase on average mAb size. This indicates that electrostatic repulsion prevents denatured mAb molecules from aggregation under acidic pH and low ionic strength, whereas neutralization reduces this repulsion and coagulation initiates. Limiting denaturation at low pH by D-sorbitol addition or temperature reduction effectively improved monomer recovery after neutralization. Our findings might be used to develop innovative viral inactivation procedures during mAb manufacturing that result in higher product yields. K E Y W O R D SANS fluorescence, downstream processing, monoclonal antibodies, protein aggregation, protein unfolding, viral inactivation
In this study we introduce three process characterization approaches toward validation of continuous twin-column capture chromatography (CaptureSMB), referred to as "standard," "model assisted," and "hybrid." They are all based on a traditional risk-based approach, using process description, risk analysis, design-of-experiments (DoE), and statistical analysis as essential elements. The first approach, the "standard" approach uses a traditional experimental DoE to explore the design space of the high-ranked process parameters for the continuous process. Due to the larger number of process parameters in the continuous process, the DoE is extensive and includes a larger number of experiments than an equivalent DoE of a single column batch capture process. In the investigated case, many of the operating conditions were practically infeasible, indicating that the design space boundaries had been chosen inappropriately. To reduce experimental burden and at the same time enhance process understanding, an alternative "model assisted" approach was developed in parallel, employing a chromatographic process model to substitute experimental runs by computer simulations. Using the "model assisted" approach only experimental conditions that were feasible in terms of process yield constraints (>90%) were considered for statistical analysis. The "model assisted" approach included an optimization part that identified potential boundaries of the design space automatically. In summary, the "model assisted" approach contributed to increased process understanding compared to the "standard" approach. In this study, a "hybrid" approach was also used containing the general concepts of the "standard" approach but substituting a number of its experiments by computer simulations. The presented approaches contain essential elements of the Food and Drug Administration's process validation guideline. K E Y W O R D S continuous capture, integrated continuous biomanufacturing, process characterization, process validation, protein A
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