Philip Lester

High‐throughput methods for miniaturization and automation of monoclonal antibody purification processes

In the last decade, high‐throughput downstream process development techniques have entered the biopharmaceutical industry. As chromatography is the standard downstream purification method, several high‐throughput chromatographic methods have been developed and applied including miniaturized chromatographic columns for utilization on liquid handling stations. These columns were used to setup a complete downstream process on a liquid handling station for the first time. In this article, a monoclonal antibody process was established in lab‐scale and miniaturized afterwards. The scale‐down methodology is presented and discussed. Liquid handling in miniaturized single and multicolumn processes was improved and applicability was demonstrated by volume balances. The challenges of absorption measurement are discussed and strategies were shown to improve volume balances and mass balances in 96‐well microtiter plates. The feasibility of miniaturizing a complete downstream process was shown. In the future, analytical bottlenecks should be addressed to gain the full benefit from miniaturized complete process development.

Use of the steric mass action model in ion-exchange chromatographic process development

Despite the preponderance of models in the literature, chromatographic process development in industrial protein purification has traditionally been based on heuristic knowledge and expertise. In this work, we explore the feasibility of using a mathematical model to guide process development and optimization. The development of an anion-exchange step to separate an antibody from its dimer is used as a paradigm to test this approach to process development. In the approach involving models, we show that the work required may be reduced to the task of determining conditions for adequate selectivity. Once these conditions are obtained, the steric mass action formalism is used to predict the preparative experimental results. Our results indicate that this model is able to accurately predict experimental results under high loadings with minimal parameter estimation. Finally, we identify ways in which such models can be used to increase productivity and process robustness.

Real-time control of purified product collection during chromatography of recombinant human insulin-like growth factor-I using an on-line assay

During preparative reversed-phase chromatography of recombinant human insulin-like growth factor-I (IGF), the separation of IGF from IGF aggregates cannot be determined using UV absorbance. An on-line reversed-phase chromatographic assay was developed that provides a quantitative measurement of IGF and IGF aggregates every 4 min, allowing real-time control of purified IGF collection. Process control using the on-line assay is a reliable and accurate method to collect purified IGF.

Binding Protein-3-Selective Insulin-Like Growth Factor I Variants: Engineering, Biodistributions, and Clearance

Insulin-like growth factor I (IGF-I) is a potent anabolic peptide that mediates most of its pleiotropic effects through association with the IGF type I receptor. Biological availability and plasma half-life of IGF-I are modulated by soluble binding proteins (IGFBPs), which sequester free IGF-I into high affinity complexes. Elevated levels of specific IGFBPs have been observed in several pathological conditions, resulting in inhibition of IGF-I activity. Administration of IGF-I variants that are unable to bind to the up-regulated IGFBP species could potentially counteract this effect. We engineered two IGFBP-selective variants that demonstrated 700- and 80,000-fold apparent reductions in affinity for IGFBP-1 while preserving low nanomolar affinity for IGFBP-3, the major carrier of IGF-I in plasma. Both variants displayed wild-type-like potency in cellular receptor kinase assays, stimulated human cartilage matrix synthesis, and retained their ability to associate with the acid-labile subunit in complex with IGFBP-3. Furthermore, pharmacokinetic parameters and tissue distribution of the IGF-I variants in rats differed from those of wild-type IGF-I as a function of their IGFBP affinities. These IGF-I variants may potentially be useful for treating disease conditions associated with up-regulated IGFBP-1 levels, such as chronic or acute renal and hepatic failure or uncontrolled diabetes. More generally, these results suggest that the complex biology of IGF-I may be clarified through in vivo studies of IGFBP-selective variants.

Quality by design approach for viral clearance by protein a chromatography.

Protein A chromatography is widely used as a capture step in monoclonal antibody (mAb) purification processes. Antibodies and Fc fusion proteins can be efficiently purified from the majority of other complex components in harvested cell culture fluid (HCCF). Protein A chromatography is also capable of removing modest levels of viruses and is often validated for viral clearance. Historical data mining of Genentech and FDA/CDER databases systematically evaluated the removal of model viruses by Protein A chromatography. First, we found that for each model virus, removal by Protein A chromatography varies significantly across mAbs, while remains consistent within a specific mAb product, even across the acceptable ranges of the process parameters. In addition, our analysis revealed a correlation between retrovirus and parvovirus removal, with retrovirus data generally possessing a greater clearance factor. Finally, we describe a multivariate approach used to evaluate process parameter impacts on viral clearance, based on the levels of retrovirus‐like particles (RVLP) present among process characterization study samples. It was shown that RVLP removal by Protein A is robust, that is, parameter effects were not observed across the ranges tested. Robustness of RVLP removal by Protein A also correlates with that for other model viruses such as X‐MuLV, MMV, and SV40. The data supports that evaluating RVLP removal using process characterization study samples can establish multivariate acceptable ranges for virus removal by the protein A step for QbD. By measuring RVLP instead of a model retrovirus, it may alleviate some of the technical and economic challenges associated with performing large, design‐of‐experiment (DoE)—type virus spiking studies. This approach could also serve to provide useful insight when designing strategies to ensure viral safety in the manufacturing of a biopharmaceutical product. Biotechnol. Bioeng. 2014;111: 95–103.

Non-flammable preparative reversed-phase liquid chromatography of recombinant human insulin-like growth factor-I.

Acetonitrile is used as an eluent for reversed-phase chromatography. However, because it is a flammable solvent, using acetonitrile on a large scale requires expensive equipment and facilities specially designed for flammable solvents. Using a non-flammable solvent as an eluent eliminates this expense. A method was developed to purify recombinant human insulin-like growth factor I by reversed-phase high-performance liquid chromatography using gradient elution with hexylene glycol, a non-flammable replacement for acetonitrile. The separation produced equivalent yield, purity and throughput as reversed-phase chromatography using elution with acetonitrile.

Porcine circovirus (PCV) removal by Q sepharose fast flow chromatography

The recently discovered contamination of oral rotavirus vaccines led to exposure of millions of infants to porcine circovirus (PCV). PCV was not detected by conventional virus screening tests. Regulatory agencies expect exclusion of adventitious viruses from biological products. Therefore, methods for inactivation/removal of viruses have to be implemented as an additional safety barrier whenever feasible. However, inactivation or removal of PCV is difficult. PCV is highly resistant to widely used physicochemical inactivation procedures. Circoviruses such as PCV are the smallest viruses known and are not expected to be effectively removed by currently‐used virus filters due to the small size of the circovirus particles. Anion exchange chromatography such as Q Sepharose® Fast Flow (QSFF) has been shown to effectively remove a range of viruses including parvoviruses. In this study, we investigated PCV1 removal by virus filtration and by QSFF chromatography. As expected, PCV1 could not be effectively removed by virus filtration. However, PCV1 could be effectively removed by QSFF as used during the purification of monoclonal antibodies (mAbs) and a log10 reduction value (LRV) of 4.12 was obtained.

The rapid identification of elution conditions for therapeutic antibodies from cation-exchange chromatography resins using high-throughput screening

Cation-exchange chromatography is widely used in the purification of therapeutic antibodies, wherein parameters such as elution pH and counterion concentration require optimization for individual antibodies across different chromatography resins. With a growing number of antibodies in clinical trials and the pressure to expedite process development, we developed and automated a high-throughput batch-binding screen to more efficiently optimize elution conditions for cation-exchange chromatography resins. The screen maps the binding behavior of antibodies and impurities as a function of pH and counterion concentration in terms of a partition coefficient (Kp). Using this approach, the binding behavior of a library of antibodies was assessed on Poros 50HS and SP Sepharose Fast Flow resins. The diversity in binding behavior between antibodies and across resins translated to the requirement of a variable counterion concentration to elute each antibody. This requirement can be met through the use of a gradient elution. However, a gradient of increasing counterion concentration spans the transition from binding to non-binding for impurities as well as the antibody, resulting in the elution of impurities within the antibody elution peak. Step elution conditions that selectively elute the antibody while retaining impurities on the resin can now be rapidly identified using our high-throughput approach. We demonstrate that by correlating antibody Kp to elution pool volume and yield on packed-bed columns and through the calculation of a separation factor, we can efficiently optimize step elution conditions that maximize impurity clearance and yield for each antibody.

Post-Licensure Purification Process Improvements for Therapeutic Antibodies: Current and Future States

Abstract Therapeutic antibodies have been manufactured since the advent of intravenous immunoglobulin in the 1950s. Rituxan was the first recombinant monoclonal antibody approved for therapeutic use by the U.S. Food and Drug Administration (FDA) in 1997. Now, over 50 recombinant monoclonal antibodies have been licensed for commercial distribution [ 1 ]. Post-licensure manufacturing process changes provide opportunities to increase manufacturing capacity, improve product quality, consistency, or safety, and may be necessary upon process transfer to a new facility [ 2 ]. Yet, the complexity of global regulatory authority oversight and approval sets a high bar for manufacturing process changes for an approved parenteral protein therapeutic. This review examines the history of antibody production, focusing on purification process technology. Strategies for post-licensure changes are discussed, including regulatory considerations. Case studies are described for several antibody process purification changes. Finally, some speculations are shared about the potential future of purification process technology and post-licensure process changes for commercial therapeutic antibody production.