Shelly Pizarro

Industrial case study: evaluation of a mixed-mode resin for selective capture of a human growth factor recombinantly expressed in E. coli.

Mixed-mode chromatography resins are gaining popularity as effective purification tools for challenging feedstocks. This study presents the development of an industrial application to selectively capture recombinant human vascular endothelial growth factor (rhVEGF) on Capto MMC from an alkaline feedstock. Capto MMC resin contains a ligand that has the potential to participate in ionic, hydrophobic, and hydrogen boding interactions with proteins and is coupled to a highly cross-linked agarose bead matrix. VEGF is a key growth factor involved in angiogenesis and has therapeutic applications for wound healing. In this process, it is expressed in Escherichia coli as inclusion bodies. Solids are harvested from the cell lysate, and the rhVEGF is solubilized and refolded at pH 9.8 in the presence of urea and redox reagents. The unique mixed-mode characteristics of Capto MMC enabled capture of this basic protein with minimal load conditioning and delivered a concentrated pool for downstream processing with >95% yields while reducing host cell protein content to <1.2%. This study explores the impact of loading conditions and residence time on the dynamic binding capacity as well as the development of elution conditions for optimal purification performance. After evaluating various elution buffers, l-arginine HCl was shown to be an effective eluting agent for rhVEGF desorption from the Capto MMC mixed-mode resin since it successfully disrupted the multiple interactions between the resin and rhVEGF. The lab scale effort produced a robust chromatography step that was successfully implemented at commercial manufacturing scale.

Evaluation of a multimodal resin for selective capture of CHO-derived monoclonal antibodies directly from harvested cell culture fluid.

This proof-of-concept study examines the applicability of using multimodal chromatography to selectively capture recombinantly produced monoclonal antibodies (mAb) directly from harvested mammalian cell culture fluid (HCCF) with minimal optimization. Capto MMC is a multimodal resin that contains a ligand with the potential to participate in ionic, hydrophobic, and hydrogen boding interactions with proteins and is coupled to a highly cross-linked agarose bead matrix. Twelve mAb HCCF feedstocks were examined for dynamic binding capacity (DBC) and then two representative feedstocks were selected to develop a systematic approach for elution buffer development. A range of dynamic binding capacities was observed for 10 feedstocks (24-53g/L) and two feedstocks had poor binding properties (<10g/L) despite load conditioning towards a more favorable pH. Analysis of the DBC versus molecular properties showed that the mAb-ligand binding interaction was predominantly charge based. Four separate elution strategies were identified to selectively recover the mAb and could be applied with minimal optimization to other mAb feedstocks. Downstream processing of the Capto MMC pools showed that it is feasible to produce material with comparable purity to a process with affinity capture after two chromatography steps.

High-yield expression of human vascular endothelial growth factor VEGF165 in Escherichia coli and purification for therapeutic applications

Vascular endothelial growth factor (VEGF(165)) is a potent mitogen that induces angiogenesis and vascular permeability in vivo and has demonstrated potential in therapeutic applications for accelerating wound healing. An industrial production method that provides high yield as well as high purity, quality, and potency is needed. The process described in this report involves a bacterial expression system capable of producing approximately 9g of rhVEGF per liter of broth and a downstream purification process consisting of protein refolding and three chromatography steps prior to formulation of the drug substance. A high cell density (HCD) fed-batch fermentation process was used to produce rhVEGF in periplasmic inclusion bodies. The inclusion bodies are harvested from the cell lysate and subjected to a single-step protein solubilization and refolding operation to extract the rhVEGF for purification. Overall recovery yields observed during development, including refolding and chromatography, were 30+/-6%. Host cell impurities are consistently cleared below target levels at both laboratory and large-scale demonstrating process robustness. The structure of the refolded and purified rhVEGF was confirmed by mass spectrometry, N-terminal sequencing, and tryptic peptide mapping while product variants were analyzed by multiple HPLC assays. Biological activity was verified by the proliferation of human umbilical vein derived endothelial cells.

Biomanufacturing process analytical technology (PAT) application for downstream processing: Using dissolved oxygen as an indicator of product quality for a protein refolding reaction.

Process analytical technology (PAT) is an initiative from the US FDA combining analytical and statistical tools to improve manufacturing operations and ensure regulatory compliance. This work describes the use of a continuous monitoring system for a protein refolding reaction to provide consistency in product quality and process performance across batches. A small‐scale bioreactor (3 L) is used to understand the impact of aeration for refolding recombinant human vascular endothelial growth factor (rhVEGF) in a reducing environment. A reverse‐phase HPLC assay is used to assess product quality. The goal in understanding the oxygen needs of the reaction and its impact to quality, is to make a product that is efficiently refolded to its native and active form with minimum oxidative degradation from batch to batch. Because this refolding process is heavily dependent on oxygen, the % dissolved oxygen (DO) profile is explored as a PAT tool to regulate process performance at commercial manufacturing scale. A dynamic gassing out approach using constant mass transfer (kLa) is used for scale‐up of the aeration parameters to manufacturing scale tanks (2,000 L, 15,000 L). The resulting DO profiles of the refolding reaction show similar trends across scales and these are analyzed using rpHPLC. The desired product quality attributes are then achieved through alternating air and nitrogen sparging triggered by changes in the monitored DO profile. This approach mitigates the impact of differences in equipment or feedstock components between runs, and is directly inline with the key goal of PAT to “actively manage process variability using a knowledge‐based approach.” Biotechnol. Bioeng. 2009; 104: 340–351

High-pressure refolding of human vascular endothelial growth factor (VEGF) recombinantly expressed in bacterial inclusion bodies: Refolding optimization, and feasibility assessment

High‐pressure has been established as an effective technique for refolding proteins at high concentrations. In this study, high hydrostatic pressure (1–3 kbar) was utilized to refold a homodimeric protein from inclusion bodies and the process was evaluated for large‐scale manufacturing feasibility. This research focused on increasing protein concentration while maximizing yield and product quality. Refolding yields of 29–42% were achieved in the absence of urea at 2 kbar and at a protein concentration of 6 g/L. Optimization of the refolding buffer composition via multivariate design of experiments and other process parameters such as refolding pressure, gas sparging, and time under pressure are discussed. Although high‐pressure refolding can be considered a viable technology for manufacturing if the gains are clearly identified, in this particular case, the benefits that the high‐pressure technology offers do not compensate for the drawbacks of implementing new equipment in an existing facility, and unknown impact of scale‐up for this molecule.

Assessing the risk of leachables from single-use bioprocess containers through protein quality characterization

Leachables from single-use bioprocess containers (BPCs) are a source of process-related impurities that have the potential to alter product quality of biotherapeutics and affect patient health. Leachables often exist at very low concentrations, making it difficult to detect their presence and challenging to assess their impact on protein quality. A small-scale stress model based on assessing protein stability was developed to evaluate the potential risks associated with storing biotherapeutics in disposable bags caused by the presence of leachables. Small-scale BPCs were filled with protein solution at high surface area–to–volume ratios (≥3× the surface area–to–volume ratio of manufacturing-scale BPCs) and incubated at stress temperatures (e.g., 25 °C or 30 °C for up to 12 weeks) along with an appropriate storage vessel (e.g., glass vial or stainless steel) as a control for side-by-side comparison. Changes in protein size variants measured by size exclusion chromatography, capillary electrophoresis, and particle formation for two monoclonal antibodies using both the small-scale stress model and a control revealed a detrimental effect of gamma-irradiated BPCs on protein aggregation and significant BPC difference between earlier and later batches. It was found that preincubation of the empty BPCs prior to protein storage improved protein stability, suggesting the presence of volatile or heat-sensitive leachables (heat-labile or thermally degraded). In addition, increasing the polysorbate 20 concentration lowered, but did not completely mitigate, the leachable-protein interactions, indicating the presence of a hydrophobic leachable. Overall, this model can inform the risk of BPC leachables on biotherapeutics during routine manufacturing and assist in making decisions on the selection of a suitable BPC for the manufacturing process by assessing changes in product quality. LAY ABSTRACT: Leachables from single-use systems often exist in small quantities and are difficult to detect with existing analytical methods. The presence of relevant detrimental leachables from single-use bioprocess containers (BPCs) can be indirectly detected by studying the stability of monoclonal antibodies via changes by size exclusion chromatography, capillary electrophoresis sodium dodecyl sulfate, and visible/sub-visible particles using a small-scale stress model containing high surface area–to–volume ratio at elevated temperature alongside with an appropriate control (e.g., glass vials or stainless steel containers). These changes in protein quality attributes allowed the evaluation of potential risks associated with adopting single-use bioprocess containers for storage as well as bag quality and bag differences between earlier and later batches. These leachables appear to be generated during the bag sterilization process by gamma irradiation. Improvements in protein stability after storage in “preheated” bags indicated that these leachables may be thermally unstable or volatile. The effect of surfactant levels, storage temperatures, surface area–to–volume ratios, filtration, and buffer exchange on leachables and protein stability were also assessed.