•Poros 50 HS, in bind and elute mode, separates empty and full CVA21 particles.•The separation is optimal at low elution pH conditions.•The separation can also be achieved when performed in ...flowthrough mode.•Multiple cation exchange resins can separate empty and full CVA21 particles.•Poros 50 HS, in bind and elute mode, separates full CVA21 particles from impurities.
Members of the enterovirus genus are promising oncolytic agents. Their morphogenesis involves the generation of both genome-packed infectious capsids and empty capsids. The latter are typically considered as an impurity in need of removal from the final product. The separation of empty and full capsids can take place with centrifugation methods, which are of low throughput and poorly scalable, or scalable chromatographic processes, which typically require peak cutting and a significant trade-off between purity and yield. Here we demonstrate the application of packed bed cation exchange (CEX) column chromatography for the separation of empty capsids from infectious virions for a prototype strain of Coxsackievirus A21. This separation was developed using high throughput chromatography techniques and scaled up as a bind and elute polishing step. The separation was robust over a wide range of operating conditions and returned highly resolved empty and full capsids. The CEX step could be operated in bind and elute or flowthrough mode with similar selectivity and returned yields greater than 70% for full mature virus particles. Similar performance was also achieved using a selection of other bead based CEX chromatography media, demonstrating general applicability of this type of chromatography for Coxsackievirus A21 purification. These results highlight the wide applicability and excellent performance of CEX chromatography for the purification of enteroviruses, such as Coxsackievirus A21.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Live virus vaccine (LVV) purification, employing chromatography, can be challenged by low binding capacities and elution yields. Alternatively, processes relying solely on enzymatic digestion steps ...and size‐based membrane separations can be limited by suboptimal reduction of process related impurities and poorly scalable unit operations. Here, we demonstrate that the combination of flowthrough mode chromatography and an ultrafiltration/diafiltration (UF/DF) unit operation delivers a purification process for two different LVV candidates, V590 and Measles, expressed in adherent Vero cells. For V590, chromatography with mixed mode cation exchange resins returned final product yields of ∼50% and logarithmic reduction values (LRVs) of 1.7–>3.4 and 2.5–3.0 for host cell DNA (hcDNA) and host cell proteins (HCPs), respectively. For Measles, chromatography with mixed mode anion exchange resins returned final product yields of ∼50% and LRVs of 1.6 and 2.2 for hcDNA and HCPs, respectively. For both V590 and Measles processing, the employed resins cleared a key HCP, fibronectin, which could foul the UF/DF unit operation, and thusly enabling it to further reduce HCPs and to formulate the final LVV products. This integrated purification process utilizes the complementary action of the two unit operations and its applicability across LVVs supports its consideration for their processing.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
During the development of a SARS‐CoV‐2 vaccine candidate, at the height of the COVID‐19 pandemic, raw materials shortages, including chromatography resins, necessitated the determination of a ...cleaning in place (CIP) strategy for a multimodal core‐shell resin both rapidly and efficiently. Here, the deployment of high throughput (HT) techniques to screen CIP conditions for cleaning Capto Core 700 resin exposed to clarified cell culture harvest (CCCH) of a SARS‐CoV‐2 vaccine candidate produced in Vero adherent cell culture are described. The best performing conditions, comprised of 30% n‐propanol and ≥0.75 N NaOH, were deployed in cycling experiments, completed with miniature chromatography columns, to demonstrate their effectiveness. The success of the CIP strategy was ultimately verified at the laboratory scale. Here, its impact was assessed across the entire purification process which also included an ultrafiltration/diafiltration step. It is shown that the implementation of the CIP strategy enabled the re‐use of the Capto Core 700 resin for up to 10 cycles without any negative impact on the purified product. Hence, the strategic combination of HT and laboratory‐scale experiments can lead rapidly to robust CIP procedures, even for a challenging to clean resin, and thus help to overcome supply shortages.
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A chimeric SARS‐CoV‐2 live virus vaccine candidate (VSVΔG‐SARS‐CoV‐2) was purified via Capto Core 700 chromatography. High Throughput (HT) scale column chromatography was deployed to screen cleaning in place (CIP) agents and to test a CIP strategy for 10 cycles. This involved the determination of product purities and yields and of the presence of foulants post CIP in resin extracts from miniature (600 μL) columns between cycles. The results were verified at 20 mL column scale for 10 batches. This work demonstrates that HT scale techniques can lead to effective CIP strategies for chromatography resins.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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