Viruses and virus-like particles (VLPs) raise unique challenges for downstream recovery and purification. In an Ask the Expert webinar on 19 September 2019, Mark Snyder (R&D applications manager of process chromatography at Bio-Rad Laboratories) discussed purification methods engineered to perform at manufacturing scales. Offering case studies featuring Nuvia resin and CHT Ceramic Hydroxyapatite chromatography support, Snyder emphasized that scalable media can help companies optimize downstream operations and address evolving manufacturing requirements.
Tremendous growth in markets for vaccines and gene therapies is compelling manufacturers to generate more drug product and to work with greater numbers of viruses and VLPs than ever before. But perennial downstream challenges remain. At manufacturing scales, the size and structural complexity of such species can diminish efforts to recover and purify drug substance. Careful selection of chromatography resins can mitigate those challenges.
CHT and Nuvia media are designed to achieve high purification at manufacturing levels. All CHT family media, including CFT Ceramic Fluorapatite, are mixed-mode media: They facilitate ion exchange on phosphate groups and metal-affinity interaction on calcium sites. Those properties enable significant tolerance of salt and phosphate, which bolsters virus and vector purification efforts.
For instance, in-house assays of CHT Type II and CFT media showed excellent purification of polio virus. Using high sodium phosphate concentrations for elution, CHT yields reached 88%. CFT yields approached 100%. Product purity exceeded 90% with both media. In-house assessments produced similar results for influenza purification.
Independent studies report equally promising findings for even more complicated manufacturing processes. One study focused on plant-based malaria vaccines. A research team loaded VLP fusions derived from malarial parasites and alfalfa mosaic virus onto columns with CHT media. Sodium phosphate concentrations began at 95 mM and increased to 150 mM during elution. Final drug product purity reached 85%. The purified product also exhibited high immunogenicity.
Aside from sharp elution capability, scalability distinguishes CHT and Nuvia media. For instance, Nuvia Q anion-exchanger resins consistently ensure high single-step purification of retrovirus-based nanoparticles. Third-party studies tracked Nuvia Q performance at six column volumes ranging from 1 to 5,100 mL — representative of scaling up from a 3- to 250-L bioreactor. Loads ranged from 0.6 to 275 L; flow rates ran between 0.3 and 1,700 mL/min. The resin maintained similar recovery profiles and elution peaks across all scales. Particle potency was retained at 100%, residual proteins and DNA were nearly undetectable, and endotoxins fell well within prescribed limits. Final virus recovery came to 65% — a solid performance considering operation scale and the use of a single purification step.
Despite the challenges that viruses and VLPs present, manufacturers must ensure high purity and strong purified potency of these molecules in the fewest number of elutions. With CHT and Nuvia media, manufacturers can achieve those ambitious goals for a rapidly growing number of therapies.
Questions and Answers
When should users prefer ion-exchange resins over CHT media for vaccine purification? Ion-exchange resins generally work well as a capture step owing to their high binding capacities and, for anion-exchange supports, clearance of DNA. CHT media are not ideal for capture steps because they can bind molecules, such as transition-state metals from fermentation media. In addition, ion-exchange resins produce considerable dewatering through the first step.
What resin is recommended for purifying plant-based vaccines? Plant-based feedlines tend to be “dirtier” than other sources. Ion-exchange resins are ideal in such cases because they can be paired with aggressive washes to remove contaminants.
What are key advantages of CHT media over other purification methods? Ultracentrifugation and tangential-flow filtration are common alternatives. But ultracentrifuges are bulky, expensive to purchase and maintain, and difficult to scale. Scalability is a key merit of CHT media. Depending on the resin, small column volumes might be sufficient to maintaining good separation.
Bio-Rad and CHT are trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions. All trademarks used herein are the property of their respective owners. Request chromatography resin samples at bio-rad.com/ATE19.
The full presentation of this webcast can be found on the BioProcess International website at the link below.