On-column virus inactivation by solvent/detergent treatment for a recombinant biological product.

Each process step in the manufacture of biological products requires expensive resources and reduces total process productivity. Since downstream processing of biologicals is the main cost driver, process intensification is a persistent topic during the entire product life cycle. We present here one approach for the intensification of bioprocesses by applying on-column virus inactivation using solvent/detergent (S/D) treatment during ion-exchange chromatography. The established purification process of a recombinant protein was used as a model to compare key process parameters (i.e., product yield, specific activity, impurity clearance) of the novel approach to the standard process protocol. Additional wash and incubation steps with and without S/D-containing buffers were introduced to ensure sufficient contact time to effectively eliminate enveloped viruses and to significantly decrease the amount of S/D reagents. Comparison of key process parameters demonstrated equivalent process performance. To assess the viral clearance capacity of the novel approach, XMuLV was spiked as model virus to the chromatographic load and all resulting fractions were analyzed by TCID50 and RT-qPCR. Data indicates the inactivation capability of on-column virus inactivation even at 10% of the nominal S/D concentration, although the mechanism of viral clearance needs further investigation.

Truly continuous low pH viral inactivation for biopharmaceutical process integration.

Continuous virus inactivation (VI) has received little attention in the efforts to realize fully continuous biomanufacturing in the future. Implementation of continuous VI must assure a specific minimum incubation time, typically 60 min. To guarantee the minimum incubation time, we implemented a packed bed continuous viral inactivation reactor (CVIR) with narrow residence time distribution (RTD) for low pH incubation. We show that the RTD does not broaden significantly over a wide range of linear flow velocities-which highlights the flexibility and robustness of the design. Prolonged exposure to acidic pH has no impact on bed stability, assuring constant RTD throughout long term operation. The suitability of the packed bed CVIR for low pH inactivation is shown with two industry-standard model viruses, that is xenotropic murine leukemia virus and pseudorabies virus. Controls at neutral pH showed no system-induced VI. At low pH, significant VI is observed, even after only 15 min. Based on the low pH inactivation kinetics, the continuous process is equivalent to traditional batch operation. This study establishes a concept for continuous low pH inactivation and, together with previous reports, highlights the versatility of the packed bed reactor for continuous VI, regardless of the inactivation method.

Hepatitis E virus and the safety of plasma products: investigations into the reduction capacity of manufacturing processes.

BACKGROUND: Hepatitis E virus (HEV) has been transmitted by transfusion of labile blood products and the occasional detection of HEV RNA in plasma pools indicates that HEV viremic donations might enter the manufacturing process of plasma products. To verify the safety margins of plasma products with respect to HEV, virus reduction steps commonly used in their manufacturing processes were investigated for their effectiveness to reduce HEV. STUDY DESIGN AND METHODS: Detection methods for HEV removal (by reverse transcription quantitative polymerase chain reaction) and inactivation (using an infectivity assay) were established. Immunoaffinity chromatography and 20-nm virus filtration for Factor (F)VIII, cold ethanol fractionation, and low-pH treatment for immunoglobulin, heat treatment for human albumin, and 35-nm nanofiltration for FVIII inhibitor-bypassing activity (FEIBA) were investigated for their capacity to reduce HEV or the physicochemically similar viruses feline calicivirus (FCV) and hepatitis A virus (HAV). RESULTS: For FVIII, HEV reduction of 3.9 and more than 3.9 log was demonstrated for immunoaffinity chromatography and 20-nm nanofiltration, respectively, and the cold ethanol fractionation for immunoglobulin removed more than 3.5 log of HEV, to below the limit of detection (LOD). Heat treatment of human albumin inactivated more than 3.1 log of HEV to below the LOD and 35-nm nanofiltration removed 4.0 log of HEV from the FEIBA intermediate. The results indicated HAV rather than FCV as the more relevant model virus for HEV. CONCLUSION: Substantial HEV reduction during processes commonly used in the manufacturing of plasma products was demonstrated, similar to that previously demonstrated for HAV.

Retrospective Evaluation of Cycled Resin in Viral Clearance Studies-A Multiple Company Collaboration.

The BioPhorum Development Group Viral Clearance Workstream performed a collaborative retrospective analysis to evaluate packed bed chromatographic resin performance after repeated cycling for two commonly used chromatography steps in biopharmaceutical manufacturing: protein A and anion exchange. Key variables evaluated in the assessment included virus type, resin type, number of reuse cycles, and virus challenge. In this retrospective analysis of viral clearance data on naïve versus cycled resin, powered by the availability of a decade's worth of accumulated industry data, clearance capability was not negatively impacted by resin cycling. This finding is consistent with publications showing that surrogates for viral clearance capabilities could be employed in lieu of testing the viral clearance of cycled resins for protein A and anion exchange chromatography. The rigorous analysis of the retrospective data supports the view that viral clearance studies for cycled resins are not necessary provided that appropriate cleaning methods are applied during repeated use of the chromatography columns.LAY ABSTRACT: The manufacturing processes for biopharmaceutical products often include reusable chromatographic resins that remove process- and product-related impurities as well as potential contaminating viruses. Typically, chromatography resin is "cycled" through repeated steps of resin conditioning, product purification, and resin cleaning. The cycling approach has been evaluated in both small- and full-scale studies that show the performance parameters are maintained. The ability to remove virus is demonstrated separately in a focused small-scale virus-spiking study that is resource-intensive and costly. This paper is a retrospective review of industry data comparing virus removal by naïve and repeatedly cycled resins that summarizes the viral clearance impact of re-using protein A and anion exchange chromatography resins. The key variables evaluated in the assessment included virus type, resin type, number of cycles, and virus challenge. In this retrospective analysis, it was found that the viral clearance capability is not negatively impacted by resin cycling. This finding is consistent with other publications and supports the view that viral clearance studies for cycled resins are not necessary if appropriate cleaning methods are applied during the repeated use of the chromatography columns.Abbreviations: AAV-2, Adeno-associated virus; A-MuLV, Amphotropic murine leukemia virus; AEX, Anion-exchange chromatography; B/E, Bind and elute; BVDV, Bovine viral diarrhea virus; C.P.G., Controlled pore glass; DEAE, Diethylaminoethanol; EMCV, Encephalomyocarditis virus; FT, Flow through; HAV, Hepatitis A virus; HSV-1, Herpes simplex virus type 1; LOD, Limit of detection; LOQ, Limit of quantification; LRF, Log10 reduction factor; mAb, Monoclonal antibody; MVM, Minute virus of mice; NaOH, Sodium hydroxide; PA, Protein A; PPV, Porcine parvovirus; QA, Quaternary amine; QP, Quaternized polyethyleneimine; qPCR, Quantitative polymerase chain reaction; Reo3, Reovirus type 3; SuHV-1, Suid herpesvirus; SV40, Simian virus 40; X-MuLV, Xenotropic murine leukemia virus.