Continuous low pH viral inactivation: Operation and scaling strategy informs viral clearance study.

A continuous viral inactivation (CVI) tubular reactor was designed for low pH viral inactivation within a continuous downstream system across multiple scales of operation. The reactors were designed to provide a minimum residence time of >60 min. The efficacy of this tubular reactor was tested with xenotropic murine leukemia virus (X-MuLV) through pulse injection experiments. It was determined that the minimum residence time of the small-scale reactor design, when operated at the target process flow rate, occurred between 63 and 67 min. Inactivation kinetics were compared between continuous operation and standard batch practices using three monoclonal antibodies. The quantification of the virus log reduction values (LRV) was similar between the two modes of operation and most of the acid-treated samples had virus concentrations below the limit of detection. However, residual infectivity was still present in the endpoint batch samples of two experiments while the continuous samples always remained below the limit of detection. This provides the foundation for leveraging a standard batch-based model to quantify the LRV for a CVI unit operation.

Rapid preparative purification of West Nile and Sindbis virus PCR products utilizing a microbore anion-exchange column.

Analysis and purification of specific PCR products from PCR reactions can be problematic due to several issues relating to amplification and low product yield. The use of HPLC as a preparative tool in PCR product analysis is common but has not replaced traditional electrophoretic techniques for purifying DNA to be used in subsequent experiments. Gel purification of PCR products can result in a net loss greater than 50% of the starting DNA amount. Thus, this method of recovery can become the limiting factor in the overall cloning protocol. This paper describes a simple and relatively inexpensive micro-preparative HPLC method to purify and analyze nM quantities of DNA. A microbore polyethyleneimine-based anion-exchange column fractionates PCR mixtures in less than 40 min with a recovery of the purified specific product as high as 80%, thus eliminating the need for gel purification. Using this method, nested PCR products from Sindbis virus differing by 18 bp in some cases and a 277 bp fragment from West Nile virus were resolved and quantified. This method differs from existing methodologies because separation is based on size and charge as well as the overall G + C content of the PCR product.