In General

Inactivation of contaminating virus and removal of this virus is an important concern in the medical industry for the production of recombinant protein. (Zou, WO/2008/036899)

Low Ph Hold: A Protein A/Virus invactivation step: is included in almost all downstream monoclonal antibody processes. This step is placed after the Protein A step because the product at this stage already is in low pH. The virus inactivation is performed during a time somewhere between 30-60 minutes, at a pH of 3.6-3.8 (IPC0m000183319D, anonymous publication dated 3/18/2009 from the IP.com electronic publication website).

Filtration: 

–Nanometer filtration: At present, sized baed nanometer filtration technology is perhaps the most robust viral removal unit operation. Based on the sizes of the mammalian virues commonly occurred in mAb production, these nanometer filters used can be divided into two classes: 50 and 20 nm pore sizes although there are also filters with a nominal pore size of 15 and 35 nm. Large pore filters are efficient in retaining large particle viruses like x-MuLV and pseudorabies virus (PRV). On the other hand, filters with a small pore sizes (20 nm) remove both large viruses and small virus particles such as MMV and respiratory enteric orphan III. (Zhou, Biotechnol. J. 2008, 3, 1185-1200)

–Aeptic filtration: For more than 30 years, aseptic filtraiton with 0.2 um membranes has remained virtually unchanged becasue of the quality, predictability and flexibility of the technology. Producign mAb at high concentraiton (up to 150 g/L) showed that aseptic filtraiton can be challenging since a limited volume of antibody therapeutics at such concentrations will readily clog most 0.2 um membranes. However, dual layer filters has been shown to correct this problem. (Zhou, Biotechnol. J. 2008, 3, 1185-1200)

–Membrane Chromatography (MC) or memrane adsorber (MA):

Q and other charged MA devices have been in development for chromatography purposes for more than 15 years. Some limitations for large production scale are distorted or poor inlet flow distribution, nonidentical membrane proe size distribution, uneven membrane thickness and lower binding capacity. The first 3 weaknesses can be improved to some degree when multiple layer configuations are ued. This configuation for the Q membrane is used in viral vaccine production. Q AEX adsorber devices have also been used for endotoxin removal at process scale. Low binding capacity is still a major disadvantage in bind/elute mode but in FT mode, the limitations are no longer considered as major issues, particularly when FT-MA is used as the polishing step for antibody purification. FT-MA chmatography is useful to remove impurities below 1% concentraiton including viruses. (Zhou, Biotechnol. J. 2008, 3, 1185-1200)

Purification Schemes which include Protein A chromatography and Virus Inactivation/Reduction

Low “future of antibody purificaition” J. Chromatography B, 848 (2007) 48-63)) teaches that although many variations exist, the vast majority of antibody purification process follow a process flow of 1. cell culture harvest, 2. protein A chromatogrpahy, 3. viral inactivation, 4. polishing steps, 5, viral filtration and 6. UF/DF.

Cell culture-Depth Filtration (DF) – Protein A-Viral inactivation-Polishing-Viral filtration-UF/DF: 

Shukla, (J. Chromatogr. B 848 (2007) 28-39) disclose an antibody purification scheme (1) cell culture harvest  as by centrifugation followed by debth filtration, (2) Protein A chromatography (3) low pH viral inactivation, (3) two polishing chromatographic steps chosen from CEX, AEX, HIC and hydroxyapatite (4) viral filtration (5) UF/DF.

(Zou, WO/2008/036899) discloses methods for remvoing viral contaminatns from a therpaeutic protein solution which includes the step of passing the solution through a protein A chromatography column before being passed through a depth filter at a pH within 1 pH unit of the isoelectric point of the virus. In one embodiment the pH is within the range of 4-6 and the virus is less than about 30 nm. 

Protein A – virus inactivation -AEX – CEX – UF/DF

Large scale purification of mAb typically consists of four steps: protein A chromatography, virus-inactivation – AEX (Q sepharose),  – CEX (CM sepharose) and UF/DF. The affinity column is generally the first step after harvest and clarification. In order to inactivate potential viruses, the eluate is typically subjected to a virus inactivaiton process followed by an AEX to reduce host cell proteins, DNA, protein A and potential viruses. Next, a CEX is typically used to further reduce the residual amounts of host cell proteins and antibody aggregates. A hydrophobic interaction column step may be used in place of the CEX. The next step is likely to be a nanofiltraiton virus removal step, using a DV20 or Planova filter. Finally, the pool is then diafiltered and further concentration (Sabbadini, WO2008/070344A2). 

Ishihara (J. Chromatography A, 1176 (2007) 149-156) disclose purification of antibodies which includes 3 sequential chromatography steps (Protein A/anion/cation and also includes a low pH virus inactivaiton step after the Protein A step.

Protein A – Viral Inactivaiton -CEX – DF

Loetscher (WO2007/068429) discloses an antibody purificaiton schemed with the steps of Protein A chromatography – Viral inactivation – CEX – flow through chromatography using Q-Sepharose FF – Diafiltration – 

Protein A – Viral Inactivation – CEX – AEX – UF/DF

Yao (US13/266860) also discloses a scheme of (1) protein A chromatography, (2) inactivating virus (3) CEX (4) AEX, (5) filtering virus particles (6)UF/DF and (7) fine filtering. 

Protein A – viral inactivation – HIC – UF/DF -AEX – NF

Ransohoff (US2013/0260419) teaches a continous process for the purification of antibody which includes clarification, Protein A, low pH viral inactivation, HIC, UF/DF, AEX, NF.

–Protein A – CEX – HIC – Viral inactivation – DF

Shadle (US 5,429,746) discloses a method for separating IgG monomers from aggregates by contacting the mixture with a hydrophobic interaction chromatographic support and selectively eluting the monomer from the support.  3 purification steps are involved (Protein A affinity, cation exchange, and hydrophobic interaction chromatograph), 2 viral inactivation steps, and a diafiltration step to exchange the product into a final buffer of choice.

Conditions for Viral Inactivation

Low pH

Brorson (Biotechnology and Bioengineering, 82(3), 2003) discloses that a 30 minute incubation time of ≥30 min, at room temperature in acetate or citrate is a robust viral inactivation step. 

Detection of Viruses

Xenotropic murine leukemia virus (X-MuLV) is often used as a model virus for clearance studies. Traditionally, cell based infectivity assay has been the standard virus quantification method. However, a real time quantitative PCR (Q-PCR) method has been developed for X-MuLV detection/quantificaiton. It is about 100 fold more sensitive than the cell based infectivity assay. (Shi, Biotechnology and Bioengineering, 87: 884-96, 2004).