Virus purification and

The unique features of tangential-flow filtration include the ability to remove cells and cell debris from the growth medium, which contains the product of interest, to concentrate the product of interest and to fractionate solutes of different size. These features have lead to numerous applications of tangential flow filtration in the purification of protein products. These same features could be exploited both for virus purification and validation of vims clearance as described in the following sections. 

Several plant vims coat proteins, including those of TMV, CPMV, AlMV and Tomato bushy stunt vims (TBSV), have been used to produce and deliver antigenic determinants from a variety of viral and bacterial pathogens. These data have been summarized in numerous publications and several reviews [12,13]. The ease of virus purification coupled with enhanced peptide immunogenicity when fused to carrier molecules makes this approach very attractive for vaccine development. 

Contaminant-clearance validation studies are of special significance in biopharmaceutical manufacture. As discussed in the previous section, downstream processing must be capable of removing contaminants such as viruses, DNA and endotoxin from the product stream. Contaminant-clearance validation studies normally entails spiking the raw material (from which the product is to be purified) with a known level of the chosen contaminant, and subjecting the contaminated material to the complete downstream processing protocol. This allows determination of the level of clearance of the contaminant achieved after each purification step, and the contaminant reduction factor for the overall process. 

Canaan-Haden, L., Ayala, M., Femandez-de-Cossio, M.E., Pedroso, I., Rodes, L., Govilondo, J.V. (1995). Purification and application a single-chain Fv antibody fragment specific to hepatitis virus surface antigen. Biotechniques, 19(4), 606-608. 

Smith JS, Roth MJ. Purification and characterization of an active human immunodeficiency virus type 1 RNase H domain. J Virol 1993 67 4037-4049. 

Bello, L.J. Bessman, M.J. The enzymology of virus-infected bacteria, IV. Purification and properties of the deoxynucleotide kinase induced by bacteriophage T2. J. Biol. Chem., 238, 1777-1787 (1963)... 

Antibody capture of viruses can be used as a preparatory step in nucleic acid amplification techniques. Immunocapture of virus particles can be used to streamline and/or optimize the concentration, purification and specificity requirements of polymerase chain reaction assays. 

K., Summerford, C., Samulski, R. J. and Muzyczka, N. (1999). Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 6, 973-985. 

Kaludov, N. et al. (2003). Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 4. Virology 306, 1-6. 

Assays for media components and purification reagents are generally specific to the particular item used and will not be discussed further here. Assays for endotoxin, live virus, mycoplasma, and live microbes are relatively standard (USP, CFR, or other regulatory procedures) and likewise will not be discussed in detail. The determination of trace levels of host cell proteins in therapeutic proteins can be a formidable task, because most commonly... 

Demi L, Schirmbeck R, Reimann J, Wolf H, Wagner R (1999), Purification and characterization of hepatitis B virus surface antigen particles produced in Drosophila Schneider-2 cells, J. Virol. Methods 79 205-217. 

Kosukegawa A, Arisaka F, Takayama M, Yajima H, Kaidow A, Handa H (1996), Purification and characterization of virus-like particles and pentamers produced by the expression of SV40 capsid proteins in insect cells, Biochim. Biophys. Acta 1290 37-45. 

In the early days of biotechnology product development, the focus was on quality issues [4] or process-related impurities.The concerns at that time were for carryover of other cellular proteins and DNA and for contamination with endotoxins, chemicals, and viruses. Of course, these concerns still exist, but methods for purification and assays for evaluation of clearance have alleviated the need for the safety assessment scientist to focus on contaminants instead they are now asked to focus on the pharmacological activity of the molecules. An ICH guidance (Q6B Specifications Test Procedures and Acceptance Criteria for Biotechnological/Biological Products) addresses the specific issues related to the manufacturing process [6], Other product-related issues such as impurities do need to be considered by the safety assessment scientist, for... 

Implicit to our recommendation for IMBPs is that viral expression system derived DNA or excipient derived contaminant carcinogens are no longer an issue. Although, for example, host cell DNA can be a risk factor for neoplasia [3], the current stringent biochemical characterization, purification, and virus inactivation steps taken with all well-characterized biopharmaceuticals adequately mitigate any potential risk from these sources. 

Mann, G. J., Gr%oslund, A., Ochiai, E., Ingemarson, R., and Thelander, L., 1991, Purification and characterization of recombinant mouse and herpes simplex virus ribonucleotide reductase R2 subunit. Biochemistry 30 1939nl947. 

Bonami, J. R., Trumper, B., Mari, J., Brehelin, M., and Lightner, D. V. (1990). Purification and characterisation of the infectious hypodermal and haematopoietic necrosis virus ofpenaeid shrimps. J. Gen. Virol. 71, 2657-2664. 

Vol. XXIX [15a]. Purification and Detection of Reverse Transcriptase in Viruses and Cells. D. L. Kacian and S. Spiegelman. 

---