Virus reduction product

The virus reduction factor of an individual purification or removal—inactivation step is defined as the log10 of the ratio of the virus load in the pre-purification material divided by the virus load in the post-purification material. A clearance factor for each stage can be calculated and the overall clearance capacity of the production process assessed. Total virus reduction is calculated as the sum of individual log reduction factors. Individual manufacturing steps must possess fundamentally different mechanisms of virus removal or inactivation in order for values to be considered cumulative. Additionally, because viruses vary greatly with regard to inactivation or removal profiles, only data for the same model virus can be cumulative. 

Plasma-derived therapeutic proteins are parenteral biologies that are purified on an industrial scale. All biologies derived from human sources, such as plasma, carry the risk of viral contamination. Thus, in order to market a medicinal product derived from human plasma, manufacturers must assure the absence of specific viral contamination. Virus validation studies are performed to evaluate the capacity of a manufacturing process to remove viral contaminants. Virus clearance across three different terminal inactivation steps, low pH incubation of immunoglobulins (IgG), pasteurization of albumin, and freeze dry/dry heat treatment of plasma-derived products (Factor VIII and Protein G), is discussed in this article. The data show that, like all other upstream virus reduction steps, the methods used for terminal inactivation are process and product dependent, and that the reduction factors for an individual step may be overestimated or underestimated due to inherent limitations or inadequate designs of viral validation studies. 

The impact of formulation on virus reduction and product recovery (e.g., potency) was evaluated during terminal freeze dry/dry heat treatment studies with an unlicensed Fraction I derived product, that will be designated Protein G. As shown in Fig. 10, the optimum formulation, that achieved >4 logio PPV inactivation and 80% product recovery, was one that contained 2% albumin, no NaCl, and <0.3%o moisture by the Karl Fischer coulometric method. In contrast, freeze dry/dry heat treatment of product formulated with no albumin, 150mM NaCl and low moisture resulted in approximately 3 logio PPV reduction and 35% product recovery. Thus, minor changes in formulations such as the addition of 2% albumin may impact virus reduction and product recovery during a freeze dry/dry heat treatment. 

Fig. 10 Effect of formulation on virus reduction and product recovery. Protein G was formulated in buffer containing albumin (0% or 2%) and NaCI (OmM or 150mM). Virus was added and a sample was immediately removed for virus titration (Pre-FD). The virus-spiked material was aseptically filled into vials, freeze dried, and then heated at 80°C, 72 hr. Mock-spiked Protein G was processed like the virus-spiked samples but was used to measure product recovery (potency).

Screening and selection of the source plasma will only avoid contamination by known pathogens. The protein purification steps and specific virus reduction methods used in production processes, however, will inactivate and/or remove both known and unknown viruses. Terminal virus inactivation treatments are applied to product in final container and must balance virus inactivation with any modifications to protein immunogenicity, activity, and yield. While many upstream virus inactivation steps rely on chemical methods that involve the addition and subsequent removal of toxic agents (e.g., solvent/detergent), physical methods for virus inactivation, such as pH and heat, are used for terminal steps. 

Manufacturing processes have evolved dramatically over the last few years. In the late 1980s, 76 /o of hemophiliacs were HCV positive, ° and between 1979 and 1985, approximately 50 /o of hemophiliacs had acquired HIV from plasma-derived FVIII. Since then, however, most U.S.-licensed plasma derivatives have not transmitted HBV, HCV, or HIV as a result of improvements in donor screening and test methods, and the inclusion of effective upstream virus-reduction and terminal virus-inactivation steps in manufacturing processes. Residual risks of virus transmission from plasma-derived products are now largely associated with non-enveloped viruses. " Thus, the need for additional terminal or upstream virus inactivation/removal steps still exists, but the current challenge is to develop cost effective methods against physico-chemically resistant non-enveloped viruses, such as human parvovirus B19. 

The most recent advance in treating HIV infections has been to simultaneously attack the virus on a second front using a protease inhibitor. Recall from Section 27.10 that proteases are enzymes that catalyze the hydrolysis of proteins at specific points. When HIV uses a cell s DNA to synthesize its own proteins, the initial product is a long polypeptide that contains several different proteins joined together. To be useful, the individual proteins must be separated from the aggregate by protease-catalyzed hydrolysis of peptide bonds. Protease inhibitors prevent this hydrolysis and, in combination with reverse transcriptase inhibitors, slow the reproduction of HIV. Dramatic reductions in the viral load in HIV-infected patients have been achieved with this approach. 

Effluent pretreatment is necessary when RO is used as tertiary treatment in order to prevent membranes filters form being blocked or abraded. UF offers a powerful tool for the reduction of fouling potential of RO/NF membranes [57]. A typical pretreatment consist of a MF allowing the removal of the large suspended solids form the WWTP effluent followed by UF unit which removes thoroughly suspended solids, colloidal material, bacteria, viruses and organic compounds from the filtrated water. The UF product is sent to the RO unit where dissolved salts are removed. 

Contaminant-clearance validation studies are of special signibcance in biopharmaceutical manufacture. As discussed in Section 7.6.4, downstream processing must be capable of removing contaminants such as viruses, DNA and endotoxin from the product steam. Contaminant-clearance validation studies normally entail spiking the raw material (from which the product is to be purihed) 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 purihcation step, and the contaminant reduction factor for the overall process. 

Apart from reduced yield, down-stream processing can cause minor or even bigger modifications in the structure of the biomolecule. Often, these modifications do not affect the activity of the product, but may change its antigenicity. Along with virus safety, the reduction of such risks is a main objective in the down-stream processing of such biomolecules. Chromatographic purification,... 

The chemical found to be the most effective to date for the control of the mealy bugs is dimefox (bisdimethylaminofluorophosphine oxide). This is the active ingredient of the commercial product Hanane produced by Pest Control, Ltd., London, which holds a contract for investigation and control of swollen shoot viruses. Hanane is applied to the soil around the roots of the trees and a very significant reduction in the mealy bug population has been obtained by this method. 

Exclusion of extraneous agents from the product. Studies including, for example, viruses with relevant physico-chemical features are undertaken, and a reduction capacity for such contaminants at each relevant stage of purification is established. 

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