Virus reduction process steps

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 overall virus reduction capacity of a manufacturing process is the sum of the individual reduction factors, and should be greater than the potential virus load in the starting material. At least one step in the process should clear significant levels of infectious virus so that the overall clearance is not made up of individual small, and possibly negligible, reductions. 

Virus reduction steps may be placed upstream or at the very end of the manufacturing process. 

The virus reduction studies of the three process steps discussed here were performed with HFV-l, Bovine viral diarrhea virus (BVDV), Pseudorabies virus (PRV), Reovirus type 3 (Reo), Hepatitis A virus (HAV), and Porcine parvovirus (PPV). HIV-1 was included as a relevant enveloped virus, while BVDV and PRV were tested as specific model viruses for HCV and HBV, respectively (Table 1). Reo was chosen as a non-specific model non-enveloped virus, HAV was included as a relevant virus and PPV was used as a surrogate for human parvovirus B19. All viruses were propagated using standard cell culture conditions. " The appropriate cell lines were infected, at a low multiplicity of infection, and incubated until 4-1- cytopathic effects were observed. The infected cells were frozen and thawed three times to release virus, centrifuged at low speed to remove cell debris and the clarified supernatants were removed for use as virus spikes. 

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. 

Certain process steps may be easier to model than others and duplicating the freeze dry/dry heat step at small scale is very difficult. The conclusions drawn from virus clearance studies are reliable only when the appropriateness of the small-scale models can be demonstrated. During the freeze dry/dry heat step of Koate -DVI, virus reduction was dependent on moisture levels so even the formulation of stoppers, which could impact the absorption of water during autoclaving and its release to freeze dried material, must be considered and tested. 

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 models can be scaled to various sizes to fit the needs of the experiment. For example, a very small-scale nanofiltration system, such as a Planova P-15 hollow-fiber cartridge with O.OOl-m surface area (Asahi Kasei Corporation, Japan), can be used to study virus retention capabilities of a virus reduction step in a biological manufacturing process, whereas a scaled-up version of the same system with a surface area of 0.01 m provides an excellent way to study the nanofiltration process variables. In a nanofiltration validation study, a feed sample is typically spiked with a known quantity of a model virus. The mixture is filtered under the expected process... 

Specific virus reduction values achieved with each individual viral removal/inactiva-tion step are cumulative, so that the final viral reduction value attained reflects multiple removal and/or inactivation procedures occurring throughout the process. In summary, the processing of rAHF-PFM is associated with a significant viral reduction capacity for both lipid-enveloped and non-lipid-enveloped viruses and provides additional assurances of reliability with respect to pathogen safety. 

Steps to be Validated for Viral Clearance. Process validation for viral clearance is conducted only on robust steps that can (1) be scaled down accurately and (2) reproducibly and effectively remove and/or inactivate a wide variety of potential viral contaminants under a wide variety of process conditions [5,41]. The number of steps selected for validation depends on estimated viral clearance effectiveness based on historical data and target clearance values [5]. The FDA demands at least two different steps for virus reduction to guarantee safety and efficacy [7]. Potentially only two steps are required for antibody processes that use serum-free medium, but additional steps might be required if viral contamination risk is increased by using serum-containing medium [7]. Due to the use of live viruses to perform clearance studies, this work usually is outsourced to reduce cross-contamination issues [14]. 

In summary, it is worth noting that the selection of an inactivation procedure will depend on the type/complexity of the material to be obtained (for instance, enveloped viruses are more difficult to inactivate than those non enveloped), as well as its compatibility with other processing steps. If the final product is not consumed during the processing steps, spontaneous inactivation due to mechanical stress or other phenomena could also be observed (for example, filtration processes involving change of phase - liquid to solid - may be traumatic for the vims particle). In this situation, an individual inactivation step would not be necessary, resulting in cost reductions. 

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. 

Virantmycin is a tetrahydroquinoline alkaloid that has inhibitory activity against DNA and RNA viruses. A total synthesis of virantmycin making use of a key type II aziridine has elucidated the absolute stereochemistry at C-2 and C-3 <1996T10609>. An intramolecular photocyclization of an azide onto a Z-alkene produces type II aziridine 351 in excellent yield. A three-step reduction/selective reoxidation procees yields key aziridine alcohol 352 in 76% overall yield (Scheme 71). The alcohol is methylated and the ester hydrolyzed without harm to the azirdine. A TFA-induced ring opening of the aziridine by chloride provides the natural product virantmycin in good yield. This entire process was also carried out with the -alkene to produce /)(-virantmycin, thus proving the stereochemistry at C-2 and C-3. 

Patel et al (1994) employed a combined process of coagulation and MF to avoid a disinfection posttreatment. The coagulation step was used to eliminate phosphorus, arsenic, and viruses, to avoid fouling, decrease particle accumulation on the membrane surface, and improve backflush characteristics. MF pilot plant studies in constant permeate flux mode showed that turbidity, particles, and faecal coliforms could be removed, but TOC removal was unreliable. Crossflow MF showed no difference to dead-end filtration, and both methods were similar to or better than sand filtration. Results with coagulation and MF improved phosphorous and turbidity removal, but the process was not optimised. The treatment lead to a reduction of chlorine demand in the product water. 

The viral clearance reduction factor, the common logarithm of the ratio between the total virus loads before and after clearance, is established for viruses known to contaminate the production process [50]. Individual step clearances are combined to obtain the total clearance reduction factor. This reduction factor is used in combination with an assessment of step robustness to classify the step as effective (>4 reduction factor and unaffected by small changes in process variables), moderately effective (4 > reduction factor > 1), or ineffective (<1 reduction factor) with respect to virus clearance [50]. Clearance factors are usually multiplied if the mechanism is different for two separate steps and sometimes are added if the mechanism is same [51]. In other cases, if two independent steps have similar mechanisms of clearance, only one step is included in the summation because virus particles removed via that mechanism would only be expected to be removed in the first step [3, 5]. A total clearance of 12-15 logs is desired for lipid-enveloped viruses and fewer logs for nonenveloped viruses (e.g., polio) [30]. 

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