Avoiding viral contamination in biotechnological and pharmaceutical processes

Almost all biological substances of value are produced from materials of animal or human origin, such as cultured cells, organs, medium components, or blood. Thus, the danger exists that manufactured biopharmaceutical products bear a risk of viral contamination. This has been shown by numerous epidemiological investigations, e.g., for classical serum hepatitis (hepatitis B virus, HBV) and for other infections by blood contamination. With the recent appearance of disease-causing human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections related to transfusions, the safety of pharmaceutical and biotechnological drugs with respect to viral contamination is now more important than ever.

Targeted selection of the starting material, as well as screening procedures such as investigation for viral markers (e.g., antibodies), are not sufficient to guarantee the safety of the products and to exclude the possibility of viral transfer. There is a need for effective elimination/inactivation methods integrated into the production process or designed as additional barriers. Inactivation of viruses is achieved by the destruction of their lipid or protein covers, making them unrecognizable to the target cell, or by destroying the viral nucleic acid, and therefore the biological activity necessary for replication. Acute concern for virus transmission from biological products derived from natural sources has led to the development of various viral clearance methods.

Viral clearance

Virus removal and inactivation strongly depend upon the specific application. Different product fluids possibly containing various viruses require viral clearance procedures that are specifically fitted to the characteristics of the system. Therefore, it is important to investigate several different clearance techniques. For each system, the narrow window of minimum product damage and maximum viral clearance has to be determined. Through laboratory experiments, process parameters will be defined that will then be used in scale-up for industrial processes. It is necessary to make a careful evaluation of the kinetics of inactivation, as well as to establish that all infectious particles are equally exposed and sensitive to the selected method.

In the past, ultrafiltration has been used successfully to remove bacteria from liquids. This method has also been used for large viruses. Virus removal by membrane filtration is relatively simple: It is fast, the product is only exposed to small shear forces, and no chemical modification takes place. Many medium components used in biotechnological processes are heat labile, and therefore may only be sterile filtered.

Little consistent data exist for virus particles <50 nm because removal efficacy strongly depends on fluid and processing conditions. Realistically, viruses the size of polio- or parvoviruses (20-25 nm) cannot be separated from similarly sized proteins by such filters, since in this size range larger proteins are already held back. Yet Maerz et al.¹ succeeded in separating nonenveloped virus-like particles (VLPs), similar in size and shape to polioviruses, from immunoglobulin M (IgM, 970 kDa) using a hollow fiber filter with 15 nm pores (Planova-15, Asahi Chemical Industries). Success in solving this separation problem may be attributed to the different static properties of viruses and antibodies. Antibodies are very flexible and can slip through the narrow membrane pores at slow flow rates, whereas small, non-enveloped viruses must be considered to be rigid molecules that are restrained by the membrane.

Chemical inactivation, such as mild pepsin processing at low pH values, or detergents in combination with solvents (e.g., TNBP/Tween 80) lead to disruption of enveloped viruses. Nonenveloped viruses like hepatitis A virus (HAV) or parvoviruses are not effectively inactivated by these methods.

Thermal inactivation methods have improved viral safety for many years, especially in the case of plasma products. Nevertheless, the required exposure times may yield conditions too rigorous for some products. As a result, biological activity is destroyed. Furthermore, viruses exhibit a wide range of sensitivity. Figure 1 shows the time course of inactivation of an enveloped (bovine herpesvirus, BHV) and a nonenveloped virus (HAV) by pasteurization of albumin at 60°C for 10 hours². The kinetics of inactivation are not easily established. Product damage can be reduced considerably by the use of stabilizers and sodium citrate, saccharose, or amino acids such as glycine. Alternatively, short time-heating procedures are available in which materials in continuous flow are heated up to a defined reaction temperature as rapidly as possible, held at this temperature for a short retention or reaction time, and afterward briefly cooled down with heat exchangers to a desired final temperature. The short heating time requires high-performance heat-transfer systems with a large ratio of transfer surface to volume. Often, fouling problems, coupled with the special demands on biotechnological systems of cleaning and validating requirements, determine the construction of such apparatus.

Figure 1

Figure 1. Inactivation of hepatitis A virus (HAV) and bovine herpesvirus (BHV) by pasteurization of albumin with 60°C for 10 h. N₀: Total amount of virus in start material; N: Total amount of virus recovered in product.

Processing of biological fluids with short-wavelength ultraviolet light (UVC) is not new. During World War II, large lots of plasma were collected and irradiated with UV. UV-treatment of blood derivatives is of special interest with respect to nonenveloped, heat-stable viruses. Chin³ and Marx⁴ have shown that the processing of plasma products with a dosage of about 1,000 J/m² leads to inactivation of infectious particles on the order of >6 logs in the case of the nonenveloped HAV and parvoviruses, pathogens that cannot be destroyed easily by other methods. However, short-wavelength UV-treatment can cause the formation of free radicals and other reactive oxygen species, thus limiting the treatment by damaging proteins and leading to aggregation and loss of biological activity. This can be minimized by the use of antioxidants/quenchers like the flavonoid Rutin⁴, or UV-lamps and filters modified to exclude the 185 nm wavelength.

A degassing of the biological fluid is also recommended in order to prevent the formation of ozone from dissolved oxygen. UV-irradiation of a molecule results in the excitation of an electron from its ground state to an excited state, from which chemical reaction pathways become probable. It is thought that the nucleic acid, and therefore the viral genome, is damaged by photochemical modifications such as the formation of pyrimidine dimers, photohydrates, and other adducts. Accumulation of a certain critical number of "hits" will overcome repair processes and result in failure of the replication process.

Immuno affinity chromatography may seem to have a good chance of removing viruses with immobilized antibodies via interaction with virus surface antigens. However, only one specific virus can be detected and depleted per immune adsorbent. Additional problems can occur if the virus (e.g., HIV) changes its surface texture often. This problem can be avoided if antibodies interacting with the desired product are immobilized, rather than antivirus antibodies, resulting in the viruses exiting the column.

Less complex cleaning methods like ion exchange chromatography or hydrophobe interaction chromatography for purification of biological products and plasma valuables are standard. Through validation studies such as virus "spiking," it can be shown that such chromatographic purification steps, which are already process-integrated, can lead to the depletion of viruses, and are therefore suitable for their removal. With the ion exchangers DEAE and CM Sepharose (Amersham Pharmacia Biotech) used in a purification process for albumin and immunglobulin G from plasma, clearance factors of >6 logs for enveloped (BHV, HIV) as well as nonenveloped viruses (encephalomyocarditis virus, EMC) can be demonstrated², without damaging sensitive proteins such as blood clotting factors.

The heart of a γ-irradiation plant is a radionuclide disintegrating with a specific half-life (e.g., cobalt-60 or cesium-137). Upon disintegration, energy in the form of electrons (b-radiation with small range) and γ-rays is transferred. Gamma-rays have a relatively small dose rate, necessitating long radiation times, but have a much higher penetration capacity through material as electrons. As temperatures in the product are kept to 35°C, inactivation of biological activity is minimized.

Gamma-rays are used successfully for virus inactivation of fetal calf serum and powdered trypsin (JRH Biosciences). Bovine viral diarrhea virus (BVDV) in serum can be depleted on the order of 6 logs, and porcine parvovirus (PPV) in trypsin can be depleted by 3 logs with minimum product damage. One exception to this method is application during plasma sterilization, since the protein products, such as coagulation factors, showed no acceptable yield of biological activity⁵. Because of the high capital expenditures and safety conditions, such irradiation devices are still rarely used in commercial viral clearance.

Validation of viral clearance

There are two prerequisites that need to be met when verifying the performance of virus elimination or inactivation methods in pharmaceutical processes. First, the functional and structural integrity of the product, most often in liquid form, has to be proven. Proteins are especially sensitive, and damage through inactivation techniques can lead to the loss of biological function. Second, the efficacy should be evaluated by challenge experiments in which a specific lot of appropriate, relevant virus is added to the starting material or to different fractions obtained during the various process stages, a technique known as "spiking."

Nevertheless, suitable proof methods for the infectivity of HBV, HCV, and many other pathogenic viruses are missing. For this reason, testing viruses similar to the relevant viruses in their properties, as well as viruses with a large bandwidth of biophysical and structural qualities, are used. Table 1 shows a survey of different model viruses. During culturing of the model viruses, high titers should be achieved in order to allow for the proof of high inactivation rates. Also desirable is a low infection risk for the laboratory staff responsible for the work, as well as dependable proof procedures for determination of the inactivation kinetics. Infectivity of a sample is proved by cytopathogenicity in cell cultures or with the aid of antivirus antibodies, where the titer is calculated using the methods of Spearman⁶ and Kaerber⁷.

Table 1 Properties of different viruses selected for validation studies.

Excellent clinical safety records have been demonstrated for the drug aprotinin (Trasylol, Bayer AG). Following downscale experiments, a full validation study of the manufacturing process of the protease inhibitor from cattle lungs was designed^8,9. Five model viruses were selected, from which inactivation data for two common bovine pathogens (bovine parvovirus and bovine viral diarrhea virus) are presented. Several process steps were selected for the virus validation study to determine the viral clearance potential. Clearance factors were calculated as the difference of virus concentration in the spiked samples and the output samples after performing the respective purification step.

The potential for virus elimination of different steps is shown in Figure 2. The overall clearance factor for BPV over the process is on the order of >11 logs, with >18 logs for BVDV clearance. Therefore, well-designed purification steps, such as extractions, chromatographic procedures, or filtration steps, can be considered as effective virus removal steps, provided that they are performed under appropriately controlled conditions.

Figure 2

Figure 2. "Spiking" scheme for virus and scrapie (BSE) clearance study in the Trasylol purification process on indicator cell lines (viruses) or in mice (prions). Samples were taken at specific purification steps (marked in red) to determine the total amount of virus/scrapie (BSE) agent before and after treatment. Samples were assayed for the presence of virus/scrapie (BSE) agent by titration in serial dilutions to end point by quantal TCID₅₀ assay on appropriate indicator cell lines. (+): clearance observed; (-): no clearance observed. Numbers in the last row represent total clearance factors over the process (log₁₀). BPV: Bovine parvovirus [nonenveloped]; BVDV: Bovine viral diarrhea virus [enveloped]; Prion: Scrapie (BSE) agent.

It is worth mentioning that inactivation methods, such as pasteurization, frequently follow a biphasic curve in which a rapid initial phase is followed by a slower phase. For this reason, it is possible that viruses escaping a first inactivation step may be more resistant to subsequent inactivation steps.

The problem of variant Creutzfeldt-Jakob disease

With the emergence of spongiform encephalopathy in sheep (scrapie) followed recently by bovine spongiform encephalopathy (BSE) in cattle, questions have arisen regarding the safety of biological products originating from or manufactured with sera (e.g., fetal calf serum) or organ tissues of such animals. It seems very likely that infectious agents from bovine origin may cause a similar disease, variant Creutzfeldt-Jakob disease (vCJD), in humans.

Although it is still a matter of debate whether infectivity is caused by a slow virus or virus-like particle, a nucleic acid, or solely by a protein complex called a prion (the protein-only hypothesis is now widely accepted), the example of the Trasylol manufacturing process shows that it is possible to demonstrate practical exclusion of risk from such product contamination by careful selection and validation. Infectious spikes of scrapie agent have served to validate single independent process steps with infectivity proofs in a suitable mouse model. The process steps—methanol extraction, two ion exchange chromatography steps, and membrane filtration—resulted in a total potential for depletion of the infectious titer of less than 18 logs.

Conclusions

Viral safety in biopharmaceuticals is a subject of continued concern. Currently, all biotechnologically produced pharmaceutical products, including the products from plasma fractionation, are subject to control by regulatory authorities. In addition to the effectiveness of the products, manufacturers also have to demonstrate their safety. Quantitative data concerning respective manufacturing steps or defined antiviral methods are obligatory. The necessity of more than one virus clearance step in the same production process is indispensable today, since not only known but also unknown, unsuspected, and harmful viruses with different biophysical and structural qualities have to be inactivated or removed. Even if technologies available for the elimination of small, non-enveloped viruses for many biofluids are limited in comparison to the control of large, enveloped viruses, there are some promising new approaches using ultra short-time high-temperature or UV-irradiation devices.