Enveloped virus inactivation by caprylate: a robust alternative to solvent-detergent treatment in plasma derived intermediates.

Solvent-detergent treatment, although used routinely in plasma product processing to inactivate enveloped viruses, substantially reduces product yield from the human plasma resource. To improve yields in plasma product manufacturing, a new viral reduction process has been developed using the fatty acid caprylate. As licensure of plasma products warrants thorough evaluation of pathogen reduction capabilities, the present study examined susceptibility of enveloped viruses to inactivation by caprylate in protein solutions with varied pH and temperature. In the immunoglobin-rich solutions from Cohn Fraction II+III, human immunodeficiency virus, Type-1, bovine viral diarrhea virus (BVDV), and pseudorabies virus were inactivated by caprylate concentrations of >/=9 mM, >/=12 mM, and >/=9 mM, respectively. Compared to solvent-detergent treatment, BVDV inactivation in Fraction II+III solution was significantly faster (20-60 fold) using 16 mM caprylate. Caprylate-mediated inactivation of BVDV was not noticeably affected by temperature within the range chosen manufacturing the immunoglobulin product. In Fraction II+III solutions, IgG solubility was unaffected by 相似文献   

辛酸盐灭活静注人免疫球蛋白中脂包膜病毒效果验证的研究

选用不同核酸类型的脂包膜病毒,其中RNA病毒为水疱性口炎病毒(VSV),DNA病毒为伪狂犬病毒(PRV),将两种指示病毒分别用于验证一定浓度的辛酸盐对某一厂家生产的人血静脉注射用丙种球蛋白(IVIG)的病毒灭活效果。结果表明,液体IVIG在辛酸钠(0.7±0.2mmol/g蛋白)、pH(5.1±0.1)、29.5~30.5℃,孵放90min可灭活VSV和PRV,两种指示病毒的灭活效果分别为≥4.00~4.12和≥5.25~5.75log TCID50/0.1ml。因此,辛酸盐是一种安全、有效、快速的灭活脂包膜病毒的灭活剂。     

Virus inactivation by protein denaturants used in affinity chromatography

Virus inactivation by a number of protein denaturants commonly used in gel affinity chromatography for protein elution and gel recycling has been investigated. The enveloped viruses Sindbis, herpes simplex-1 and vaccinia, and the non-enveloped virus polio-1 were effectively inactivated by 0.5 M sodium hydroxide, 6 M guanidinium thiocyanate, 8 M urea and 70% ethanol. However, pH 2.6, 3 M sodium thiocyanate, 6 M guanidinium chloride and 20% ethanol, while effectively inactivating the enveloped viruses, did not inactivate polio-1. These studies demonstrate that protein denaturants are generally effective for virus inactivation but with the limitation that only some may inactivate non-enveloped viruses. The use of protein denaturants, together with virus reduction steps in the manufacturing process should ensure that viral cross contamination between manufacturing batches of therapeutic biological products is prevented and the safety of the product ensured.     

Characterization of ionic strength for X-MuLV inactivation by low pH treatment for monoclonal antibody purification

In the production of monoclonal antibodies (mAbs) intended for use in humans, it is a global regulatory requirement that the manufacturing process includes unit operations that are proven to inactivate or remove adventitious agents to ensure viral safety. Viral inactivation by low pH hold (LPH) is typically used to ensure this viral safety in the purification process of mAbs and other biotherapeutics derived from mammalian cell lines. To ascertain the effectiveness of the LPH step, viral clearance studies have evaluated LPH under worst-case conditions of pH above the manufacturing set point and hold duration at or below the manufacturing minimum. Highly acidic conditions (i.e., pH < 3.60) provide robust and effective enveloped virus inactivation but may lead to reduced product quality of the therapeutic protein. However, when viral inactivation is operated above pH 3.60 to ensure product stability, effective (>4 log₁₀ reduction factor) viral inactivation may not be observed under these worst-case pH conditions in viral clearance studies. A multivariate design of experiments was conducted to further characterize the operating space for low pH viral inactivation of a model retrovirus, xenotropic murine leukemia virus (X-MuLV). The statistically designed experiment evaluated the effect of mAb isotype, pH, temperature, acid titrant, sodium chloride (NaCl) concentration, virus spike timing, and post-spike filtration on X-MuLV inactivation. Data from the characterization study were used to generate predictive models to identify conditions that reliably achieve effective viral inactivation at pH ≥ 3.60. Results of the study demonstrated that NaCl concentration has the greatest effect on virus inactivation in the range studied, and pH has a large effect when the load material has no additional NaCl. Overall, robust and effective inactivation of X-MuLV at pH 3.65–3.80 can be achieved by manipulating either the pH or the NaCl concentration of the load material. This study contributes to the understanding of ionic strength as an influential parameter in low pH viral inactivation studies.     

Arginine-enveloped virus inactivation and potential mechanisms

Arginine synergistically inactivates enveloped viruses at a pH or temperature that does little harm to proteins, making it a desired process for therapeutic protein manufacturing. However, the mechanisms and optimal conditions for inactivation are not fully understood, and therefore, arginine viral inactivation is not used industrially. Optimal solution conditions for arginine viral inactivation found in the literature are high arginine concentrations (0.7–1 M), a time of 60 min, and a synergistic factor of high temperature (≥40°C), low pH (≤pH 4), or Tris buffer (5 mM). However, at optimal conditions full inactivation does not occur over all enveloped viruses. Enveloped viruses that are resistant to arginine often have increased protein stability or membrane stabilizing matrix proteins. Since arginine can interact with both proteins and lipids, interaction with either entity may be key to understanding the inactivation mechanism. Here, we propose three hypotheses for the mechanisms of arginine induced inactivation. Hypothesis 1 describes arginine-induced viral inactivation through inhibition of vital protein function. Hypothesis 2 describes how arginine destabilizes the viral membrane. Hypothesis 3 describes arginine forming pores in the virus membrane, accompanied by further viral damage from the synergistic factor. Once the mechanisms of arginine viral inactivation are understood, further enhancement by the addition of functional groups, charges, or additives may allow the inactivation of all enveloped viruses in mild conditions.     

Partitioning and inactivation of viruses by the caprylic acid precipitation followed by a terminal pasteurization in the manufacturing process of horse immunoglobulins

Caprylic acid (octanoic acid), has been used for over 50 years as a stabilizer of human albumin during pasteurization. In addition caprylic acid is of great interest, by providing the advantage of purifying mammalian immunoglobulins and clearing viruses infectivity in a single step. Exploiting these two properties, we sequentially used the caprylic acid precipitation and the pasteurization to purify horse hyperimmune globulins used in the manufacturing of Sérocytol. To evaluate the effectiveness of the process for the removal/inactivation of viruses, spiking studies were carried out for each dedicated step. Bovine viral diarrhoea virus (BVDV), pseudorabies virus (PRV), encephalomyocarditis virus (EMCV) and minute virus of mice (MVM) were used for the virological validation. Our data show that the treatment with caprylic acid 5% (v/v) can effectively be used as well to purify or to ensure viral safety of immunoglobulins. Caprylic acid precipitation was very efficient in removing and/or inactivating enveloped viruses (PRV, BVDV) and moderately efficient against non-enveloped viruses (MVM, ECMV). However the combination with the pasteurization ensured an efficient protection against both enveloped and non-enveloped viruses. So that viruses surviving to the caprylic acid precipitation will be neutralized by pasteurization. Significant log reduction were achieved > or =9 log(10) for enveloped viruses and 4 log(10) for non-enveloped viruses, providing the evidence of a margin of viral safety achieved by our manufacturing process. Its a simple and non-expensive manufacturing process of immunoglobulins easily validated that we have adapted to a large production scale with a programmable operating system.     

Inactivation of lipid enveloped viruses by octanoic Acid treatment of immunoglobulin solution.

Inactivation of lipid enveloped viruses by treatment with octanoic acid has been investigated for three intravenous immunoglobulin preparations, using Human Immunodeficiency Virus, Bovine Viral Diarrhoea Virus, Sindbis Virus and Pseudorabies Virus as test viruses. At a concentration of 7.45 g octanoic acid per kg solution complete inactivation of lipid enveloped viruses to below detectable level (>5.36, >4.68, >6.25 and >5.55 log(10), respectively) was achieved within the first minutes of treatment. Octanoic acid treatment as described here, has been demonstrated as an effective and rapid virus inactivation procedure, which shows high robustness at the tested ranges of temperature, pH and protein content of the test material. However, pH must be considered as a critical parameter of treatment, as octanoic acid fails to inactivate lipid coated viruses at basic pH. At suitable conditions, e.g. pH<6.0 and a concentration of >3.7 g/kg, octanoic acid treatment gives reliable and highly effective inactivation of lipid enveloped viruses.     

Characterization of operating parameters for XMuLV inactivation by low pH treatment

To ensure the viral safety of protein therapeutics made in mammalian cells, purification processes include dedicated viral clearance steps to remove or inactivate adventitious and endogenous viruses. One such dedicated step is low pH treatment, a robust and effective method commonly used in monoclonal antibody production to inactivate enveloped viruses. To characterize the operating space for low pH viral inactivation, we performed a statistically designed experiment evaluating the effect of pH, temperature, hold duration, acid type, and buffer concentration on inactivation of the retrovirus model, XMuLV. An additional single factor experiment was performed to study the effect of protein concentration. These data were used to generate predictive models of inactivation at each time point studied, which can be used to identify conditions for robust and effective XMuLV inactivation. At pH 3.6, XMuLV inactivation was rapid, robust, and relatively unaffected by the other factors studied, providing support for this as a generic viral inactivation condition for products that can tolerate this low pH. At pH 3.7 and 3.8, other factors besides pH affected XMuLV inactivation. By understanding the impact of each factor on inactivation, the factors can be manipulated within the operating space to ensure effective inactivation while achieving desired product quality goals. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:89–97, 2016     

Enveloped virus inactivation using neutral arginine solutions and applications in therapeutic protein purification processes

For the manufacturing of recombinant protein therapeutics produced from mammalian cell culture, demonstrating the capacity of the purification process to effectively clear infectious viruses is a regulatory requirement. At least two process steps, using different mechanisms of virus removal and/or inactivation, should be validated in support of the regulatory approval process. For example, exposure of the product stream to low pH, detergents or solvent/detergent combinations is commonly incorporated in protein purification processes for the inactivation of lipid‐enveloped viruses. However, some proteins have limited stability at low pH or in the presence of the detergents, and alternative techniques for achieving the inactivation of enveloped viruses would be beneficial. We present here an alternative and novel approach for the rapid inactivation of enveloped viruses using pH‐neutral buffer solutions containing arginine. The implementation of this approach in a monoclonal antibody or Fc‐fusion protein purification process is described and illustrated with several different therapeutic proteins. The use of the neutral pH arginine solution was able to effectively inactivate two enveloped model viruses, with no measurable effect on the product quality of the investigated proteins. Thus, the use of pH‐neutral arginine containing buffer solutions provides an alternative means of virus inactivation where other forms of virus inactivation, such as low pH and/or solvent/detergent treatments are not possible or undesirable due to protein stability limitations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:108–112, 2014     

Virus study for continuous low pH viral inactivation inside a coiled flow inverter

Continuous processing for the production of monoclonal antibodies (mAb) gains more and more importance. Several solutions exist for all the necessary production steps, leading to the possibility to build fully continuous processes. Low pH viral inactivation is a part of the standard platform process for mAb production. Consequently, Klutz et al. introduced the coiled flow inverter (CFI) as a tool for continuous low pH viral inactivation. Besides theoretical calculations of viral reduction, no viral clearance study has been presented so far. In addition, the validation of continuous viral clearance is often neglected in the already existing studies for continuous processing. This study shows in detail the development and execution of a virus study for continuous low pH viral inactivation inside a CFI. The concept presented is also valid for adaptation to other continuous viral clearance steps. The development of this concept includes the technical rationale for an experimental setup, a valid spiking procedure, and finally a sampling method. The experimental results shown represent a viral study using xenotropic murine leukemia virus as a model virus. Two different protein A (ProtA) chromatography setups with varying pH levels were tested. In addition, one of these setups was tested against a batch experiment utilizing the same process material. The results show that sufficient low pH viral inactivation (decadic logarithm reduction value >4) was achieved in all experiments. Complete viral inactivation took place within the first 14.5 min for both continuous studies and the batch study, hence showing similar results. This study therefore represents a successful virus study concept and experiment for a continuous viral inactivation step. Moreover, it was shown that the transfer from batch results to the continuous process is possible. This is accomplished by the narrow residence time distribution of the CFI, showing how close the setup approaches the ideal plug flow and with that batch operation.     

Use of lipid solvents for viral inactivation in factor VIII concentrates

Model virus inactivation studies with lipid solvents were carried our in antihemophilic factor concentrates. The procoagulant activity obtained was >/=80% recovery with 20% amyl acetate-0.1% deoxycholate. A concurrent reduction of four logs of virus titer was obtained for model viruses provided the viral mass contained significant amounts (>/=20%) of lipid. From this preliminary study it appears that further investigations in animal models may be warranted to demonstrate the inactivation of hepatitis B virus, non-A-non-B virus, and AIDS virus with 20% amyl acetate-0.1% deoxycholate in antihemophilic factor concentrates.     

Lack of correlation between virus barosensitivity and the presence of a viral envelope during inactivation of human rotavirus, vesicular stomatitis virus, and avian metapneumovirus by high-pressure processing

High-pressure processing (HPP) is a nonthermal technology that has been shown to effectively inactivate a wide range of microorganisms. However, the effectiveness of HPP on inactivation of viruses is relatively less well understood. We systematically investigated the effects of intrinsic (pH) and processing (pressure, time, and temperature) parameters on the pressure inactivation of a nonenveloped virus (human rotavirus [HRV]) and two enveloped viruses (vesicular stomatitis virus [VSV] and avian metapneumovirus [aMPV]). We demonstrated that HPP can efficiently inactivate all tested viruses under optimal conditions, although the pressure susceptibilities and the roles of temperature and pH substantially varied among these viruses regardless of the presence of a viral envelope. We found that VSV was much more stable than most food-borne viruses, whereas aMPV was highly susceptible to HPP. When viruses were held for 2 min under 350 MPa at 4°C, 1.1-log, 3.9-log, and 5.0-log virus reductions were achieved for VSV, HRV, and aMPV, respectively. Both VSV and aMPV were more susceptible to HPP at higher temperature and lower pH. In contrast, HRV was more easily inactivated at higher pH, although temperature did not have a significant impact on inactivation. Furthermore, we demonstrated that the damage of virion structure by disruption of the viral envelope and/or capsid is the primary mechanism underlying HPP-induced viral inactivation. In addition, VSV glycoprotein remained antigenic although VSV was completely inactivated. Taken together, our findings suggest that HPP is a promising technology to eliminate viral contaminants in high-risk foods, water, and other fomites.     

Inactivation of viruses using novel protein A wash buffers

Low pH viral inactivation is typically performed in the eluate pool following the protein A capture step during the manufacturing of monoclonal antibodies and Fc‐fusion proteins. However, exposure to low pH has the potential to alter protein quality. To avoid these difficulties, novel wash buffers capable of inactivating viruses while antibodies or Fc‐fusion proteins were bound to protein A or mixed mode resins were developed. By equilibrating the column in high salt buffer (2 M ammonium sulfate or 3 M sodium chloride) after loading, the hydrophobic interactions between antibodies and protein A ligands were increased enough to prevent elution at pH 3. The ammonium sulfate was also found to cause binding of an antibody to a mixed mode cation exchange and a mixed mode anion exchange resin at pH values that caused elution in conventional cation and anion exchange resins (pH 3.5 for Capto Adhere and pH 8.0 for Capto MMC), indicating that retention was due to enhanced hydrophobic interactions. The potential of the 2 M ammonium sulfate pH 3 buffer, a 1 M arginine buffer, and a buffer containing the detergent LDAO to inactivate XMuLV virus when used as protein A wash buffers with a 1 hour contact time were studied. The high salt and detergent containing wash buffers provided about five logs of removal, determined using PCR, and complete combined removal and inactivation (> 6 logs), determined by measuring infectivity. The novel protein A washes could provide more rapid, automated viral inactivation steps with lower pool conductivities. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:406–413, 2015     

Optimization of non-detergent treatment for enveloped virus inactivation using the Taguchi design of experimental methodology (DOE)

Abstract

In mammalian cell culture technology, viral contamination is one of the main challenges; and, so far, various strategies have been taken to remove or inactivate viruses in the cell-line production process. The suitability and feasibility of each method are determined by different factors including effectiveness in target virus inactivation, maintaining recombinant protein stability, easiness—in terms of the process condition, cost-effectiveness, and eco-friendliness. In this research, Taguchi design-of-experiments (DOE) methodology was used to optimize a non-detergent viral inactivation method via considering four factors of temperature, time, pH, and alcohol concentration in an unbiased (orthogonal) fashion with low influence of nuisance factors. Herpes Simplex Virus-1 (HSV1) and Vero cell-line were used as models for enveloped viruses and cell-line, respectively. Examining the cytopathic effects (CPE) in different dilutions showed that pH (4), alcohol (15%), time (120?min), and temperature (25?°C) were the optimal points for viral inactivation. Evaluating the significance of each parameter in the HSV-1 inactivation using Taguchi and ANOVA analyses, the contributions of pH, alcohol, temperature and time were 56.5%, 19.2%, 12%, and 12%, respectively. Examining the impact of the optimal viral treatment condition on the stability of model recombinant protein-recombinant human erythropoietin, no destabilization was detected.     

Factors influencing virus inactivation and retention of platelet properties following treatment with aminomethyltrimethylpsoralen and ultraviolet A light.

A wide variety of viruses are inactivated by psoralen compounds in the presence of ultraviolet A light (UVA). Use of aminomethyltrimethylpsoralen (AMT) and UVA is being evaluated as a method to inactivate viruses that may be present in platelet suspensions prepared for transfusion. Studies have been conducted to assess how variation in various environmental parameters influences the extent of viral inactivation and the retention of platelet properties. Most notably, it was determined that increasing levels of plasma progressively inhibited the inactivation of model viruses. As a result, experiments were routinely conducted at a plasma level of approximately 14.5%, using 40 micrograms/ml AMT, which was determined to be optimal when using this reduced plasma level. The reduced plasma level was achieved by dilution with a nonplasma medium that has been shown to be satisfactory for storage of platelets. Under these conditions, about 5 logs of vesicular stomatitis virus (VSV), pseudorabies, and phi 6 inactivation were achieved. Variation of platelet and leukocyte counts, within normal levels, had a minimal effect on extent of viral inactivation. Although oxygen level (mean levels, 97.9 mm Hg versus 19.2 mm Hg) had only a small influence on viral inactivation with 2.4, 4.8, and 7.2 J/cm2 of UVA (equivalent to 1-3 minutes of exposure), in vitro platelet properties, such as medium pH, morphology characteristics, and aggregation response, were better retained with a longer exposure time at the reduced oxygen level. With normal oxygen (97.9 mm Hg), platelet properties declined substantially relative to untreated controls (no UVA, no AMT) on exposure to 4.8 J/cm2. Our studies have identified two sets of conditions that provide about 5 logs of virus inactivation without extensively altering platelet in vitro properties.     

Rabies virus-induced membrane fusion.

Rabies virus is a member of the rhabdovirus family. It enters cells by a process of receptor mediated endocytosis. Following this step, the viral envelope fuses with the endosomal membrane to allow release of the viral nucleocapsid into the cytoplasm. Fusion is induced by the low pH of the endosomal compartment and is mediated by the single viral glycoprotein G, a homotrimeric integral membrane protein. Rabies virus fusion properties are related to different conformational states of G. By different biochemical and biophysical approaches, it has been demonstrated that G can assume at least three different states: the native (N) state detected at the viral surface above pH 7, the activated (A) hydrophobic state which interacts with the target membrane as a first step of the fusion process, and the fusion inactive (I) conformation. Differently from other fusogenic viruses for which low pH-induced conformational changes are irreversible, there is a pH dependent equilibrium between these states, the equilibrium being shifted toward the I-state at low pH. The objective of this review is to detail recent findings on rhabdovirus-induced membrane fusion and to underline the differences that exist between this viral family and influenza virus which is the best known fusogenic virus. These differences have to be taken into consideration if one wants to have a global understanding of virus-induced membrane fusion.     

Infectivity and antigenicity reduction rates of human rotavirus strain Wa in fresh waters.

The rates of inactivation of human rotavirus type 2 (strain Wa) (HRV-Wa) and poliovirus type 1 (strain CHAT) were compared in polluted waters (creek water and secondary effluent before chlorination) and nonpolluted waters (lake water, groundwater, and chlorinated tap water). Viral infectivity titers were determined by plaque assays, while HRV-Wa antigenicity also was monitored by an enzyme-linked immunosorbent assay. Both viruses persisted longest in lake water and shortest in tap water. The actual inactivation times (i.e., times required for two-log10 reductions of initial viral titers) for the two viruses were significantly different in all waters except tap water. With the exception of the groundwater and secondary effluent results, the HRV-Wa inactivation times in the fresh waters tested were significantly different. Owing perhaps to aggregation, HRV-Wa appeared less susceptible to the effects of chlorine than previously reported for this virus and for the simian rotavirus SA11. HRV-Wa displayed prolonged survival in lake water and groundwater exceeding that previously reported for the SA11 virus. The HRV-Wa infectivity reduction rate (ki) was significantly correlated with the water pH (i.e., as pH increased, ki increased). The water pH may have influenced viral aggregation and thereby HRV-Wa susceptibility to other virucidal factors in the water. Enzyme-linked immunosorbent assay results showed similar inactivation patterns with the most significant reduction in HRV-Wa antigenicity occurring in polluted waters and tap water. In all waters, particularly tap water, infectivity declined at a faster rate than antigenicity. It is proposed that HRV-Wa can be used as a model for future studies of rotaviral persistence in the aquatic environment.     

Assessment of the viral safety of antivenoms fractionated from equine plasma.

Antivenoms are preparations of intact or fragmented (F(ab')2 or Fab) immunoglobulin G (IgG) used in human medicine to treat the severe envenomings resulting from the bites and stings of various animals, such as snakes, spiders, scorpions, or marine animals, or from the contact with poisonous plants. They are obtained by fractionating plasma collected from immunized horses or, less frequently, sheep. Manufacturing processes usually include pepsin digestion at acid pH, papain digestion, ammonium sulphate precipitation, caprylic acid precipitation, heat coagulation and/or chromatography. Most production processes do not have deliberately introduced viral inactivation or removal treatments, but antivenoms have never been found to transmit viruses to humans. Nevertheless, the recent examples of zoonotic diseases highlight the need to perform a careful assessment of the viral safety of antivenoms. This paper reviews the characteristics of equine viruses of antivenoms and discusses the potential of some manufacturing steps to avoid risks of viral contamination. Analysis of production parameters indicate that acid pH treatments and caprylic acid precipitations, which have been validated for the manufacture of some human IgG products, appear to provide the best potential for viral inactivation of antivenoms. As many manufacturers of antivenoms located in developing countries lack the resources to conduct formal viral validation studies, it is hoped that this review will help in the scientific understanding of the viral safety factors of antivenoms, in the controlled implementation of the manufacturing steps with expected impact on viral safety, and in the overall reinforcement of good manufacturing practices of these essential therapeutic products.     

Comparable virus inactivation by bovine or vegetable derived Tween 80 during solvent/detergent treatment.

A mixture of Tri-n-butyl phosphate (TNBP) and Polysorbate 80 (Tween 80) is often used for virus inactivation during the manufacture of medicinal products derived from human plasma. This procedure, known as solvent/detergent treatment, is of high effectiveness for inactivation of enveloped viruses. Tween 80 can be manufactured from bovine tallow or from vegetable material. As the bovine-derived Tween 80 is normally used for the solvent/detergent treatment, the question has been raised whether vegetable-derived Tween 80 can be applied as an alternative substance for the solvent/detergent treatment. Comparable inactivation studies were therefore performed using Vesicular Stomatitis Virus (VSV), Pseudorabiesvirus (PRV), Semliki Forest Virus (SFV) and Bovine Diarrhoea Virus (BVDV). In principle, no differences were observed in the effectiveness of the solvent/detergent treatment when bovine or vegetable-derived Tween 80 was used. The comparability in the efficiency of both detergents for virus inactivation was shown to be independent of solvent/detergent concentration, of temperature (16 degrees C and 6 degrees C vs. 27 degrees C and 25 degrees C) and protein concentration (10% and 5% human albumin). In summary, vegetable-derived Tween 80 is of the same effectiveness as bovine-derived Tween 80, when used for virus inactivation by the solvent/detergent treatment.