Virus elimination during the recycling of chromatographic columns used during the manufacture of coagulation factors

Various chromatographic procedures are used during the purification and manufacture of plasma products such as coagulation factors. These steps contribute to the overall safety of such products by removing potential virus contamination. Virus removal by two affinity chromatography procedures, i.e. monoclonal antibody chromatography and metal chelate chromatography (immobilised metal ion affinity chromatography), used during the manufacture of the high purity factor VIII (Replenate^®) and factor IX (Replenine^®-VF), respectively, has been investigated. In addition, as these columns are recycled after use, the effectiveness of the sanitisation procedures for preventing possible cross-contamination, has also been investigated.Both chromatographic steps proved effective for eliminating a range of model enveloped and non-enveloped viruses by 4 to >6 and 5 to >8 log for the monoclonal and metal chelate columns, respectively. The effectiveness of the relatively mild column sanitisation conditions used, i.e. ethanol for factor IX and acetic acid for factor VIII, was confirmed using non-spiked column runs. The chemicals used contributed to virus elimination by inactivation and/or by physical removal of the virus. In summary, these studies demonstrate that potential virus contamination between chromatographic runs can be prevented when an effective column recycling and sanitisation procedure is included.     

Virus reduction in an intravenous immunoglobulin by solvent/detergent treatment, ion-exchange chromatography and terminal low pH incubation

Virus reduction by several steps in the manufacturing process for the intravenous immunoglobulin Vigam^®, has been investigated. The solvent/detergent step based on treatment with 0.3% tri-n-butyl phosphate and 1% polysorbate 80 at 37 °C, was confirmed to be effective for a range of enveloped viruses. Virus infectivity was undetectable i.e. >6 log inactivation within 30 min of the standard 6 h process. This was consistent over the range of conditions tested i.e. solvent/detergent and protein concentration, temperature and pH. The ion-exchange chromatography step in the process was also able to remove some viruses. Virus spiked followed by blank column runs confirmed the effectiveness of the sanitisation step for ensuring there was no virus cross contamination between column runs. The terminal low pH incubation step was also able to inactivate enveloped viruses, as well as some non-enveloped viruses. The combination of these three steps ensures a high margin of virus safety for this product.     

Isolation of proteins by gel filtration in 6M guanidinium chloride: application to RNA tumor viruses

Separation of RNA tumor virus proteins by gel filtration in 6m guanidinium chloride indicates that this method will effectively separate polypeptides in milligram amounts whose molecular weights differ by as little as 15 percent. Such separations are achieved because proteins in 6m guanidinium chloride have a random coil conformation with diffusion coefficients considerably smaller than the corresponding native proteins. Consequently, protein bands that emerge from a gel filtration column in 6m guanidinium chloride are remarkably sharp. A 60–70% yield of renatured RNA tumor virus proteins could be obtained following dialysis against a dilute solution of mercaptoethanol. The major proteins of avian RNA tumor viruses were obtained as pure components by gel filtration in 6m guanidinium chloride. Mammalian RNA tumor viruses appear to have a more complex protein structure, since, in some cases, additional purification was required to obtain the pure proteins. This method of protein separation might be used as the initial stop in the isolation of components from other biological macrostructures.     

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     

Removal and inactivation of viruses during the manufacture of a high-purity antihemophilic factor IX from human plasma

The purpose of this study was to evaluate the efficacy and mechanisms of the solvent/detergent (S/D) treatment, DEAE-toyopearl 650M anion-exchange column chromatography, heparin-sepharose 6FF affinity column chromatography, and Viresolve NFP filtration steps employed in the manufacture of high-purity antihemophilic factor IX (Green-Nine VF) from human plasma, with regard to removal and/or inactivation of blood-borne viruses. A variety of experimental model viruses for human pathogenic viruses, including human immunodeficiency virus (HIV), bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), hepatitis A virus (HAV), murine encephalomyocarditis virus (EMCV), and porcine parvovirus (PPV), were all selected for this study. Samples from relevant stages of the production process were spiked with each virus and subjected to scale-down processes mimicking the manufacture of high-purity factor IX. Samples were collected at each step, immediately titrated using a 50% tissue culture infectious dose (TCID₅₀), and virus reduction factors were evaluated. S/D treatment using the organic solvent, tri (n-butyl) phosphate (TNBP), and the detergent, Tween 80, was a robust and effective step in inactivation of enveloped viruses. Titers of HIV, BHV, and BVDV were reduced from the initial titer of 6.06, 7.72, and 6.92 log₁₀ TCID₅₀, respectively, reaching undetectable levels within 1 min of S/D treatment. DEAE-toyopearl 650M anion-exchange column chromatography was found to be a moderately effective step in the removal of HAV, EMCV, and PPV with log reduction factors of 1.12, 2.67, and 1.38, respectively. Heparin-sepharose 6FF affinity column chromatography was also moderately effective for partitioning BHV, BVDV, HAV, EMCV, and PPV with log reduction factors of 1.55, 1.35, 1.08, 1.19, and 1.61, respectively. The Viresolve NFP filtration step was a robust and effective step in removing all viruses tested, since HIV, BHV, BVDV, HAV, EMCV, and PPV were completely removed during the filtration step with log reduction factors of ≥ 5.51, ≥ 5.76, ≥ 5.18, ≥ 5.34, ≥ 6.13, and ≥ 4.28, respectively. Cumulative log reduction factors of HIV, BHV, BVDV, HAV, EMCV, and PPV were ≥ 10.52, ≥ 12.07, ≥ 10.49, ≥ 7.54, ≥ 9.99, and ≥ 7.24, respectively. These results indicate that the production process for GreenNine VF has a sufficient virus reduction capacity for achievement of a high margin of virus safety.     

Investigations of prion and virus safety of a new liquid IVIG product.

A highly purified, liquid, 10% immunoglobulin product stabilized with proline, referred to as IgPro10 has recently been developed. IgG was purified from human plasma by cold ethanol fractionation, octanoic acid precipitation and anion-exchange chromatography. The manufacturing process includes two distinctly different partitioning steps and virus filtration, which were also assessed for the removal of prions. Prion removal studies used different spike preparations (brain homogenate, microsomes, purified PrP(sc)) and three different detection methods (bioassay, Western blot, conformation-dependent immunoassay). All of the investigated production steps were shown to reduce significantly all different spike preparations, resulting in an overall reduction of >10log(10). Moreover, the biochemical assays proved equally effective to the bioassay for the demonstration of prion elimination. Four of the manufacturing steps cover three different mechanisms of virus clearance. These are: i) virus inactivation; ii) virus filtration; and iii) partitioning. These mechanisms were assessed for their virus reduction capacity. Virus validation studies demonstrated overall reduction factors of >18log(10) for enveloped and >7log(10) for non-enveloped model viruses. In conclusion, the IgPro10 manufacturing process has a very high reduction potential for prions and for a wide variety of viruses resulting in a state-of-the-art product concerning safety towards known and emerging pathogens.     

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.     

Bracketed generic inactivation of rodent retroviruses by low pH treatment for monoclonal antibodies and recombinant proteins

Viral safety is a predominant concern for monoclonal antibodies (mAbs) and other recombinant proteins (RPs) with pharmaceutical applications. Certain commercial purification modules, such as nanofiltration and low-pH inactivation, have been observed to reliably clear greater than 4 log(10) of large enveloped viruses, including endogenous retrovirus. The concept of "bracketed generic clearance" has been proposed for these steps if it could be prospectively demonstrated that viral log(10) reduction value (LRV) is not impacted by operating parameters that can vary, within a reasonable range, between commercial processes. In the case of low-pH inactivation, a common step in mAb purification processes employed after protein A affinity chromatography, these parameters would include pH, time and temperature of incubation, the content of salts, protein concentration, aggregates, impurities, model protein pI, and buffer composition. In this report, we define bracketed generic clearance conditions, using a prospectively defined bracket/matrix approach, where low-pH inactivation consistently achieves >or=4.6 log(10) clearance of xenotropic murine leukemia virus (X-MLV), a model for rodent endogenous retrovirus. The mechanism of retrovirus inactivation by low-pH treatment was also investigated.     

Removal of Viruses from Human Intravenous Immune Globulin by 35 nm Nanofiltration

Viral safety is an important prerequisite for clinical immunoglobulin preparations. A common manufacturing practice is to utilize several virus removal/inactivation process steps to ensure the safety of human intravenous immunoglobulin (IVIg). In this regard, we examined the use of Planova 35 nm filters to reduce potential loads of both non-enveloped and enveloped viruses prior to end-stage solvent detergent treatment. The nanofiltration process was validated for removal of a variety of enveloped and non-enveloped viruses ranging in size from 70 nm to 18 nm including: Sindbis virus, Simian Virus 40 (SV40), Bovine Viral Diarrhoea virus (BVDV), Feline Calicivirus, Encephalomyocarditis virus (EMC), Hepatitis A virus (HAV), Bovine Parvovirus (BPV) and Porcine Parvovirus (PPV). The filtration procedure was carried out by first spiking a 7% solution of IVIg with < 10(8) virus. The spiked IVIg solution was then filtered through a 75 nm Planova filter followed by two Planova 35 nm filters in series (75/35/35). The 75 nm prefilter is incorporated into this process to increase the capacity of the 35 nm viral removal filters. As a result of the inclusion of the 75 nm pre-filtration step it was possible to assess the removal of virus by the 35 nm filters independent of possible aggregation of the initial viral spiking material. Samples were collected at each step and immediately titred by viral plaque assay. A process control sample of the spiked load solution was held at the same conditions for the duration of the filtration process and then titred to determine the extent to which antibody neutralization may have contributed to overall viral reduction. Control assays of spiked IVIg were performed to establish the degree of toxicity of the IVIg solution to the indicator cell lines and the extent to which the IVIg interfered with plaque formation in the assay system. This combined data was used to establish assay sensitivity for the calculation of log removal by the filtration process. It was noted that toxicity/interference effects could have a significant effect upon apparent log reductions, and these effects could vary greatly, even within viruses of the same family. The results of these studies indicate that 35 nm filtration is very effective for removing substantial quantities of both non-enveloped and enveloped viruses from IVIg. Complete clearance (to the limits of detection of the assay) was obtained for all viruses larger than 35 nm. Interestingly, viruses reported to have mean diameters of less than 35 nm (EMC and HAV) were at least partially removed by the filtration (4.3 and > 4.7 logs removal, respectively). Even small viruses such as PPV were to some extent removed from the IVIg solution by the filters (2.6 logs removal). Reduction of BPV would not be assessed due to extensive neutralization and interference with plaque formation by the IVIg. Sindbis and SV40 also were subject to neutralization and assay interference due to the IVIg, though to a lesser extent. We conclude from these studies that the 35 nm mean pore size is functionally efficient in removal of smaller size viruses from spiked IVIg concentrates.     

Identification of trimeric peptides that bind porcine parvovirus from mixtures containing human blood plasma

Virus contamination in human therapeutics is of growing concern as more therapeutic products from animal or human sources come into the market. All biopharmaceutical processes are required to have at least two distinct viral clearance steps to remove viruses. Most of these steps work well for enveloped viruses and large viruses, whether enveloped or not. That leaves a class of small non-enveloped viruses, like parvoviruses and hepatitis A, which are not easily removed by these typical steps. In this study, we report the identification of trimeric peptides that bind specifically to porcine parvovirus (PPV) and their potential use to remove this virus from process solutions. All of the trimeric peptides isolated completely removed all detectable PPV from buffer in the first nine column volumes, corresponding to a clearance of 4.5-5.5 log of infectious virus. When the virus was spiked into a more complex matrix consisting of 7.5% human blood plasma, one of the trimers, WRW, was able to remove all detectable PPV in the first three column volumes, after which human blood plasma began to interfere with the binding of the virus to the peptide resin. These trimer resins removed considerably more virus than weak ion exchange resins. The results of this work indicate that small peptide ligand resins have the potential to be used in virus removal processes where removal of contaminating virus is necessary to ensure product safety.     

Viral clearance during the manufacture of urokinase from human urine

The purpose of the present study was to evaluate the efficacies and mechanisms of the PAB (para-amino benzamidine) affinity column chromatography, virus filtration, pasteurization (60°C heat treatment for 10 h), and lyophilization steps employed in the manufacture of urokinase from human urine with regard to the removal and/or inactivation of human immunodeficiency virus (HIV), bovine viral diarrhoea virus (BVDV), bovine herpes virus (BHV), and murine encephalomyocarditis virus (EMCV). Samples from relevant stages of the production process were spiked with each virus and subjected to scale-down processes mimicking the manufacture of urokinase. Samples were collected at each step, immediately titrated using a 50% tissue culture infectious dose (TCID₅₀), and the virus reduction factors evaluated. PAB chromatography was found to be an effective step for removing BVDV, BHV, and EMCV with log reduction factors of 2.79, 6.50, and 5.96, respectively. HIV, BVDV, BHV, and EMCV were completely removed during the Viresolve NFP filtration step with log reduction factors of ≥6.06, ≥4.60, ≥5.44, and ≥6.87, respectively. Pasteurization was also found to be a robust and effective step in inactivating all the viruses tested, since there were no residual viruses detected after the pasteurization process. The log reduction factors achieved by pasteurization were ≥5.73 for HIV, ≥3.86 for BVDV, ≥6.75 for BHV, and ≥5.92 for EMCV. Lyophilization showed significant efficacy for inactivating BVDV, BHV, and EMCV with log reduction factors of 2.69, 1.37, and 4.70, respectively. These results indicate that the production process for urokinase exhibited a sufficient viral reducing capacity to achieve a high margin of virus safety.     

Evaluation of viral clearance in the production of HPV-16 L1 virus-like particles purified from insect cell cultures

Biopharmaceutical products produced from cell cultures have a potential for viral contamination from cell sources or from adventitious introduction during production. The objective of this study was to assess viral clearance in the production of insect cell-derived recombinant human papillomavirus (HPV)-16 type L1 virus-like particles (VLPs). We selected Japanese encephalitis virus (JEV), bovine viral diarrhea virus (BVDV), and minute virus of mice (MVM) as relevant viruses to achieve the aim of this study. A downstream process for the production of purified HPV-16 L1 VLPs consisted of detergent lysis of harvested cells, sonication, sucrose cushion centrifugation, and cesium chloride (CsCl) equilibrium density centrifugation. The capacity of each purification/treatment step to clear viruses was expressed as reduction factor by measuring the difference in log virus infectivity of sample pools before and after each process. As a result, detergent treatment (0.5% v/v, Nonidet P-40/phosphate-buffered saline) was effective for inactivating enveloped viruses such as JEV and BVDV, but no significant reduction (< 1.0 log(10)) was observed in the non-enveloped MVM. The CsCl equilibrium density centrifugation was fairly effective for separating all three relevant adventitious viruses with different CsCl buoyant density from that of HPV-16 L1 VLPs (JEV, BVDV, and MVM = 4.30, 3.10, > or = 4.40 log(10) reductions). Given the study conditions we used, overall cumulative reduction factors for clearance of JEV, BVDV, and MVM were > or = 10.50, > or = 9.20, and > or = 6.40 log(10) in 150 ml of starting cell cultures, respectively.     

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.     

Purification of human B cell growth factor

Human B cell growth factor (BCGF, 12,000 to 14,000 daltons) has been purified from lectin-stimulated, peripheral blood mononuclear cell-conditioned medium. The purification procedure involves a series of column chromatographic steps incorporating ion exchange, affinity binding, and gel filtration. This procedure is centered around a relatively high yield single chromatographic step, for the removal of co-eluting cytokines from BCGF, that is based on differential binding characteristics to the weak ion-exchange matrix, hydroxylapatite. Reverse-phase high-pressure liquid chromatographic separation on a C18-Bondapak column effectively separates the BCGF and TCGF moieties, yet is characterized by poor yields. High-pressure liquid chromatographic procedures on anion exchange and size exclusion provided the final purification step for BCGF, at an analytical level, resulting in a single band with a m.w. of 12,000 on a SDS-polyacrylamide gel.     

Viral clearance studies on new and used chromatography resins: critical review of a large dataset.

Viral clearance studies for na?ve and maximally cycled chromatographic resins used for cGMP recombinant protein production are reviewed for three products, comprising 10 different chromatographic steps, including affinity, ion exchange, immobilized metal ion affinity, and hydrophobic interaction modes. Thirty-two separate studies were conducted (over 90 runs in total). No consistent reductions in model virus clearance were observed with used resins. The results address the reproducibility of virus clearance studies conducted by different scientists over several years at multiple contract labs. The log reduction values (LRVs) are typically within 0.5 LRVs for new and used resin, but varied as much as 2 LRVs for resins showing no functional deterioration. This relatively large difference is not believed to reflect resin changes, but highlights the challenges encountered in modeling column clearance. Production column performance and cleaning efficacy are demonstrated for these steps by trending mock runs, impurity removal and product recovery. No deterioration in cGMP column performance is seen over the established resin lifetimes, confirming that the resin regeneration and sanitization procedures restore the resins to a suitable initial state without damage. It is proposed that for some chromatography steps, the combination of lab-scale cycling studies confirming consistent performance throughout the resin lifetime and monitoring of cGMP manufacturing preclude the need for virus clearance studies on maximally cycled resin.     

Limits in virus filtration capability? Impact of virus quality and spike level on virus removal with xenotropic murine leukemia virus

Virus filtration (VF) is a key step in an overall viral clearance process since it has been demonstrated to effectively clear a wide range of mammalian viruses with a log reduction value (LRV) > 4. The potential to achieve higher LRV from virus retentive filters has historically been examined using bacteriophage surrogates, which commonly demonstrated a potential of > 9 LRV when using high titer spikes (e.g. 10¹⁰ PFU/mL). However, as the filter loading increases, one typically experiences significant decreases in performance and LRV. The 9 LRV value is markedly higher than the current expected range of 4‐5 LRV when utilizing mammalian retroviruses on virus removal filters (Miesegaes et al., Dev Biol (Basel) 2010;133:3‐101). Recent values have been reported in the literature (Stuckey et al., Biotech Progr 2014;30:79‐85) of LRV in excess of 6 for PPV and XMuLV although this result appears to be atypical. LRV for VF with therapeutic proteins could be limited by several factors including process limits (flux decay, load matrix), virus spike level and the analytical methods used for virus detection (i.e. the Limits of Quantitation), as well as the virus spike quality. Research was conducted using the Xenotropic‐Murine Leukemia Virus (XMuLV) for its direct relevance to the most commonly cited document, the International Conference of Harmonization (ICH) Q5A (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland, 1999) for viral safety evaluations. A unique aspect of this work is the independent evaluation of the impact of retrovirus quality and virus spike level on VF performance and LRV. The VF studies used XMuLV preparations purified by either ultracentrifugation (Ultra 1) or by chromatographic processes that yielded a more highly purified virus stock (Ultra 2). Two monoclonal antibodies (Mabs) with markedly different filtration characteristics and with similar levels of aggregate (<1.5%) were evaluated with the Ultra 1 and Ultra 2 virus preparations utilizing the Planova 20 N, a small virus removal filter. Impurities in the virus preparation ultimately limited filter loading as measured by determining the volumetric loading condition where 75% flux decay is observed versus initial conditions (V₇₅). This observation occurred with both Mabs with the difference in virus purity more pronounced when very high spike levels were used (>5 vol/vol %). Significant differences were seen for the process performance over a number of lots of the less‐pure Ultra 1 virus preparations. Experiments utilizing a developmental lot of the chromatographic purified XMuLV (Ultra 2 Development lot) that had elevated levels of host cell residuals (vs. the final Ultra 2 preparations) suggest that these contaminant residuals can impact virus filter fouling, even if the virus prep is essentially monodisperse. Process studies utilizing an Ultra 2 virus with substantially less host cell residuals and highly monodispersed virus particles demonstrated superior performance and an LRV in excess of 7.7 log₁₀. A model was constructed demonstrating the linear dependence of filtration flux versus filter loading which can be used to predict the V₇₅ for a range of virus spike levels conditions using this highly purified virus. Fine tuning the virus spike level with this model can ultimately maximize the LRV for the virus filter step, essentially adding the LRV equivalent of another process step (i.e. protein A or CEX chromatography). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:135–144, 2015     

Analysis of viral clearance unit operations for monoclonal antibodies

Demonstration of viral clearance is a critical step in assuring the safety of biotechnology products. We generated a viral clearance database that contains product information, unit operation process parameters, and viral clearance data from monoclonal antibody and antibody‐related regulatory submissions to FDA. Here we present a broad overview of the database and resulting analyses. We report that the diversity of model viruses tested expands as products transition to late‐phase. We also present averages and ranges of viral clearance results by Protein A and ion exchange chromatography steps, low pH chemical inactivation, and virus filtration, focusing on retro‐ and parvoviruses. For most unit operations, an average log reduction value (LRV, a measure of clearance power) for retrovirus of >4 log₁₀ were measured. Cases where clearance data fell outside of the anticipated range (i.e., outliers) were rationally explained. Lastly, a historical analysis did not find evidence of any improvement trend in viral clearance over time. The data collectively suggest that many unit operations in general can reliably clear viruses. Biotechnol. Bioeng. 2010;106: 238–246. Published 2010 Wiley Periodicals, Inc.     

Inactivation and clearance of viruses during the manufacture of high purity factor IX.

Haemophilia is a bleeding disorder characterised by a deficiency in Factor IX. Replacement therapy in the form of a Factor IX concentrate is a widely accepted practice. In this paper we describe a double virus inactivated chromatographic process for producing a high purity Factor IX product, MonoFIX((R))-VF. The process involves separation of the prothrombin complex by cryoprecipitation, fraction I precipitation and DEAE-cellulose adsorption, further ion-exchange chromatography of crude Factor IX, followed by solvent/detergent treatment. Heparin affinity chromatography is then used to further purify Factor IX. Final nanofiltration is sequential through 35 nm then 15 nm membrane filters. The principal virus inactivation/removal steps are solvent/detergent treatment and nanofiltration and the partitioning of relevant and model viruses provides further reduction in virus load through the production process.Solvent/detergent treatment was shown to achieve log reduction factors of 4.5 for HIV-1, 5.1 for Sindbis virus, 6.1 for vesicular stomatitis virus (VSV), 5.1 for bovine viral diarrhoea virus (BVDV) and 5.3 for pseudorabies virus (PRV). BVDV is a model for hepatitis C virus (HCV), and pseudorabies virus (PRV), like hepatitis B virus (HBV) is an enveloped DNA virus. Using scaled down models of the production process, we have also demonstrated the neutralization/partitioning of at least 6 logs of hepatitis A virus (HAV) during cryoprecipitation, Fraction I precipitation, and the DEAE adsorption and elution step, and a further 1.6 log reduction in HAV load as a result of heparin affinity chromatography. The log reduction factors for HAV as a result of the second ion-exchange chromatography step and as a result of enhanced neutralisation associated with solvent/detergent treatment were not significant. Nanofiltration was shown to contribute a further log reduction factor of 6.7 for HAV and 5.8 for BVDV indicating that log reduction factors of this order would be obtained with other viruses of a similar or larger size, such as HIV, HBV and HCV.Overall, these studies indicate that MonoFIX-VF is a product with an extremely high level of viral safety.     

The effect of protein a cycle number on the performance and lifetime of an anion exchange polishing step

Most mAb platform purification processes consist of an affinity capture step followed by one or two polishing steps. An understanding of the performance linkages between the unit operations can lead to robust manufacturing processes. In this study, a weak‐partitioning anion‐exchange chromatography polishing step used in a mAb purification process was characterized through high‐throughput screening (HTS) experiments, small‐scale experiments including a cycling study performed on qualified scale‐down models, and large‐scale manufacturing runs. When material from a Protein A column that had been cycled <10× was loaded on the AEX resin, early breakthrough of impurities and premature loss of capacity was observed. As the cycle number on the Protein A resin increased, the capacity of the subsequent AEX step increased. Different control strategies were considered for preventing impurity breakthrough and improving AEX resin lifetimes. Depth filtration of the Protein A peak pool significantly improved the AEX resin capacity, robustness, and lifetime. Further, the turbidity of the Protein A pool has the potential for use as an in‐process control parameter for monitoring the performance of the AEX step. Biotechnol. Bioeng. 2013; 110: 1142–1152. © 2012 Wiley Periodicals, Inc.