Planova 20N Research Articles

Effect of pH, NaCl concentration, and mAb concentration of feed solution on the filterability of Planova™ 20N and Planova™ BioEX.

Virus filtration is one of the most important steps in ensuring viral safety during the purification of monoclonal antibodies (mAbs) and other biotherapeutics derived from mammalian cell cultures. Regarding the various virus retentive filters, including Planova filters, a great deal of data has been reported on the virus retention capability and its mechanism. Along with the virus retention capability, filterability is a key performance indicator for designing a robust and high-throughput virus filtration step. In order to obtain higher filterability, optimization of the feed solution conditions, and filter selection is essential; however, limited data are available regarding the filtration characteristics of Planova filters. Furthermore, for Planova 20N and Planova BioEX, the virus retention characteristics were reported to differ due to their respective membrane materials and layer structures. Whether these filters differ in their filtration characteristics is an interesting question, but no comparative evaluations have been reported. In this study, the filterability of the two filters was investigated and compared using 15 feed mAb solutions of a single mAb selected by design of experiments with different combinations of pH, NaCl concentration, and mAb concentration. The filterability of Planova 20N was affected not only by the feed solution viscosity, but also by the mAb aggregate content of the feed mAb solution and mAb-membrane electrostatic interactions. In contrast, the filterability of Planova BioEX decreased under some buffer conditions. These findings and the established design spaces of these filters provide valuable insights into the process optimization of virus filtration.

Integration of Planova filters in manufacturing processes of biologicals improve the virus safety effectively: A review of publicly available data.

The capacity to remove viruses by Planova filters produced by Asahi Kasei, primarily by small virus-retentive filters, were compiled from data in peer-reviewed publications and, partly, publicly available data from presentations at conferences (Planova workshops). Data from more than 100 publications and presentations at conferences covering Planova filters were assessed. The data were grouped according to the different virus filters regarding mean pore sizes and viruses of different sizes for plasma and cell culture derived products. Planova 15N and 20N filters removed parvoviruses below the limit of detection of viruses in the filtrate in approx. 50% of all studies and mean LRFs (log reduction factors) for viruses detected in the filtrate were above 4, demonstrating effective parvovirus reduction. Parvovirus removal capacity increased for Planova BioEX filters as well as for 2 Planova 20N in series. Large viruses as retroviruses (e.g., HIV and MuLV), herpesviruses, flaviviruses and togaviruses were removed effectively by Planova 15N, 20N and BioEX filters and also by Planova 35N filters. Flow interruption, transmembrane pressure, volume and protein concentration per filter area had had no substantial impact on virus removal capacity at manufacturing specification. In conclusion, the incorporation of Planova filters in manufacturing processes of biologicals remove, depending on the filter pore size, small and large viruses from the feed stream reliably. This virus reduction step with an orthogonal mechanism integrated in the manufacturing processes of biologicals, based primarily on size exclusion of viruses, improves the virus safety of these biopharmaceutical products considerably.

Size-based analysis of virus removal filter fouling using fractionated protein aggregates.

Fouling by protein aggregates reduces virus removal filter performance. In the present study, we investigated the effects of different-sized protein aggregates on fouling and aggregate retention in order to better understand the fouling mechanisms. Human immunoglobulin G was denatured by heating to produce aggregates of various sizes and then fractionated by size exclusion chromatography into different-sized aggregates with a narrow size distribution. The fractionated aggregates were filtered on Planova 20N, a virus removal filter known for its stable filtration capability. Analysis of flux behavior demonstrated different flux decrease patterns for different-sized aggregates. Observation of aggregate retention by staining revealed that larger aggregates were captured closer to the inner surface of the membrane while smaller aggregates penetrated farther into the membrane. These findings demonstrate that Planova 20N has a gradient structure with decreasing pore size from the inner to the outer surface of the membrane. This structure minimizes fouling and enables stable filtration by protecting the smaller pores located closer to the outer surface from clogging by large aggregates. Applying the predominant clogging models to the present filtrations revealed that clogging behavior transitioned from complete blocking to cake filtration as filtration progressed. In this combination model, after a certain number of pores are blocked by complete blocking, newly arrived aggregates begin to accumulate on previously captured aggregates, generating cake between capture layers within the membrane. Application of the approaches described here will facilitate elucidation of membrane fouling and virus removal mechanisms.

Determination of optimum nanofiltration conditions for the manufacturing process of human normal immunoglobulin G for intravenous administration

Scientific relevance. Medicinal products based on immunoglobulin class G (IgG) from human plasma are widely used in clinical practice to treat bacterial and viral infections, primary and secondary immunodeficiencies, and autoimmune diseases. Nanofiltration is a way to mitigate the risk of in-process contamination of raw materials with various pathogens, including viruses. Therefore, it is relevant to investigate the development and implementation of additional viral inactivation and/or elimination steps.Aim. This study aimed to develop and validate optimum nanofiltration conditions and to scale up the nanofiltration step for the manufacturing of human IgG for intravenous administration.Materials and methods. The study used a solution of IgG from plasma fractions II and III. The authors paired nanofilters manufactured by Planova 20N and BioEx (Asahi Kasei, Japan), Viresolve Pro (Merck Millipore, USA), Virosart HC and HF (Sartorius, Germany), and Pegasus SV4 and Prime (Pall, USA) with Sartopore polyethersulphone prefilters by Sartorius (Germany), Virosart MAX polyamide prefilters by Sartorius (Germany), and EKX-P regenerated cellulose prefilters by Pall (Germany). Virus reduction validation studies were performed with model viruses (human immunodeficiency virus type 1, porcine transmissible gastroenteritis virus, porcine parvovirus, murine encephalomyocarditis virus, and bovine viral diarrhoea virus) in the laboratories of the N.F. Gamaleya centre. The sample data analysis involved calculating mean values with 95% confidence intervals.Results. For all the selected combinations of prefilters and filters, the maximum nanofiltration throughput depended on the IgG concentration in the test solution. With the combination of an EKX-P filter with a Pegasus SV4 nanofilter, the maximum throughput and the IgG yield reached 6300 g/m2 and 95%, respectively. When combined with a Planova 20N nanofilter, EKX-P and Sartopore (polyethersulphone) filters provided a maximum throughput of up to 2980 g/m2 and an IgG yield of almost 100%, provided that the test solution had an IgG concentration of 10 g/L. With different filter combinations, virus reduction levels ranged from 4.00±0.05 to 4.75±0.04 log10 for human immunodeficiency virus type 1, from 4.30±0.04 to 4.55±0.06 log10 for porcine transmissible gastroenteritis virus, from 5.38±0.08 log10 to 5.57±0.04 log10 for murine encephalomyocarditis virus, 5.12±0.10 log10 to 5.25±0.08 log10 for porcine parvovirus, and exceeded 5.00 log10 for bovine viral diarrhoea virus. The virus reduction levels achieved were not statistically associated with prefilter brands.Conclusions. The study demonstrated that nanofiltration was effective at removing viruses with various virion sizes and physicochemical characteristics, including viruses as small as parvovirus B19. The levels of virus reduction exceeded 4 log10 and met the acceptance criteria.The laboratory-scale nanofiltration parameters and the corresponding filtration times, as well as IgG yields, did not change when the process was scaled up. Therefore, nanofiltration is an effective and productive technique that helps eliminate various types of viruses and considerably improve viral safety without affecting the quality of biological medicinal products.

Visualizing protein fouling and its impact on parvovirus retention within distinct filter membrane morphologies

Virus filtration is considered an effective and robust method to remove viral contaminants that may enter the manufacturing process of biotherapeutics. However, insights into the retention mechanism of viruses under the influence of different operating conditions have been limited so far. In this work, we visualize the impact of filter fouling and flow decay on the retention of fluorescently labeled minute virus of mice (MVM) in asymmetric Planova 20N and nearly homogeneous Pegasus SV4 filter membranes. Filtration of feedstreams containing polyclonal human immunoglobulin G (IgG) revealed a complex interplay of different fouling mechanisms depending on high- or low-fouling solution properties, and characteristic for the distinct filter types. The asymmetric filter morphology – which allowed gradual foulant deposition across membrane zones with different pore sizes – provided a larger capacity for capture of fouling particulates as well as robust virus retention under challenging feedstream conditions. Taken together, our results demonstrate that different filtration conditions can lead to a combination of various, even opposing effects on virus retention depending on the membrane structure and pore size characteristics. The phenomena visualized in this work contribute to a better understanding of the underlying molecular mechanisms and provide cues for specific optimization of virus filtration processes.

Removal of minute virus of mice-mock virus particles by nanofiltration of culture growth medium supplemented with 10% human platelet lysate

Background aimsPlatelet concentrates (PCs) are pooled to prepare human platelet lysate (HPL) supplements of growth media to expand primary human cells for transplantation; this increases the risk of contamination by known, emerging, and unknown viruses. This possibility should be of concern because viral contamination of cell cultures is difficult to detect and may have detrimental consequences for recipients of cell therapies. Viral reduction treatments of chemically defined growth media have been proposed, but they are not applicable when media contain protein supplements currently needed to expand primary cell cultures. Recently, we successfully developed a Planova 35NPlanova 20N nanofiltration sequence of growth media supplemented with two types of HPL. The nanofiltered medium was found to be suitable for mesenchymal Stromal cell (MSC) expansion. MethodsHerein, we report viral clearance achieved by this nanofiltration process used for assessing a new experimental model using non-infectious minute virus of mice-mock virus particle (MVM-MVP) and its quantification by an immunoqPCR. Then, high doses of MVM-MVP (1012 MVPs/mL) were spiked to obtain a final concentration of 1010 MVPs/mL in Planova 35N-nanofiltered growth medium supplemented with both types of HPLs [serum converted platelet lysate SCPL) and intercept human platelet lysate (I-HPL)] at 10% (v/v) and then filtering through Planova 20N. ResultsNo substantial interference of growth medium matrices by the immune-qPCR assay was first verified. Log reduction values (LRVs) were ≥ 5.43 and ≥ 5.36 respectively, SCPL and I-HPL media. MVM-MVPs were also undetectable by dynamic light scattering and transmission electron microscopy. ConclusionsThe nanofiltration of growth media supplemented with 10% HPL provides robust removal of small nonenveloped viruses, and is an option to improve the safety of therapeutic cells expanded using HPL supplements.

Direct visualization of virus removal process in hollow fiber membrane using an optical microscope

Virus removal filters developed for the decontamination of small viruses from biotherapeutic products are widely used in basic research and critical step for drug production due to their long-established quality and robust performance. A variety of imaging techniques have been employed to elucidate the mechanism(s) by which viruses are effectively captured by filter membranes, but they are limited to ‘static’ imaging. Here, we propose a novel method for detailed monitoring of ‘dynamic process’ of virus capture; specifically, direct examination of biomolecules during filtration under an ultra-stable optical microscope. Samples were fluorescently labeled and infused into a single hollow fiber membrane comprising cuprammonium regenerated-cellulose (Planova 20N). While proteins were able to pass through the membrane, virus-like particles (VLP) accumulated stably in a defined region of the membrane. After injecting the small amount of sample into the fiber membrane, the real-time process of trapping VLP in the membrane was quantified beyond the diffraction limit. The method presented here serves as a preliminary basis for determining optimum filtration conditions, and provides new insights into the structure of novel fiber membranes.

Introduction and clearance of beta-glucan in the downstream processing of monoclonal antibodies.

β‐Glucan process‐related impurities can be introduced into biopharmaceutical products via upstream or downstream processing or via excipients. This study obtained a comprehensive process‐mapping dataset for five monoclonal antibodies to assess β‐glucan introduction and clearance during development and production runs at various scales. Overall, 198 data points were available for analysis. The greatest β‐glucan concentrations were found in the depth‐filtration filtrate (37–2,745 pg/ml). Load volume correlated with β‐glucan concentration in the filtrate, whereas flush volume was of secondary importance. Cation‐exchange chromatography significantly cleared β‐glucans. Furthermore, β‐glucan leaching from the Planova 20N virus removal filter was reduced by increasing the flush volume (1 vs. 10 L/m2). β‐glucan concentrations after filter flush with 10 L/m2 were consistently <10 pg/ml. No or only limited β‐glucan clearance was attained via ultrafiltration/diafiltration (UF/DF). However, during the first run with monoclonal antibody (mAb) 4, β‐glucan concentration in the UF/DF retentate was 10.8 pg/mg, potentially due to β‐glucan leaching from the first run with a regenerated cellulose membrane. Overall, β‐glucan levels in the final mAb drug substance were 1–12 pg/mg. Assuming high doses of 1,000–5,000 mg, a β‐glucan contamination at 20 pg/mg would translate to 20–100 ng/dose, which is below the previously suggested threshold for product safety (≤500 ng/dose).

Visualizing effects of protein fouling on capture profiles in the Planova BioEX and 20N virus filters

One of the challenges in applying virus filtration in bioprocessing is the high level of membrane fouling seen with many process streams. A number of previous studies have examined the nature of the resulting flux decline, but much less is known about the effect of protein fouling on the virus retention characteristics. The objective of this work was to use confocal and electron microscopy to evaluate changes in the capture of 20 nm nanoparticles, comparable in size to small parvovirus, within the depth of PlanovaTM 20N and BioEX hollow fiber virus filters after fouling with serum IgG. Confocal images of fluorescently-labeled IgG showed protein capture throughout the virus filter, with the greatest intensity seen more than half-way through the depth of the filter. IgG fouling of the PlanovaTM 20N led to clustering in nanoparticle capture, likely due to changes in the flow distribution through the filter. This effect was not seen with the BioEX membrane. Instead, IgG fouling shifted the location of the captured nanoparticles towards the inlet (lumen) surface of the filter. These results provide important insights into the effects of protein fouling on the flow and virus capture behaviour of these important hollow fiber virus filters.

Development of small-scale models to understand the impact of continuous downstream bioprocessing on integrated virus filtration.

We designed small‐scale virus filtration models to investigate the impact of the extended process times and dynamic product streams present in continuous manufacturing. Our data show that the Planova 20N and BioEX virus filters are capable of effectively removing bacteriophage PP7 (>4 log) when run continuously for up to 4 days. Additionally, both Planova 20N and BioEX filters were able to successfully process a mock elution peak of increased protein, salt, and bacteriophage concentrations with only an increase in filtration pressure observed during the higher protein concentration peak. These experiments demonstrated that small‐scale viral clearance studies can be designed to model a continuous virus filtration step with specific process parameters.

New insights into the performance characteristics of the Planova-series hollow-fiber parvovirus filters using confocal and electron microscopy.

Virus filtration remains a critical step in the downstream process for the production of monoclonal antibodies and other mammalian cell-derived biotherapeutics. Recent studies have shown large differences in virus capture behavior of different virus filters, although the origin of these differences is still unclear. The objective of this study was to use confocal and scanning electron microscopy to directly evaluate the capture of virus-size nanoparticles in Planova 20N and BioEX hollow-fiber virus filters. Confocal images of fluorescent nanoparticles were quantified using ImageJ image processing software based on the measured fluorescence intensity of the labeled nanoparticles. Nanoparticle capture by the Planova BioEX was independent of transmembrane pressure from 10 to 45 psi. In contrast, the Planova 20N showed significant differences in nanoparticle capture profile at low pressure, consistent with literature data showing virus breakthrough under these conditions. Images obtained after a process interruption show significant migration of previously captured nanoparticles in the Planova 20N filters but not in the BioEX. These results provide important insights into the nature of virus capture in different virus filters and its dependence on the underlying structure of the virus filtration membranes.

Characterizing the impact of pressure on virus filtration processes and establishing design spaces to ensure effective parvovirus removal.

Virus filtration provides robust removal of potential viral contaminants and is a critical step during the manufacture of biotherapeutic products. However, recent studies have shown that small virus removal can be impacted by low operating pressure and depressurization. To better understand the impact of these conditions and to define robust virus filtration design spaces, we conducted multivariate analyses to evaluate parvovirus removal over wide ranges of operating pressure, solution pH, and conductivity for three mAb products on Planova™ BioEX and 20N filters. Pressure ranges from 0.69 to 3.43 bar (10.0–49.7 psi) for Planova BioEX filters and from 0.50 to 1.10 bar (7.3 to 16.0 psi) for Planova 20N filters were identified as ranges over which effective removal of parvovirus is achieved for different products over wide ranges of pH and conductivity. Viral clearance at operating pressure below the robust pressure range suggests that effective parvovirus removal can be achieved at low pressure but that Minute virus of mice (MVM) logarithmic reduction value (LRV) results may be impacted by product and solution conditions. These results establish robust design spaces for Planova BioEX and 20N filters where high parvovirus clearance can be expected for most antibody products and provide further understanding of viral clearance mechanisms. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:1294–1302, 2017

Anti-Human Platelet Antigen-1a Immunoglobulin G Preparation Intended to Prevent Fetal and Neonatal Alloimmune Thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe disease that is caused by maternal alloantibodies generated during pregnancy or at delivery as a result of incompatibility between maternal and fetal human platelet antigens (HPAs) inherited from the father. Antibody-mediated immune suppression using anti-HPA-1a immunoglobulins is thought to be able to prevent FNAIT caused by HPA-1a. A fractionation process to prepare anti-HPA-1a immunoglobulin (Ig) G (IgG) from human plasma was therefore developed. Anti-HPA-1a plasma was obtained from volunteer mothers who underwent alloimmunization against HPA-1a during a previous pregnancy. Plasma was cryoprecipitated and the supernatant treated with caprylic acid and solvent/detergent (S/D), purified by chromatography, nanofiltered, concentrated, and sterile-filtered. The anti-HPA-1a immunoglobulin fraction was characterized for purity and safety. PAK12 and quantitative monoclonal antibody immobilization of platelet antigen (MAIPA) assays were used to detect anti-HPA-1a IgG. Hepatitis C virus (HCV) removal during nanofiltration was assessed by spiking experiments, using cell culture-derived reporter HCV and luciferase analysis. The caprylic acid treatment precipitated non-Ig proteins yielding a 90% pure Ig supernatant. S-HyperCel chromatography of the S/D-treated supernatant followed by HyperCel STAR AX provided high IgG recovery (>80%) and purity (>99.5%), and efficient IgA and IgM removal. Concentrations of complement factors C3 and C4 were < 0.5 and < 0.4 mg/dL, respectively. The final IgG could be nanofiltered on Planova 20N under conditions removing more than 3 log HCV infectivity to baseline mock infection level, and concentrated to ca. 30 g/L. Proteolytic activity and thrombin generation were low in the final fraction. The Pak12 and MAIPA assays showed good recovery of anti-HPA-1a throughout the process. Clinical-grade HPA-1a IgG can be prepared using a process compliant with current quality requirements opening perspectives for the prevention of FNAIT.

Prediction of viral filtration performance of monoclonal antibodies based on biophysical properties of feed.

Controlling viral contamination is an important issue in the process development of monoclonal antibodies (MAbs) produced from mammalian cell lines. Virus filtration (VF) has been demonstrated to be a robust and effective clearance step which can provide ≥4 logs of reduction via size exclusion. The minimization of VF area by increasing flux and filter loading is critical to achieving cost targets as VFs are single use and often represent up to 10% of total purification costs. The research presented in this publication describes a development strategy focused on biophysical attributes of product streams that are directly applicable to VF process performance. This article summarizes a case study where biophysical tools (high-pressure size exclusion chromatography, dynamic light scattering, and absolute size exclusion chromatography) were applied to a specific MAb program to illustrate how changes in feed composition (pH, sodium chloride concentration, and buffer salt type) can change biophysical properties which correlate with VF performance. The approach was subsequently refined and expanded over the course of development of three MAbs where performance metrics (i.e., loading and flux) were evaluated for two specific virus filters (Viresolve Pro and Planova 20N) during both unspiked control runs and virus clearance experiments. The analyses of feed attributes can be applied to a decision tree to guide the recommendation of a VF filter and operating conditions for use in future MAb program development. The understanding of the biophysical properties of the feed can be correlated to virus filter performance to significantly reduce the mass of product, time, and costs associated with virus filter step development.

A novel approach to achieving modular retrovirus clearance for a parvovirus filter

Viral filtration is routinely incorporated into the downstream purification processes for the production of biologics produced in mammalian cell cultures (MCC) to remove potential viral contaminants. In recent years, the use of retentive filters designed for retaining parvovirus (~20 nm) has become an industry standard in a conscious effort to further improve product safety. Since retentive filters remove viruses primarily by the size exclusion mechanism, it is expected that filters designed for parvovirus removal can effectively clear larger viruses such as retroviruses (~100 nm). In an attempt to reduce the number of viral clearance studies, we have taken a novel approach to demonstrate the feasibility of claiming modular retrovirus clearance for Asahi Planova 20N filters. Porcine parvovirus (PPV) and xenotropic murine leukemia virus (XMuLV) were co-spiked into six different feedstreams and then subjected to laboratory scale Planova 20N filtration. Our results indicate that Planova 20N filters consistently retain retroviruses and no retrovirus has ever been detected in the filtrates even when significant PPV breakthrough is observed. Based on the data from multiple in-house viral validation studies and the results from the co-spiking experiments, we have successfully claimed a modular retrovirus clearance of greater than 6 log10 reduction factors (LRF) to support clinical trial applications in both USA and Europe.

Multiple Dedicated Measures Result in a Plasma-Derived FVIII Concentrate with High Margin of Safety Regarding Pathogen Transmission

Abstract 4355A minimal load of infectious virus, if any, in human plasma, the starting material for the production of a FVIII concentrate, is achieved by selecting donor centers and donors, by testing donations for viral markers and genomic material of HAV, HBV, HCV, HIV-1 and high titers of parvovirus B19 (B19V), and by releasing plasma pools for fractionation when non-reactive for these blood-borne viruses. Two dedicated virus reduction steps and further steps of the manufacturing process of this FVIII concentrate [Beriate] were investigated: heat treatment in aqueous, stabilized solution (“pasteurization”), virus filtration (filter with a mean pore size of approx. 19 nm (Planova 20N)), and a chromatography step. The virus and prion reduction capacity of the manufacturing process of this FVIII concentrate was evaluated in in vitro studies.Product intermediates from selected steps of the manufacturing process for the FVIII concentrate, derived from different production lots, were spiked with enveloped and non-enveloped viruses of diverse physico-chemical characteristics and processed according to a scaled-down, validated manufacturing process. The virus panel employed in these studies consisted of HIV, BVDV (bovine viral diarrhea virus, a specific model virus for HCV and WNV), HAV, parvoviruses (B19V and CPV (canine parvovirus)) and PRV (pseudorabies virus, a non-specific enveloped DNA virus). The prion reduction capacity was studied by spiking cryoprecipitate with two different spike preparations and performing the whole manufacturing process including the virus filtration according to a valid down scaled process.Pasteurization (heat treatment) inactivates all blood-borne viruses or their specific model viruses effectively, i.e. HIV, BVDV, HAV and B19V were inactivated in the order of 4 log10 or more. Virus filtration removed effectively all enveloped viruses and HAV and removed CPV, a small non-enveloped virus, to a very high degree. Further manufacturing steps reliably contribute to the overall virus reduction capacity of the manufacturing process of this FVIII concentrate.Two different prion preparations prepared from brain homogenate of 263K infected hamsters were used in prion evaluation studies: a membrane-associated preparation of prion material (microsomes) and a PrPSc preparation without membranes (purified PrPSc) were used to evaluate the prion reduction capacity of the FVIII production process. Studying the whole manufacturing process in a combined step approach documented the removal of both spike preparations below the limit of detection. Prion material in the different fractions was quantified using a biochemical/serological method, the Conformation-Dependent Immunoassay (CDI). Spiking different manufacturing steps independently and adding the individual reduction factors to the overall prion reduction factor was considered not appropriate as conditioning of the spiked prion material by the production process may impact the reduction capacity.The pathogen reduction capacity for the FVIII concentrate Beriate is shown in the following table:Based on the epidemiology in the donor population and donation frequency, the sensitivity of the NAT assay, the amount of plasma needed to produce one vial of FVIII concentrate and the virus reduction factors demonstrated, it can be concluded that the measures taken result in a FVIII concentrate [Beriate] with a very high margin of safety for a wide range of viruses and other pathogens. Disclosures:Groener:CSL Behring: Employment. Nowak:CSL Behring: Employment. Popp:CSL Behring: Employment. Schäfer:CSL Behring: Employment.Manufacturing stepsPathogen Reduction Factor [log10]PrionsHIVBVDVPRVHAVParvoviruses*Microsomespurified PrPScCryoprecipitationAdsorption to Al(OH)3/IEX resin≥ 3.6≥ 4.1Heat treatment (Pasteurization)≥6.8≥9.34.73.9≥3.81IEX chromatography3.33.02.11.33.4220N virus filtration≥6.0≥5.8≥7.2≥5.53.42Formulation/sterile filtration/lyophilizationOverall Reduction≥16.1≥18.1≥14.0≥10.7≥10.6≥3.6≥4.1*parvoviruses used in virus validation studies1B19V (human parvovirus B19),2CPV (animal parvovirus: canine parvovirus)

Achieving high mass‐throughput of therapeutic proteins through parvovirus retentive filters

Parvovirus retentive filters that assure removal of viruses and virus-like particles during the production of therapeutic proteins significantly contribute to total manufacturing costs. Operational approaches that can increase throughput and reduce filtration area would result in a significant cost savings. A combination of methods was used to achieve high throughputs of an antibody or therapeutic protein solution through three parvovirus retentive filters. These methods included evaluation of diatomaceous earth or size-based prefilters, the addition of additives, and the optimization of protein concentration, temperature, buffer composition, and solution pH. An optimum temperature of 35°C was found for maximizing throughput through the Virosart CPV and Viresolve Pro filters. Mass-throughput values of 7.3, 26.4, and 76.2 kg/m(2) were achieved through the Asahi Planova 20N, Virosart CPV, and Viresolve Pro filters, respectively, in 4 h of processing. Mass-throughput values of 73, 137, and 192 kg/m(2) were achieved through a Millipore Viresolve Pro filter in 4.0, 8.8, and 22.1 h of processing, respectively, during a single experiment. However, large-scale parvovirus filtration operations are typically controlled to limit volumetric throughput to below the level achieved during small-scale virus spiking experiments. The virus spike may cause significant filter plugging, limiting throughput. Therefore newer parvovirus filter spiking strategies should be adopted that may lead to more representative viral clearance data and higher utilization of large-scale filter capacity.

Effect of antibody solution conditions on filter performance for virus removal filter Planova™ 20N

We investigated the effect of antibody solution conditions (ionic strength, pH, IgG concentration, buffer composition, and aggregate level (dimer content)) on filter performance for a virus removal filtration process using the Planova 20N, a virus removal filter. Ionic strength and pH affected the filter flux. A consistent high flux was maintained at an ionic strength greater than 10 mM and at pH 4-8 under a typical buffer composition (sodium chloride, citrate, acetate, and phosphate). Optimum IgG concentration was 10-20 mg/mL allowing for high throughput (kg/m(2) of IgG). Dimer content negligibly affected the flux level. Under high throughput conditions, virus spiking did not affect flux whereas a parvovirus logarithmic reduction value greater than 5 was maintained. From the results of zeta potential analyses for IgG and the membrane, we considered that electrostatic interactions between antibodies and the membrane affect filter performance (flux level and throughput). These results indicate that the Planova 20N filter is applicable for a wide range of solution conditions typically used in antibody processing.

Second‐generation nanofiltered plasma‐derived mannan‐binding lectin product: process and characteristics

Mannan-binding lectin (MBL) is an important component of the innate immune defence; it binds to carbohydrate structures on pathogenic micro-organisms resulting in complement activation and opsonization. Individuals with low MBL levels are at risk of recurrent and severe infections. Substitution therapy with plasma-derived MBL is a promising treatment of diseases associated with MBL deficiency. A first-generation MBL product has been shown to be safe and well tolerated, and patients have benefited from MBL treatment. Following is a description of the development of a nanofiltered second-generation MBL product from Cohn fraction III, with the use of a new affinity matrix for MBL purification and the characteristics of this improved product. Carbohydrate-based gels were comparatively screened as affinity matrices. MBL was extracted from fraction III, and affinity purified on a Superdex 200 pg column. The eluted material underwent two virus reduction steps: filtration through Planova 20N and solvent/detergent treatment. It was further purified by anion-exchange and gel-filtration chromatography. The affinity eluate and the final MBL fraction were characterized by protein chemical, immunological, and functional assays. In production scale, Superdex 200 pg was found to be superior to other carbohydrate-based matrices, and MBL was affinity purified from fraction III with a yield of 70%. The viral safety was increased by performing a nanofiltration of the affinity eluate through Planova 20N with a minimal loss of MBL. The purity of the final MBL fraction was 53% excluding the MBL-associated serine proteases (MASP). The product consisted of high-oligomeric MBL, with two dominating forms, and with MASP-1, -2, -3 and 19 kDa MBL-associated protein (MAp19). Only a few protein impurities were present, the major being alpha2-macroglobulin. MBL formed complexes with alpha2-macroglobulin bridged by MASP-1 covalently attached to the latter. The functional activity, assessed by mannan-binding activity and opsonic function, was intact, whereas half of the C4 activating capacity was lost during the production process. A second-generation MBL process was developed with an average yield of 50%. It was possible to nanofilter the MBL-MASP complexes through Planova 20N with only a minor loss resulting in an increased safety profile of this MBL product.