Titers In Cell Culture Research Articles

Definition and dynamic control of a continuous chromatography process independent of cell culture titer and impurities

Advances in cell culture technology have enabled the production of antibody titers upwards of 30g/L. These highly productive cell culture systems can potentially lead to productivity bottlenecks in downstream purification due to lower column loadings, especially in the primary capture chromatography step. Alternative chromatography solutions to help remedy this bottleneck include the utilization of continuous processing systems such as periodic counter-current chromatography (PCC).Recent studies have provided methods to optimize and improve the design of PCC for cell culture titers up to about 3g/L. This paper defines a continuous loading strategy for PCC that is independent of cell culture background and encompasses cell culture titers up to about 31g/L. Initial experimentation showed a challenge with determining a difference in change in UV280nm signal (ie. ΔUV) between cell culture feed and monoclonal antibody (mAb) concentration. Further investigation revealed UV280nm absorbance of the cell culture feedstock without antibody was outside of the linear range of detection for a given cell pathlength. Additional experimentation showed the difference in ΔUV for various cell culture feeds can be either theoretically predicted by Beer’s Law given a known absorbance of the media background and impurities or experimentally determined using various UV280nm cell pathlengths. Based on these results, a 0.35mm pathlength at UV280nm was chosen for dynamic control to overcome the background signal. The pore diffusion model showed good agreement with the experimental frontal analysis data, which resulted in definition of a ΔUV setpoint range between 20 and 70% for 3C-PCC experiments. Product quality of the elution pools was acceptable between various cell culture feeds and titers up to about 41g/L. Results indicated the following ΔUV setpoints to achieve robust dynamic control and maintain 3C-PCC yield: ∼20-45% for titers greater than 10g/L depending on UV absorbance of the HCCF and ∼45-70% for titers of up to 10g/L independent of UV absorbance of the HCCF. The strategy and results presented in this paper show column loading in a continuous chromatography step can be dynamically controlled independent of the cell culture feedstock and titer, and allow for enhanced process control built into the downstream continuous operations.

Transgene expression in the genome of Middle East respiratory syndrome coronavirus based on a novel reverse genetics system utilizing Red-mediated recombination cloning.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a high-priority pathogen in pandemic preparedness research. Reverse genetics systems are a valuable tool to study viral replication and pathogenesis, design attenuated vaccines and create defined viral assay systems for applications such as antiviral screening. Here we present a novel reverse genetics system for MERS-CoV that involves maintenance of the full-length viral genome as a cDNA copy inserted in a bacterial artificial chromosome amenable to manipulation by homologue recombination, based on the bacteriophage λ Red recombination system. Based on a full-length infectious MERS-CoV cDNA clone, optimal genomic insertion sites and expression strategies for GFP were identified and used to generate a reporter MERS-CoV expressing GFP in addition to the complete set of viral proteins. GFP was genetically fused to the N-terminal part of protein 4a, from which it is released during translation via porcine teschovirus 2A peptide activity. The resulting reporter virus achieved titres nearly identical to the wild-type virus 48 h after infection of Vero cells at m.o.i. 0.001 (1×105 p.f.u. ml-1 and 3×105 p.f.u. ml-1, respectively), and allowed determination of the 50 % inhibitory concentration for the known MERS-CoV inhibitor cyclosporine A based on fluorescence readout. The resulting value was 2.41 µM, which corresponds to values based on wild-type virus. The reverse genetics system described herein can be efficiently mutated by Red-mediated recombination. The GFP-expressing reporter virus contains the full set of MERS-CoV proteins and achieves wild-type titres in cell culture.

A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection

ABSTRACT: Few studies have described enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against porcine circovirus type 2 (PCV2) based on antigens produced in cell culture. Furthermore, few articles have described viral purification techniques for members of the family Circoviridae. This occurs because circoviruses are difficult to isolate, noncytopathogenic, and produce low viral titres in cell culture. Thus, for overcoming these difficulties in the cultivation of PCV2, this study aimed to develop a double-antibody sandwich ELISA based on the cell culture antigen PCV2b for the quantification of anti-PCV2 antibodies. A 20% and 50% discontinuous sucrose cushion was used for viral purification, which enabled the separation of cell culture proteins in the 20% sucrose cushion and a greater viral concentration in the 50% sucrose cushion. Following isopycnic centrifugation, PCV2 was concentrated in the band with density values from 1.330 to 1.395g/cm3. Viral purification was assessed using SDS-PAGE, indirect ELISA and electron microscopy. The standardised ELISA revealed a strong linear correlation (r= 0.826, p<0.001) when compared with a commercial ELISA kit. The assay exhibited low variability (inter-assay coefficient of variation of 4.24% and intra-assay of 1.80%) and excellent analytical specificity conferred by the capture antibody produced in rabbit. Thus, this ELISA is a rapid, specific and convenient method for the detection of antibodies against PCV2 in studies of experimental and natural infection, and in monitoring the response to vaccination on commercial farms.

Pool-less processing to streamline downstream purification of monoclonal antibodies.

With cell culture titers and productivity increasing in the last few years, pressure has been placed on downstream purification to look at alternative strategies to meet the demand of biotech products with high dose requirements. Even when the upstream process is not continuous (perfusion based), adopting a more productive and/or continuous downstream process can be of significant advantage. Due to the recent trend in exploring continuous processing options for biomolecules, several enabling technologies have been assessed at Biogen. In this paper, we evaluate the capability of one of these technologies to streamline and improve our downstream mAb purification platform. Current conventional downstream polishing steps at Biogen are operated in flow-through mode to achieve higher loadings while maintaining good selectivity. As titers increase, this would result in larger columns and larger intermediate product pool holding tanks. A semicontinuous downstream process linking the second and third chromatography steps in tandem can reduce/eliminate intermediate holding tanks, reduce overall processing time, and combine unit operations to reduce validation burdens. A pool-less processing technology utilizing inline adjustment functionality was evaluated to address facility fit challenges for three high titer mAbs. Two different configurations of polishing steps were examined: (i) anion exchange and hydrophobic interaction and (ii) anion exchange and mixed mode chromatography. Initial laboratory scale proof of concept studies showed comparable performance between the batch purification process and the pool-less process configuration.

Genetic Modification of Oncolytic Newcastle Disease Virus for Cancer Therapy.

Clinical development of a mesogenic strain of Newcastle disease virus (NDV) as an oncolytic agent for cancer therapy has been hampered by its select agent status due to its pathogenicity in avian species. Using reverse genetics, we have generated a lead candidate oncolytic NDV based on the mesogenic NDV-73T strain that is no longer classified as a select agent for clinical development. This recombinant NDV has a modification at the fusion protein (F) cleavage site to reduce the efficiency of F protein cleavage and an insertion of a 198-nucleotide sequence into the HN-L intergenic region, resulting in reduced viral gene expression and replication in avian cells but not in mammalian cells. In mammalian cells, except for viral polymerase (L) gene expression, viral gene expression is not negatively impacted or increased by the HN-L intergenic insertion. Furthermore, the virus can be engineered to express a foreign gene while still retaining the ability to grow to high titers in cell culture. The recombinant NDV selectively replicates in and kills tumor cells and is able to drive potent tumor growth inhibition following intratumoral or intravenous administration in a mouse tumor model. The candidate is well positioned for clinical development as an oncolytic virus. Avian paramyxovirus type 1, NDV, has been an attractive oncolytic agent for cancer virotherapy. However, this virus can cause epidemic disease in poultry, and concerns about the potential environmental and economic impact of an NDV outbreak have precluded its clinical development. Here we describe generation and characterization of a highly potent oncolytic NDV variant that is unlikely to cause Newcastle disease in its avian host, representing an essential step toward moving NDV forward as an oncolytic agent. Several attenuation mechanisms have been genetically engineered into the recombinant NDV that reduce chicken pathogenicity to a level that is acceptable worldwide without impacting viral production in cell culture. The selective tumor replication of this recombinant NDV, both in vitro and in vivo, along with efficient tumor cell killing makes it an attractive oncolytic virus candidate that may provide clinical benefit to patients.

Verapamil has Antiviral Activities that Target Different Steps of the Influenza Virus Replication Cycle

Safe and efficacious therapeutic options for infections caused by highly pathogenic influenza viruses (IV) remain limited. Viruses resistant to approved antiviral drugs are increasingly emerging and, in some cases, existing vaccines are insufficient to prevent seasonal epidemics or pandemics caused by these viruses. As a consequence, alternative antiviral strategies that, for example, target cellular factors or mechanisms essential for IV propagation have attracted increasing interest over the past years. Here, we characterized antiviral effects of the calcium channel blocker Verapamil on different steps of the IV replication cycle. Our data suggest that Verapamil (i) efficiently reduces influenza A and B virus titres in cell culture at non-toxic concentrations, (ii) does not influence virus entry, (iii) impairs the replication and transcription activity of the viral polymerase, (iv) decreases viral protein production, (v) reduces virus-induced NF-κB activation and (vi) does not induce the emergence of resistant IV variants, resulting in strongly reduced infectious particle formation.

Synonymous Deoptimization of Foot-and-Mouth Disease Virus Causes Attenuation In Vivo while Inducing a Strong Neutralizing Antibody Response.

Codon bias deoptimization has been previously used to successfully attenuate human pathogens, including poliovirus, respiratory syncytial virus, and influenza virus. We have applied a similar technology to deoptimize the capsid-coding region (P1) of foot-and-mouth disease virus (FMDV). Despite the introduction of 489 nucleotide changes (19%), synonymous deoptimization of the P1 region rendered a viable FMDV progeny. The resulting strain was stable and reached cell culture titers similar to those obtained for wild-type (WT) virus, but at reduced specific infectivity. Studies in mice showed that 100% of animals inoculated with the FMDV A12 P1 deoptimized mutant (A12-P1 deopt) survived, even when the animals were infected at doses 100 times higher than the dose required to cause death by WT virus. All mice inoculated with the A12-P1 deopt mutant developed a strong antibody response and were protected against subsequent lethal challenge with WT virus at 21 days postinoculation. Remarkably, the vaccine safety margin was at least 1,000-fold higher for A12-P1 deopt than for WT virus. Similar patterns of attenuation were observed in swine, in which animals inoculated with A12-P1 deopt virus did not develop clinical disease until doses reached 1,000 to 10,000 times the dose required to cause severe disease in 2 days with WT A12. Consistently, high levels of antibody titers were induced, even at the lowest dose tested. These results highlight the potential use of synonymous codon pair deoptimization as a strategy to safely attenuate FMDV and further develop live attenuated vaccine candidates to control such a feared livestock disease. Foot-and-mouth disease (FMD) is one of the most feared viral diseases that can affect livestock. Although this disease appeared to be contained in developed nations by the end of the last century, recent outbreaks in Europe, Japan, Taiwan, South Korea, etc., have demonstrated that infection can spread rapidly, causing devastating economic and social consequences. The Global Foot-and-Mouth Disease Research Alliance (GFRA), an international organization launched in 2003, has set as part of their five main goals the development of next-generation control measures and strategies, including improved vaccines and biotherapeutics. Our work demonstrates that newly developed codon pair bias deoptimization technologies can be applied to FMD virus to obtain attenuated strains with potential for further development as novel live attenuated vaccine candidates that may rapidly control disease without reverting to virulence.

Concentrated fed-batch cell culture increases manufacturing capacity without additional volumetric capacity

Biomanufacturing factories of the future are transitioning from large, single-product facilities toward smaller, multi-product, flexible facilities. Flexible capacity allows companies to adapt to ever-changing pipeline and market demands. Concentrated fed-batch (CFB) cell culture enables flexible manufacturing capacity with limited volumetric capacity; it intensifies cell culture titers such that the output of a smaller facility can rival that of a larger facility. We tested this hypothesis at bench scale by developing a feeding strategy for CFB and applying it to two cell lines. CFB improved cell line A output by 105% and cell line B output by 70% compared to traditional fed-batch (TFB) processes. CFB did not greatly change cell line A product quality, but it improved cell line B charge heterogeneity, suggesting that CFB has both process and product quality benefits. We projected CFB output gains in the context of a 2000-L small-scale facility, but the output was lower than that of a 15,000-L large-scale TFB facility. CFB’s high cell mass also complicated operations, eroded volumetric productivity, and showed our current processes require significant improvements in specific productivity in order to realize their full potential and savings in manufacturing. Thus, improving specific productivity can resolve CFB’s cost, scale-up, and operability challenges.

A novel multiplex RT-qPCR method based on dual-labelled probes suitable for typing all known genotypes of viral haemorrhagic septicaemia virus.

Viral haemorrhagic septicaemia (VHS) is a notifiable fish disease, whose causative agent is a rhabdovirus isolated from a wide range of fish species, not only in fresh but also in marine and brackish waters. Phylogenetic studies have identified four major genotypes, with a strong geographical relationship. In this study, we have designed and validated a new procedure--named binary multiplex RT-qPCR (bmRT-qPCR)--for simultaneous detection and typing of all four genotypes of VHSV by real-time RT-PCR based on dual-labelled probes and composed by two multiplex systems designed for European and American/Asiatic isolates, respectively, using a combination of three different fluorophores. The specificity of the procedure was assessed by including a panel of 81 VHSV isolates covering all known genotypes and subtypes of the virus, and tissue material from experimentally infected rainbow trout, resulting in a correct detection and typing of all strains. The analytical sensitivity was evaluated in a comparative assay with titration in cell culture, observing that both methods provided similar limits of detection. The proposed method can be a powerful tool for epidemiological analysis of VHSV by genotyping unknown samples within a few hours.

Growth kinetics and transmission potential of existing and emerging field strains of infectious laryngotracheitis virus.

Attenuated live infectious laryngotracheitis virus (ILTV) vaccines are widely used in the poultry industry to control outbreaks of disease. Natural recombination between commercial ILTV vaccines has resulted in virulent recombinant viruses that cause severe disease, and that have now emerged as the dominant field strains in important poultry producing regions in Australia. Genotype analysis using PCR—restriction fragment length polymorphism has shown one recombinant virus (class 9) has largely replaced the previously dominant class 2 field strain. To examine potential reasons for this displacement we compared the growth kinetics and transmission potential of class 2 and class 9 viruses. The class 9 ILTV grew to higher titres in cell culture and embryonated eggs, but no differences were observed in entry kinetics or egress into the allantoic fluid from the chorioallantoic membrane. In vivo studies showed that birds inoculated with class 9 ILTV had more severe tracheal pathology and greater weight loss than those inoculated with the class 2 virus. Consistent with the predominance of class 9 field strains, birds inoculated with 102 or 103 plaque forming units of class 9 ILTV consistently transmitted virus to in-contact birds, whereas this could only be seen in birds inoculated with 104 PFU of the class 2 virus. Taken together, the improved growth kinetics and transmission potential of the class 9 virus is consistent with improved fitness of the recombinant virus over the previously dominant field strain.

Induction of antibodies and T cell responses by a recombinant influenza virus carrying an HIV-1 TatΔ51-59 protein in mice.

Recombinant influenza viruses hold promise as vectors for vaccines to prevent transmission of mucosal pathogens. In this study, we generated a recombinant WSN/TatΔ51–59 virus in which Tat protein lacking residues 51 to 59 of the basic domain was inserted into the N-terminus of the hemagglutinin (HA) of A/WSN/33 virus. The TatΔ51–59 insertion into the viral HA caused a 2-log reduction in viral titers in cell culture, compared with the parental A/WSN/33 virus, and severely affected virus replication in vivo. Nevertheless, Tat-specific antibodies and T cell responses were elicited upon a single intranasal immunization of BALB/c mice with WSN/TatΔ51–59 virus. Moreover, Tat-specific immune responses were also detected following vaccine administration via the vaginal route. These data provide further evidence that moderately large HIV antigens can be delivered by chimeric HA constructs and elicit specific immune responses, thus increasing the options for the potential use of recombinant influenza viruses, and their derivatives, for prophylactic and therapeutic vaccines.

Discrimination of infectious hepatitis A virus and rotavirus by combining dyes and surfactants with RT-qPCR.

BackgroundHuman enteric viruses are major agents of foodborne diseases. Because of the absence of a reliable cell culture method for most of the enteric viruses involved in outbreaks, real-time reverse transcriptase PCR is now widely used for the detection of RNA viruses in food samples. However this approach detects viral nucleic acids of both infectious and non infectious viruses, which limits the impact of conclusions with regard to public health concern. The aim of the study was to develop a method to discriminate between infectious and non-infectious particles of hepatitis A virus (HAV) and two strains of rotavirus (RV) following thermal inactivation by using intercalating dyes combined with RT-qPCR.ResultsOnce the binding of propidium monoazide (PMA) or ethidium monoazide (EMA) was shown to be effective on the viral ssRNA of HAV and dsRNA of two strains of RV (SA11 and Wa), their use in conjunction with three surfactants (IGEPAL CA-630, Tween 20, Triton X-100) prior to RT-qPCR assays was evaluated to quantify the infectious particles remaining following heat treatment. The most promising conditions were EMA (20 μM) and IGEPAL CA-630 (0.5%) for HAV, EMA (20 μM) for RV (WA) and PMA (50 μM) for RV (SA11). The effectiveness of the pre-treatment RT-qPCR developed for each virus was evaluated with three RT-qPCR assays (A, B, C) during thermal inactivation kinetics (at 37°C, 68 C, 72°C, 80°C) through comparison with data obtained by RT-qPCR and by infectious titration in cell culture. At 37°C, the quantity of virus (RV, HAV) remained constant regardless of the method used. The genomic titers following heat treatment at 68°C to 80°C became similar to the infectious titers only when a pre-treatment RT-qPCR was used. Moreover, the most effective decrease was obtained by RT-qPCR assay A or B for HAV and RT-qPCR assay B or C for RV.ConclusionsWe concluded that effectiveness of the pre-treatment RT-qPCR is influenced by the viral target and by the choice of the RT-qPCR assay. Currently, it would be appropriate to further develop this approach under specific conditions of inactivation for the identification of infectious viruses in food and environmental samples.

Live attenuated rubella vectors expressing SIV and HIV vaccine antigens replicate and elicit durable immune responses in rhesus macaques

BackgroundLive attenuated viruses are among our most potent and effective vaccines. For human immunodeficiency virus, however, a live attenuated strain could present substantial safety concerns. We have used the live attenuated rubella vaccine strain RA27/3 as a vector to express SIV and HIV vaccine antigens because its safety and immunogenicity have been demonstrated in millions of children. One dose protects for life against rubella infection. In previous studies, rubella vectors replicated to high titers in cell culture while stably expressing SIV and HIV antigens. Their viability in vivo, however, as well as immunogenicity and antibody persistence, were unknown.ResultsThis paper reports the first successful trial of rubella vectors in rhesus macaques, in combination with DNA vaccines in a prime and boost strategy. The vectors grew robustly in vivo, and the protein inserts were highly immunogenic. Antibody titers elicited by the SIV Gag vector were greater than or equal to those elicited by natural SIV infection. The antibodies were long lasting, and they were boosted by a second dose of replication-competent rubella vectors given six months later, indicating the induction of memory B cells.ConclusionsRubella vectors can serve as a vaccine platform for safe delivery and expression of SIV and HIV antigens. By presenting these antigens in the context of an acute infection, at a high level and for a prolonged duration, these vectors can stimulate a strong and persistent immune response, including maturation of memory B cells. Rhesus macaques will provide an ideal animal model for demonstrating immunogenicity of novel vectors and protection against SIV or SHIV challenge.

A Plant Extract of Ribes nigrum folium Possesses Anti-Influenza Virus Activity In Vitro and In Vivo by Preventing Virus Entry to Host Cells

Infections with influenza A viruses (IAV) are still amongst the major causes of highly contagious severe respiratory diseases not only bearing a devastating effect to human health, but also significantly impact the economy. Besides vaccination that represents the best option to protect from IAV infections, only two classes of anti-influenza drugs, inhibitors of the M2 ion channel and the neuraminidase, often causing resistant IAV variants have been approved. That is why the need for effective and amply available antivirals against IAV is of high priority. Here we introduce LADANIA067 from the leaves of the wild black currant (Ribes nigrum folium) as a potent compound against IAV infections in vitro and in vivo. LADANIA067 treatment resulted in a reduction of progeny virus titers in cell cultures infected with prototype avian and human influenza virus strains of different subtypes. At the effective dose of 100 µg/ml the extract did not exhibit apparent harming effects on cell viability, metabolism or proliferation. Further, viruses showed no tendency to develop resistance to LADANIA067 when compared to amantadine that resulted in the generation of resistant variants after only a few passages. On a molecular basis the protective effect of LADANIA067 appears to be mainly due to interference with virus internalisation. In the mouse infection model LADANIA067 treatment reduces progeny virus titers in the lung upon intranasal application. In conclusion, an extract from the leaves of the wild black currant might be a promising source for the development of new antiviral compounds to fight IAV infections.

Virus quantitation by transmission electron microscopy, TCID50, and the role of timing virus harvesting: A case study of three animal viruses

Quantitation of viruses is practised widely in both basic and applied virology. Infectious titration in cell cultures, the most common approach to it, is quite labour-intensive and alternative protocols are therefore sought. One of the alternatives is transmission electron microscope (TEM) quantitation using latex particles at a known concentration as a reference for counting virus particles. If virus TCID50 is determined in parallel, the ratio of infectious to non-infectious virus particles may be established. This study employs such an approach to compute the number of virus particles and TCID50, and establish their correlation for three viruses: Canine adenovirus 1 (CAdV-1), Feline calicivirus (FCV) and Bovine herpesvirus 1 (BoHV-1). Each of the viruses was grown in five replicates until complete cytopathology was recorded (time 0), then frozen. They were thawed, filter-sterilised and left for additional periods of 16, 32 and 48h at 37°C. At each time point, the infectious ability of the virus was characterised by TCID50 and the number of virions quantified by TEM, in order to evaluate the influence of timing on virus harvest. The virus particle count determined by TEM did not change for any of the viruses throughout the experiment. The relationship between virus particle counts with TCID50 at time 0 showed good linearity response; their ratio was almost constant. The virus particle-to-TCID50 ratio varied between 146 and 426 (mean±SD: 282±103) for CAdV-1, between 36 and 79 (57±18) for FCV and between 110 and 249 (167±53) for BoHV-1. The proportion of non-infectious particles did not change throughout the experiment for either CAdV-1 or BoHV-1. However, a decrease in virus infectious ability disclosed by TCID50 indicated that the fraction of non-infectious particles in FCV increased 300,000 times when time 0 and 48h were compared. The quantitation of viruses with TEM is a simple and rapid protocol for virus quantitation but account must be taken of the type of virus and harvesting time as virus counts need not necessarily correlate with virus infectious ability.

Integration of stochastic simulation with multivariate analysis: Short-term facility fit prediction

This article describes a decision-support tool to help pinpoint the potential root causes of sub-optimal short-term facility fit issues in biopharmaceutical facilities. This was achieved by creating a tool that integrated stochastic simulation with advanced multivariate statistical analysis. Process fluctuations in product titers in cell culture, step yields, and chromatography eluate volumes were mimicked using Monte Carlo simulation data derived using a stochastic discrete-event simulation model. The resulting stochastic datasets, with the computed consequences on key metrics such as product mass loss and cost of goods, were examined using advanced multivariate statistical techniques. Principal component analysis combined with clustering algorithms was used to analyze the complex datasets from complete industrial batch processes for biopharmaceuticals. The challenge of visualizing the multidimensional nature of the dataset was addressed using hierarchical and k-means clustering as well as stacked parallel co-ordinate plots to help identify process fingerprints and characteristics of clusters leading to sub-optimal facility fit issues. Industrially-relevant case studies are presented that focus on technology transfer challenges for therapeutic antibodies moving from early phase to late phase clinical trials. The case study details how sub-optimal facility fit can be alleviated by allocating alternative product pool tanks. The impact of this operational change is then assessed by reviewing an updated process fingerprint.

Evaluation of the effectiveness of semen processing techniques to remove bovine viral diarrhea virus from experimentally contaminated semen samples

The aim of this study was to evaluate the capacity of three semen processing techniques, Percoll gradient centrifugation, Swim-up and a combination of Swim-up and Percoll gradient centrifugation, to reduce the viral load of bovine viral diarrhea virus (BVDV) in experimentally infected semen samples. The evaluation was performed using two approaches: first, searching for the presence of virus in the processed samples (via virus titration and RT-PCR) and second, ascertaining the possible interference on in vitro embryo production. The sperm count and DNA integrity (Comet assay) of the processed samples were analyzed (Experiment 1). The amount of virus in the processed samples was determined by titration in cell culture (Experiment 2). The samples processed by Swim up/Percoll gradient centrifugation were utilized for in vitro embryo production, and the embryos produced were tested for BVDV by RT-PCR (Experiment 3). Sperm concentration, Comet assay and embryo production were analyzed by chi-squared tests (P<0.05). There was a significant difference between sperm separation techniques when the sperm count and Comet assay were analyzed. The sperm count obtained from the Swim up/Percoll gradient centrifugation group was lower than that obtained in either of the two other groups (Swim up and Percoll gradient centrifugation), and the Comet assay showed that the combination of the two semen processing techniques (Swim up/Percoll gradient) produced a 1.1% prevalence of Comet level 2, which was not observed in the other groups. The BVDV titer (10(6.68)TCID(50)/mL) added to experimentally infected semen samples decreased after Percoll gradient centrifugation to 10(2.3)-10(1)TCID(50)/mL; for the Swim up group, the titer range was 10(3.3)-10(1.87)TCID(50)/mL, and in the Swim up/Percoll gradient centrifugation group, BVDV was undetectable. The decreases in titer varied from 99.9% in the Swim up-processed group to 100% in the Swim up/Percoll gradient centrifugation group. In vitro embryo production displayed similar blastocyst development rates among all groups, and RT-PCR was negative for the produced embryos. The data showed that the combination of Swim up/Percoll gradient centrifugation promoted the elimination of BVDV from the semen samples without damaging spermatozoa cells and also allowed successful in vitro embryo production free of BVDV. Hence, the risk of BVDV contamination is negligible for the embryo recipient.

Decisional tool to assess current and future process robustness in an antibody purification facility

Increases in cell culture titers in existing facilities have prompted efforts to identify strategies that alleviate purification bottlenecks while controlling costs. This article describes the application of a database-driven dynamic simulation tool to identify optimal purification sizing strategies and visualize their robustness to future titer increases. The tool harnessed the benefits of MySQL to capture the process, business, and risk features of multiple purification options and better manage the large datasets required for uncertainty analysis and optimization. The database was linked to a discrete-event simulation engine so as to model the dynamic features of biopharmaceutical manufacture and impact of resource constraints. For a given titer, the tool performed brute force optimization so as to identify optimal purification sizing strategies that minimized the batch material cost while maintaining the schedule. The tool was applied to industrial case studies based on a platform monoclonal antibody purification process in a multisuite clinical scale manufacturing facility. The case studies assessed the robustness of optimal strategies to batch-to-batch titer variability and extended this to assess the long-term fit of the platform process as titers increase from 1 to 10 g/L, given a range of equipment sizes available to enable scale intensification efforts. Novel visualization plots consisting of multiple Pareto frontiers with tie-lines connecting the position of optimal configurations over a given titer range were constructed. These enabled rapid identification of robust purification configurations given titer fluctuations and the facility limit that the purification suites could handle in terms of the maximum titer and hence harvest load.

Functional Analysis of Conserved Motifs in Influenza Virus PB1 Protein

The influenza virus RNA polymerase complex is a heterotrimer composed of the PB1, PB2, and PA subunits. PB1, the catalytic core and structural backbone of the polymerase, possesses four highly conserved amino acid motifs that are present among all viral RNA-dependent RNA polymerases. A previous study demonstrated the importance of several of these conserved amino acids in PB1 for influenza polymerase activity through mutational analysis. However, a small number of viruses isolated in nature possesses non-consensus amino acids in one of the four motifs, most of which have not been tested for their replicative ability. Here, we assessed the transcription/replication activities of 25 selected PB1 mutations found in natural isolates by using minireplicon assays in human and avian cells. Most of the mutations tested significantly reduced polymerase activity. One exception was mutation K480R, observed in several pandemic (H1N1) 2009 viruses, which slightly increased polymerase activity relative to wild-type. However, in the background of the pandemic A/California/04/2009 (H1N1) virus, this mutation did not affect virus titers in cell culture. Our results further demonstrate the functional importance of the four conserved PB1 motifs in influenza virus transcription/replication. The finding of natural isolates with non-consensus PB1 motifs that are nonfunctional in minireplicon assays suggests compensatory mutations and/or mixed infections which may have ‘rescued’ the inactive PB1 protein.

Single pass tangential flow filtration to debottleneck downstream processing for therapeutic antibody production

As the therapeutic monoclonal antibody (mAb) market continues to grow, optimizing production processes is becoming more critical in improving efficiencies and reducing cost-of-goods in large-scale production. With the recent trends of increasing cell culture titers from upstream process improvements, downstream capacity has become the bottleneck in many existing manufacturing facilities. Single Pass Tangential Flow Filtration (SPTFF) is an emerging technology, which is potentially useful in debottlenecking downstream capacity, especially when the pool tank size is a limiting factor. It can be integrated as part of an existing purification process, after a column chromatography step or a filtration step, without introducing a new unit operation. In this study, SPTFF technology was systematically evaluated for reducing process intermediate volumes from 2× to 10× with multiple mAbs and the impact of SPTFF on product quality, and process yield was analyzed. Finally, the potential fit into the typical 3-column industry platform antibody purification process and its implementation in a commercial scale manufacturing facility were also evaluated. Our data indicate that using SPTFF to concentrate protein pools is a simple, flexible, and robust operation, which can be implemented at various scales to improve antibody purification process capacity.