Characterizing clearance of helper adenovirus by a clinical rAAV1 manufacturing process.

Recombinant adeno-associated viral vectors (rAAV) are being developed as gene therapy delivery vehicles and as genetic vaccines, and some of the most scaleable manufacturing methods for rAAV use live adenovirus to induce production. One aspect of establishing safety of rAAV products is therefore demonstrating adequate and reliable clearance of this helper virus by the vector purification process. The ICH Q5A regulatory guidance on viral safety provides recommendations for process design and characterization of viral clearance for recombinant proteins, and these principles were adapted to a rAAV serotype 1 purification process for clinical vectors. Specific objectives were to achieve overall adenovirus clearance factors significantly greater than input levels by using orthogonal separation and inactivation methods, and to segregate adenovirus from downstream operations by positioning a robust clearance step early in the process. Analytical tools for process development and characterization addressed problematic in-process samples, and a viral clearance validation study was performed using adenovirus and two non-specific model viruses. Overall clearance factors determined were >23 LRV for adenovirus, 11 LRV for BVDV, and >23 LRV for AMuLV.     

Anion exchange chromatography provides a robust, predictable process to ensure viral safety of biotechnology products

The mammalian cell-lines used to produce biopharmaceutical products are known to produce endogenous retrovirus-like particles and have the potential to foster adventitious viruses as well. To ensure product safety and regulatory compliance, recovery processes must be capable of removing or inactivating any viral impurities or contaminants which may be present. Anion exchange chromatography (AEX) is a common process in the recovery of monoclonal antibody products and has been shown to be effective for viral removal. To further characterize the robustness of viral clearance by AEX with respect to process variations, we have investigated the ability of an AEX process to remove three model viruses using various combinations of mAb products, feedstock conductivities and compositions, equilibration buffers, and pooling criteria. Our data indicate that AEX provides complete or near-complete removal of all three model viruses over a wide range of process conditions, including those typically used in manufacturing processes. Furthermore, this process provides effective viral clearance for different mAb products, using a variety of feedstocks, equilibration buffers, and different pooling criteria. Viral clearance is observed to decrease when feedstocks with sufficiently high conductivities are used, and the limit at which the decrease occurs is dependent on the salt composition of the feedstock. These data illustrate the robust nature of the AEX recovery process for removal of viruses, and they indicate that proper design of AEX processes can ensure viral safety of mAb products.     

Suitability of a generic virus safety evaluation for monoclonal antibody investigational new drug applications

Biologics produced from CHO cell lines with endogenous virus DNA can produce retrovirus-like particles in cell culture at high titers, and other adventitious viruses can find their way through raw materials into the process to make a product. Therefore, it is the industry standard to have controls to avoid introduction of viruses into the production process, to test for the presence of viral particles in unclarified cell culture, and to develop purification procedures to ensure that manufacturing processes are robust for viral clearance. Data have been accumulated over the past four decades on unit operations that can inactivate and clear adventitious virus and provide a high degree of assurance for patient safety. During clinical development, biological products are traditionally tested at process set points for viral clearance. However, the widespread implementation of platform production processes to produce highly similar IgG antibodies for many indications makes it possible to leverage historical data and knowledge from representative molecules to allow for better understanding and control of virus safety. More recently, individualized viral clearance studies are becoming the rate-limiting step in getting new antibody molecules to clinic, particularly in Phase 0 and eIND situations. Here, we explore considerations for application of a generic platform virus clearance strategy that can be applied for relevant investigational antibodies within defined operational parameters in order to increase speed to the clinic and reduce validation costs while providing a better understanding and assurance of process virus safety.     

Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors.

Engineered derivatives of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) possessing a unique restriction site provide a source of viral DNA that can be linearized by digestion with a specific endonuclease. Circular or linearized DNA from two such viruses were compared in terms of their infectivity and recombinogenic activities. The linear forms were 15- to 150-fold less infectious than the corresponding circular forms, when transfected into Spodoptera frugiperda cells using the calcium phosphate method. Linear viral DNA was, however, proficient at recombination on co-transfection with an appropriate transfer vector. Up to 30% of the progeny viruses were recombinant, a 10-fold higher fraction of recombinants than was obtained from co-transfections with circular AcMNPV DNA. The isolation of a recombinant baculovirus expression vector from any of the AcMNPV transfer vectors currently in use can thus be facilitated by linearization of the viral DNA at the appropriate location.     

Benefits and risks of antibody and vaccine production in transgenic plants

Phytopharming, the production of protein biologicals in recombinant plant systems, has shown great promise in studies performed over the past 13 years. A secretory antibody purified from transgenic tobacco was tested successfully in humans, and prevented bacterial re-colonization after topical application in the mouth. Rapid production of patient-tailored anti-lymphoma antibodies in recombinant Tobamovirus-infected tobacco may provide effective cancer therapy. Many different candidate vaccines from bacterial and viral sources have been expressed in transgenic plants, and three human clinical trials with oral delivery of transgenic plant tissues have shown exciting results. The use of crop plants with agricultural practice could allow cheap production of valuable proteins, while providing enhanced safety by avoidance of animal viruses or other contaminants. However development of this technology must carefully consider the means to ensure the separation of food and medicinal products when crop plants are used for phytopharming.     

Cation exchange chromatography performed in overloaded mode is effective in removing viruses during the manufacturing of monoclonal antibodies

Viral safety is a critical concern with regard to monoclonal antibody (mAb) products produced in mammalian cells such as Chinese hamster ovary cells. Manufacturers are required to ensure the safety of such products by validating the clearance of viruses in downstream purification steps. Cation exchange (CEX) chromatography is widely used in bind/elute mode as a polishing step in mAb purification. However, bind/elute modes require a large volume of expensive resin. To reduce the production cost, the use of CEX chromatography in overloaded mode has recently been investigated. The viral clearance ability in overloaded mode was evaluated using murine leukemia virus (MLV). Even under high-load conditions such as 2,000 g mAb/L resin, MLV was removed from mAb solutions. This viral clearance ability was not significantly affected by resin type or mAb type. The overloaded mode can also remove other types of viruses such as pseudorabies virus and reovirus Type 3 from mAb solutions. Based on these results, this cost-effective overloaded mode is comparable to the bind-elute mode in terms of viral removal.     

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.     

Application of methods for viral clearance in stem cell production

Regenerative medicine therapies will allow in the future the transplant of cells of human origin in some diseases that until now have been incurable. The assurance of the safety and quality, especially from a microbiological point of view, is very important for these therapeutic products. Depending on the starting material, there are several sources of pathogen presence, mainly human viruses. Also, the use of feeders of animal origin as layers in which the stem cells can grow may permit the transmission of animal pathogens to these cells. However, cell sources are limited due to the low availability of spare in vitro fecundation human embryos and the low rate of success in the derivation of human stem cell lines. Thus, in several cases, it will be necessary to evaluate the possibility of removing or inactivating these microorganisms. In this paper, we summarize the main methods of viral clearance and we have provided an overview of the main features taking into account in the viral clearance techniques.     

Production scale insect cell culture

Insect cells in culture are currently commanding great interest as superior hosts for the efficient production of biologicals with applications in health care and in agriculture. Insect cell culture is ripe for scale-up technologies, in order to meet future projected production requirements of (a) insect viruses used as bioinsecticides and (b) recombinant proteins of therapeutic potential for humans and animals. The single most prominent system used in research-based and in commercial insect cell culture today involves lepidopteran cells transfected with baculovirus expression vectors for abundant formation of recombinant biologicals. However, dipteran insect cell lines also are beginning to emerge as useful tools in biotechnology. Current practices in bioprocess development using insect cell culture, advances in media formulation and in insect cell bioreactor design, and emerging trends are presented and critically evaluated.     

High-frequency homologous recombination in vaccinia virus DNA.

A recombinant vaccinia virus genome was constructed in which the viral thymidine kinase (tk) gene was placed between direct repeats of a 1.5-kilobase-pair DNA sequence of heterologous origin. When forced to replicate in tk- cells in the presence of methotrexate (i.e., under tk+-selective conditions), the recombinant maintained its tk+ phenotype. Under nonselective conditions, however, the tk gene was frequently excised by both inter- and intramolecular recombination events because the repeated sequences provided substantial targets for homologous DNA recombination. Unique DNA products of intramolecular recombination were detected in the cytoplasm of infected cells soon after the onset of viral DNA replication, and their appearance was blocked by inhibitors of DNA synthesis. During repeated passage of the virus under nonselective conditions, the tk+ fraction decreased with first-order kinetics at a rate that reflected the frequency of recombination per cycle of virus replication. Eventually, a residual population of stable tk+ viruses remained, and analyses of the genome structures of individual members of this population showed that some of them appeared to be the products of nonhomologous DNA recombination.     

Conservation and progressive methylation of Epstein-Barr viral DNA sequences in transformed cells.

The structure of intracellular viral DNA from a number of cell lines arising by clonal transformation of human lymphocytes in vitro with Epstein-Barr virus was analyzed. Intracellular viral DNAs were partially purified and digested with several restriction endonucleases, and the products of digestion were separated by electrophoresis in agarose gels. The viral fragments were detected by transferring the DNA from the gel to nitrocellulose sheets, hybridizing radiolabeled recombinant vectors carrying fragments of viral DNA to those transfers, and visualizing the hybrids by autoradiography. These analyses indicated that: (i) regions of repetitious viral DNA do undergo expansion and contraction although one size predominates; (ii) novel sequence arrangements appear in the intracellular viral DNA of different clones but are not found in clones analyzed serially and propagated extensively; (iii) the viral DNA is increasingly methylated upon cell propagation. We have not identified a transformed cell phenotype or a viral phenotype that segregates with the observed progressive methylation. We have not detected in Epstein-Barr viral plasmids analogs of the gross rearrangements of viral DNAs observed after lytic infections with high multiplicities of papova-, adeno-, or herpes simplex viruses.     

Understanding the mechanism of virus removal by Q sepharose fast flow chromatography during the purification of CHO‐cell derived biotherapeutics

During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non‐electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science‐based process validation strategies to ensure viral safety of biotechnology products. Biotechnol. Bioeng. 2009; 104: 371–380 © 2009 Wiley Periodicals, Inc.     

Multiplex RT Q-PCR assay for simultaneous quantification of three viruses used for validation of virus clearance by biopharmaceutical production

Virus removal studies are used to insure the safety of biopharmaceutical products by quantitatively estimating the viral clearance capacity by the manufacturing process. Virus quantification assays are used to measure the log₁₀ clearance factor of individual purification unit operations in spike recovery studies. We have developed a multiplex RT Q-PCR assay that detects and quantifies three commonly used model viruses X-MuLV, SV40, and MMV simultaneously. This RT Q-PCR multiplex assay has a 6 log₁₀ dynamic range with a limit of detection (LOD) of ≈1 genome copy/μL. Amplification profiles are similar to existing singleplex assays. Overall, this RT Q-PCR multiplex assay is highly quantitative, accurately identifies multiple viruses simultaneously, and may prove useful to validate viral clearance of biological products in small scale studies.     

Biosimilars--global issues, national solutions

Biotechnology derived medicinal products are presently the best characterized biologicals with considerable production and clinical experience, and have revolutionized the treatment of some of the most difficult-to-treat diseases, prolonging and improving the quality of life and patient care. They are also currently one of the fastest growing segments of the pharmaceutical industry market. The critical challenge that the biopharmaceutical industry is facing is the expiry of patents for the first generation of biopharmaceuticals, mainly recombinant DNA derived products, such as interferons, growth hormone and erythropoetin. The question that immediately arose was how should such copies of the originator products be licensed, bearing in mind that they are highly complex biological molecules produced by equally complex biological production processes with their inherent problem of biological variability. Copying biologicals is much more complex than copying small molecules and the critical issue was how to handle the licensing of products if relying in part on data from an innovator product. Since 2004 there has been considerable international consultation on how to deal with biosimilars and biological copy products. This has led to a better understanding of the challenges in the regulatory evaluation of the quality, safety and efficacy of "biosimilars", to the exchange of information between regulators, as well as to the identification of key issues. The aim of this article is to provide a brief overview of the scientific and regulatory challenges faced in developing and evaluating similar biotherapeutic products for global use. It is intended as an introduction to the series of articles in this special issue of Biologicals devoted to similar biotherapeutic products.     

Genetic requirements for homologous recombination in Autographa californica nucleopolyhedrovirus

It is known that baculovirus infection promotes high-frequency recombination between its genomes and plasmid DNA during the construction of recombinant viruses for foreign gene expression. However, little is known about the viral genes necessary to promote homologous recombination (HR). We developed an assay to identify viral genes that are necessary to stimulate HR. In this assay, we used two plasmids containing extensive sequence homology that yielded a visible and quantifiable phenotype if HR occurred. The plasmids contained the green fluorescent protein gene (gfp) that was mutated at either the N or the C terminus and a viral origin of DNA replication. When the plasmids containing these mutant gfp genes were transfected into insect cells alone or together, few green fluorescent protein (GFP)-positive cells were observed, confirming that the host cell machinery alone was not able to promote high levels of HR. However, if viral DNA or viral genes involved in DNA replication were cotransfected into cells along with the mutant gfp-containing plasmids, a dramatic increase in GFP-positive cells was observed. The viral genes ie-1, ie-2, lef-7, and p35 were found to be important for efficient HR in the presence of all other DNA replication genes. However, ie-1 and ie-2 were sufficient to promote HR in the absence of other viral genes. Recombination substrates lacking a viral origin of replication had similar genetic requirements for recombination but were less dependent on ie-1. Interestingly, even though HR was stimulated by the presence of a viral origin of DNA replication, virally stimulated HR could proceed in the presence of the DNA synthesis inhibitor aphidicolin.     

Real time quantitative PCR as a method to evaluate simian virus 40 removal during pharmaceutical protein purification.

Continuous cell lines used for pharmaceutical protein manufacturing have the potential to be contaminated by viruses. To ensure the safety of pharmaceutical proteins derived from continuous cell lines, validation of the ability of the manufacturing process to clear potential contaminating viruses is required for product registration. In this paper, a real time quantitative PCR method has been applied to the evaluation of simian virus 40 (SV40) removal during chromatography and filtration procedures. This method takes advantage of the 5'-3' exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 sequence detection system of PE Applied Biosystems for automated SV40 DNA quantification through a dual-labeled fluorogenic probe. This method provides accurate and reproducible quantification of SV40 DNA. The SV40 clearance during chromatography and filtration procedures determined by this method is highly comparable with that determined by the cell-based infectivity assay. This method offers significant advantages over cell-based infectivity assays, such as higher sensitivity, greater reliability, higher sample throughput and lower cost. This method can be potentially used to evaluate the clearance of all model viruses during chromatography and filtration procedures. This method can be used to substitute cell-based infectivity assays for process validation of viral removal procedures and the availability of this method should greatly facilitate and reduce the cost of viral clearance evaluations required for new biologic product development.     

Rescue of functional replication origins from embedded configurations in a plasmid carrying the adenovirus genome.

A variant of the adenovirus type 5 genome which lacks EcoRI sites has been cloned in a bacterial plasmid after the addition of EcoRI oligonucleotide linkers to its ends. Closed circular forms of the recombinant viral genome were not infectious upon their introduction into permissive eucaryotic cells. The linear genome released by digestion of the 39-kilobase recombinant plasmid (pXAd) with EcoRI produced infectious virus at about 5% of the level of wild-type controls. The viruses which arose were indistinguishable from the parental strain, and the normal termini of the viral genome had been restored. Marker rescue experiments demonstrate that provision of a DNA fragment with a normal viral end improves infectivity. When a small fragment carrying a wild-type left end (the 0 to 2.6% ClaI-B fragment) was ligated to ClaI-linearized pXAd, virus was produced with efficiencies comparable to a similar reconstitution of the two ClaI fragments of the wild-type genome. These viruses stably carry the left-end fragment at both ends, leaving the normal right end embedded in 950 base pairs of DNA. The embedded right origin is inactive. The consensus of the analyses reported here is that a free end is a necessary configuration for the sequences which make up the adenovirus origin of replication.     

Development of a modular virus clearance package for anion exchange chromatography operated in weak partitioning mode

Anion exchange chromatography (AEX) operated under weak partitioning mode has been proven to be a powerful polishing step as well as a robust viral clearance step in Pfizer's monoclonal antibody (mAb) platform purification process. A multivariate design of experiment (DoE) study was conducted to understand the impact of operating parameters and feedstream impurity levels on viral clearance by weak partitioning mode AEX. Bacteriophage was used initially as a surrogate for neutral and acidic isoelectric point mammalian viruses (e.g., retrovirus and parvovirus). Five different mAbs were used in the evaluation of process parameters such as load challenge (both product and impurities), load pH, load conductivity, and contact time (bed height and flow‐rate). The operating ranges obtained from phage clearance studies and Pfizer's historical data were used to define an appropriate operating range for a subsequent clearance study with model retrovirus and parvovirus. Both phage and virus clearance evaluations included feedstreams containing different levels of impurities such as high molecular mass species (HMMS), host cell proteins (HCPs), and host cell DNA. For all the conditions tested, over 5 log₁₀ of clearance for both retrovirus and parvovirus was achieved. The results demonstrated that weak partitioning mode AEX chromatography is a robust step for viral clearance and has the potential to be included as part of the modular viral clearance approach. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:750–757, 2015