Nucleic acid impurity reduction in viral vaccine manufacturing

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... Benzonase® (Merck KGaA), a recombinant endonuclease produced in Escherichia coli, is commonly used in various bioprocessing applications to remove host-cell DNA [8,9] and/or plasmid DNA [10]. The manufacturer of Benzonase reports that this enzyme is capable of digesting all nucleic acids into short oligonucleotides (< 10 base pairs) under optimum processing conditions [11]. Other notable recombinant endonucleases include Denarase® (c-LEcta), which is expressed in Bacillus sp. and has been used in the downstream purification of Influenza A virus [12,13], and Turbonu-clease™ (Accelagen), which is expressed in Serratia marcescens and has been used in the downstream purification of adeno-associated virus [14,15]. ...
... This is a concern because, as other researchers have pointed out, the use of high concentrations of endonuclease significantly increases production costs [8]. Furthermore, given safety concerns related to the presence of residual endonuclease in the final virus product, it is considered good practice to add them in early stages of the process so they can be removed during the subsequent downstream purification steps [11]. With regards to incubation time, endonuclease manufacturers typically report enzyme activity in terms of the amount of DNA that is completely digested after 30 min of incubation at 37°C. ...
... Initially, we were quite surprised by the large fragments of DNA in the digested samples, as each of the various enzyme manufacturers claim that optimum digestion conditions will result in DNA fragments in the size range of a few base pairs [11]. Although an early study by Janning et al. [32] is often cited in the literature to support this claim, that study was performed using pre-purified calf-thymus DNA, a combination of Benzonase (25 U/mg of DNA) and alkaline phosphatase (1.75 U/mg of DNA), and an overnight incubation step. ...
High titer and purity levels are key requirements for virus-based cancer and gene therapy biopharmaceuticals. However, the task of removing enough host-cell DNA from therapeutic viruses in order to comply with the FDA’s 10 ng/dose limit is particularly challenging. In a previous study, we demonstrated the advantages of using laterally-fed membrane chromatography (LFMC) for adenovirus purification. Although this approach achieved >90% DNA removal, significant amounts of DNA remained in the product due to the poor performance of a pre-LFMC DNA digestion step. In the present study, we attempt to improve upon this outcome by employing an integrated approach to process development that examines the interactions between different downstream steps (i.e., clarification, enzymatic DNA digestion, and membrane chromatography (MC)). First, we identified the most efficient process sequence involving a clarification step followed by a DNA digestion step, as well as three endonucleases (Benzonase®, Denarase®, and Turbonuclease™) that perform similarly with respect to DNA digestion. Next, a factorial design of experiments (DOE) was used to evaluate how enzyme (Benzonase and Denarase) concentration and time impact DNA and virus concentrations after DNA digestion. Since Denarase showed slightly better efficiency, it was used along with a subset of the DOE conditions to evaluate the removal of DNA via MC with Sartobind Q 96-well filter plates. The lowest amount of DNA per dose was achieved using MC in conjunction with feed that had been digested with 10 U/mL of Denarase for 4 hours; as such, this approach was used to prepare lysates, which were then purified using an LFMC device containing 1 mL of Sartobind Q membrane. This process enabled a virus-recovery rate of 73%, and residual DNA levels of 77 ng/dose. Ultimately, the proposed integrated process development approach resulted in an approximately 80-fold improvement in DNA removal.
... Further, the genomic DNA contamination in RNA samples always is a troublesome component in reverse transcription PCR, especially for RNA abundance detection. However, Benzonase, the most widely used nuclease from Serratia marcescens, is active on both DNA and RNA [19,20]. From this point of view, DNA-specific nuclease must be superior to Benzonase for pretreatment of RNA samples. ...
... The protein-DNA interactions were mainly governed by electrostatic forces and depended on salt concentration [22], and higher salt levels of NaCl (300 mM) completely eliminated observable protein binding to DNA [23]. Although Benzonase nuclease is effective to remove nucleic acids both in the laboratory-and industrial-scale processes [20], its activity was inhibited seriously by salt when the concentration reached above 50 mM [24]. A high salt-tolerant nuclease might be more promising for DNA elimination in protein production process. ...
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Endonuclease I is a widely distributed periplasmic or extracellular enzyme. A method for the high-level production of recombinant AsEndI (endonuclease I from Aliivibrio salmonicida) in Escherichia coli with secretion expression is investigated. The coding sequence of AsEndI gene was assembled according to the E. coli codon usage bias, and AsEndI was expressed in the periplasm of E. coli TOP10 with a C-terminal 6× His-tagged fusion. The recombinant AsEndI (His-AsEndI) was purified by Ni-NTA resin with a yield of 1.29 × 10⁷ U from 1-L LB medium. His-AsEndI could be classified into Ca²⁺/Mg²⁺-dependent nucleases and showed highest nuclease activity to dsDNA at pH 8.0 and 37 °C. His-AsEndI is highly active in a broad range of salt concentration range up to 1.0 M with optimal NaCl concentration at 0.4 M. His-AsEndI can effectively remove DNA contamination in RNA sample or in PCR reagents to the level that cannot be detected by highly sensitive nested PCR and without adverse effects on the subsequent PCR reaction. His-AsEndI can remove DNA contamination at high salt conditions, especially for the DNA that may be shielded by DNA-binding protein at low salt conditions.
... Nuclease treatment is commonly used to reduce DNA size and decrease bulk viscosity [69]. During biotherapeutic particles manufacturing, and in order to achieve the HC DNA levels imposed by regulatory authorities, the nuclease is usually used before the clarification step since it is a process additive that should be removed during the next purification processes [70]. ...
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Introduction: The development of novel complex biotherapeutics led to new challenges in biopharmaceutical industry. The potential of these particles has been demonstrated by the approval of several products, in the different fields of gene therapy, oncolytic therapy, and tumor vaccines. However, their manufacturing still presents challenges related to the high dosages and purity required. Areas covered: The main challenges that biopharmaceutical industry faces today and the most recent developments in the manufacturing of different biotherapeutic particles are reported here. Several unit operations and downstream trains to purify virus, virus-like particles and extracellular vesicles are described. Innovations on the different purification steps are also highlighted with an eye on the implementation of continuous and integrated processes. Expert opinion: Manufacturing platforms that consist of a low number of unit operations, with higher-yielding processes and reduced costs will be highly appreciated by the industry. The pipeline of complex therapeutic particles is expanding and there is a clear need for advanced tools and manufacturing capacity. The use of single-use technologies, as well as continuous integrated operations, are gaining ground in the biopharmaceutical industry and should be supported by more accurate and faster analytical methods.
... It also has to be considered that nuclease treatment enhances safety of vaccines preparations regarding possible contamination with adventitious agents. In a subsequent process step, either ligand-activated core chromatography [28] or tangential flow filtration [30] could be used to remove the nucleases. On the other hand, He et al. [31] reported a residual DNA amount of 0.03 ng per 15 mg HA. ...
The continuously increasing demand for potent and safe vaccines and the intensifying economic pressure on health care systems underlines the need for further optimization of vaccine manufacturing. Here, we focus on downstream processing of human influenza vaccines, investigating the purification of serum-free cell culture-derived influenza virus (A/PR/8/34 H1N1) using continuous chromatography. Therefore, quaternary amine anion exchange monoliths (CIM® QA) were characterized for their capacity to capture virus particles from animal cells cultivated in different media and their ability to separate virions from contaminating host cell proteins and DNA. The continuous chromatography was implemented as simulated moving bed chromatography (SMB) in a three zone open loop configuration with a detached high salt zone for regeneration.
... Sometimes losses as high as 30% can be observed at the lysate clarification step (Wolf and Reichl, 2011). The addition of Benzonase at the lysis step helps solve the filtration problem (Gousseinov et al., 2014). Washing the cells with phosphate buffered saline after the harvest and a quick "freezethaw" in high salt (500 mM NaCl) containing lysis buffer help to remove the aggregates. ...
Vaccines are derived from a variety of sources including tissue extracts, bacterial cells, virus particles, recombinant mammalian, yeast and insect cell produced proteins and nucleic acids. The most common method of vaccine production is based on an initial fermentation process followed by purification. Production of vaccines is a complex process involving many different steps and processes. Selection of the appropriate purification method is critical to achieving desired purity of the final product. Clarification of vaccines is a critical step that strongly impacts product recovery and subsequent downstream purification. There are several technologies that can be applied for vaccine clarification. Selection of a harvesting method and equipment depends on the type of cells, product being harvested, and properties of the process fluids. These techniques include membrane filtration (microfiltration, tangential-flow filtration), centrifugation, and depth filtration (normal flow filtration). Historically vaccine harvest clarification was usually achieved by centrifugation followed by depth filtration. Recently membrane based technologies have gained prominence in vaccine clarification. The increasing use of single-use technologies in upstream processes necessitated a shift in harvest strategies. This review offers a comprehensive view on different membrane based technologies and their application in vaccine clarification, outlines the challenges involved and presents the current state of best practices in the clarification of vaccines.
There is a strong and growing interest in the development and production of gene therapy products, including those utilizing adeno-associated virus (AAV) particles. This is evident with the increase in number of clinical trials and agency approvals for AAV therapeutics. As bioproduction of AAV viral vectors matures, a quality by design (QbD) approach to process development can aid in process robustness and product quality. Furthermore, it may become a regulatory expectation. The first step in any QbD approach is to determine what physical, chemical, biological or microbiological property or characteristic product attributes should be controlled within an appropriate limit, range, or distribution to ensure the desired product quality. Then predefined goals are set to allow proactive process development to design in quality. This review lists typical quality attributes used for release testing of AAV viral vectors and discusses these and selected attributes important to extended characterization studies in terms of safety, efficacy and impact upon the patient immune response. This article is protected by copyright. All rights reserved.
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