Implementing high-temperature short-time media treatment in commercial-scale cell culture manufacturing processes

The production of therapeutic proteins by mammalian cell culture is complex and sets high requirements for process, facility, and equipment design, as well as rigorous regulatory and quality standards. One particular point of concern and significant risk to supply chain is the susceptibility to contamination such as bacteria, fungi, mycoplasma, and viruses. Several technologies have been developed to create barriers for these agents to enter the process, e.g. filtration, UV inactivation, and temperature inactivation. However, if not implemented during development of the manufacturing process, these types of process changes can have significant impact on process performance if not managed appropriately. This article describes the implementation of the high-temperature short-time (HTST) treatment of cell culture media as an additional safety barrier against adventitious agents during the transfer of a large-scale commercial cell culture manufacturing process. The necessary steps and experiments, as well as subsequent results during qualification runs and routine manufacturing, are shown.     

Design of a UV-C irradiation process for the inactivation of viruses in protein solutions.

The ability of ultraviolet (UV) light to inactivate viruses is well established. However, attempts to apply this to the manufacture of pharmaceutical proteins have been limited by incomplete treatment, low capacity or excessive dilution. Effective processing of large-scale batches of UV-opaque protein solutions has been achieved using a continuous-flow device. The operation of this device has been modelled and a design equation derived to relate the processing conditions and product characteristics to the degree of virus inactivation obtained. Variables included in the model are UV-absorbance at 254 nm (A(254)), hydrodynamic properties of the protein solution, residence time, intensity of UV light and diameter and length of irradiation tube. With this information a specific constant was calculated for each virus which denotes its relative sensitivity to UV and from which the degree of virus inactivation expected can be estimated.     

UV inactivation of Cryptosporidium hominis as measured in cell culture

The Cryptosporidium spp. UV disinfection studies conducted to date have used Cryptosporidium parvum oocysts. However, Cryptosporidium hominis predominates in human cryptosporidiosis infections, so there is a critical need to assess the efficacy of UV disinfection of C. hominis. This study utilized cell culture-based methods to demonstrate that C. hominis oocysts displayed similar levels of infectivity and had the same sensitivity to UV light as C. parvum. Therefore, the water industry can be confident about extrapolating C. parvum UV disinfection data to C. hominis oocysts.     

Inactivation of feline calicivirus and adenovirus type 40 by UV radiation

Little information regarding the effectiveness of UV radiation on the inactivation of caliciviruses and enteric adenoviruses is available. Analysis of human calicivirus resistance to disinfectants is hampered by the lack of animal or cell culture methods that can determine the viruses' infectivity. The inactivation kinetics of enteric adenovirus type 40 (AD40), coliphage MS-2, and feline calicivirus (FCV), closely related to the human caliciviruses based on nucleic acid organization and capsid architecture, were determined after exposure to low-pressure UV radiation in buffered demand-free (BDF) water at room temperature. In addition, UV disinfection experiments were also carried out in treated groundwater with FCV and AD40. AD40 was more resistant than either FCV or coliphage MS-2 in both BDF water and groundwater. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in BDF water were 109, 55, and 16 mJ/cm(2), respectively. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in groundwater were slightly lower than those in BDF water. FCV was inactivated by 99% by 13 mJ/cm(2) in treated groundwater. A dose of 103 mJ/cm(2) was required for 99% inactivation of AD40 in treated groundwater. The results of this study indicate that if FCV is an adequate surrogate for human caliciviruses, then their inactivation by UV radiation is similar to those of other single-stranded RNA enteric viruses, such as poliovirus. In addition, AD40 appears to be more resistant to UV disinfection than previously reported.     

Bioreactor technology: a novel industrial tool for high-tech production of bioactive molecules and biopharmaceuticals from plant roots

Plants are the richest source for different bioactive molecules. Because of the vast number of side effects associated with synthetic pharmaceuticals, medical biotechnologists turned to nature to provide new promising therapeutic molecules from plant biofactories. The large-scale availability of the disease- and pesticide-free raw material is, however, restricted in vivo. Many bioactive plant secondary metabolites are accumulated in roots. Engineered plants can also produce human therapeutic proteins. Vaccines and diagnostic monoclonal antibodies can be won from their roots, so that engineered plants hold immense potential for the biopharmaceutical industry. To obtain sufficient amounts of the plant bioactive molecules for application in human therapy, adventitious and hairy roots have to be cultured in in vitro systems. High-tech pilot-scale bioreactor technology for the establishment of a long-term adventitious root culture from biopharmaceutical plants has recently been established. In this review, I briefly discuss a technology for cultivating bioactive molecule-rich adventitious and hairy roots from plants using a high-tech bioreactor system, as well as the principles and application of genome-restructuring mechanisms for plant-based biopharmaceutical production from roots. High-tech bioreactor-derived bioactive phytomolecules and biopharmaceuticals hold the prospect of providing permanent remedies for improving human well-being.     

Inactivation of Feline Calicivirus and Adenovirus Type 40 by UV Radiation

Little information regarding the effectiveness of UV radiation on the inactivation of caliciviruses and enteric adenoviruses is available. Analysis of human calicivirus resistance to disinfectants is hampered by the lack of animal or cell culture methods that can determine the viruses' infectivity. The inactivation kinetics of enteric adenovirus type 40 (AD40), coliphage MS-2, and feline calicivirus (FCV), closely related to the human caliciviruses based on nucleic acid organization and capsid architecture, were determined after exposure to low-pressure UV radiation in buffered demand-free (BDF) water at room temperature. In addition, UV disinfection experiments were also carried out in treated groundwater with FCV and AD40. AD40 was more resistant than either FCV or coliphage MS-2 in both BDF water and groundwater. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in BDF water were 109, 55, and 16 mJ/cm², respectively. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in groundwater were slightly lower than those in BDF water. FCV was inactivated by 99% by 13 mJ/cm² in treated groundwater. A dose of 103 mJ/cm² was required for 99% inactivation of AD40 in treated groundwater. The results of this study indicate that if FCV is an adequate surrogate for human caliciviruses, then their inactivation by UV radiation is similar to those of other single-stranded RNA enteric viruses, such as poliovirus. In addition, AD40 appears to be more resistant to UV disinfection than previously reported.     

Assessment of DNA damage and repair in Mycobacterium terrae after exposure to UV irradiation

AIM: Ultraviolet (UV) irradiation for drinking water treatment was examined for inactivation and subsequent dark and photo-repair of Mycobacterium terrae. METHODS AND RESULTS: UV sources tested were low pressure (monochromatic, 254 nm) and medium pressure (polychromatic UV output) Hg lamps. UV exposure resulted in inactivation, and was followed by dark or photo-repair experiments. Inactivation and repair were quantified utilizing a molecular-based endonuclease sensitive site (ESS) assay and conventional colony forming unit (CFU) viability assay. Mycobacterium terrae was more resistant to UV disinfection compared to many other bacteria, with approximately 2-log reduction at a UV fluence of 10 mJ cm(-2) ; similar to UV inactivation of M. tuberculosis. There was no difference in inactivation between monochromatic or polychromatic UV lamps. Mycobacterium terrae did not undergo detectable dark repair. Photo-repair resulted in recovery from inactivation by approximately 0.5-log in less than 30 min for both UV lamp systems. CONCLUSIONS: Mycobacterium terrae is able to photo-repair DNA damage within a short timeframe. The number of pyrimidine dimers induced by UV light were similar for Escherichia coli and M. terrae, however, this similarity did not hold true for viability results. SIGNIFICANCE AND IMPACT OF THE STUDY: There is no practical difference between UV sources for disinfection or prevention of DNA repair for M. terrae. The capability of M. terrae to photo-repair UV damage fairly quickly is important for wastewater treatment applications where disinfected effluent is exposed to sunlight. Finally, molecular based assay results should be evaluated with respect to differences in the nucleic acid content of the test micro-organism.     

UV Inactivation of Cryptosporidium hominis as Measured in Cell Culture

The Cryptosporidium spp. UV disinfection studies conducted to date have used Cryptosporidium parvum oocysts. However, Cryptosporidium hominis predominates in human cryptosporidiosis infections, so there is a critical need to assess the efficacy of UV disinfection of C. hominis. This study utilized cell culture-based methods to demonstrate that C. hominis oocysts displayed similar levels of infectivity and had the same sensitivity to UV light as C. parvum. Therefore, the water industry can be confident about extrapolating C. parvum UV disinfection data to C. hominis oocysts.     

Enhanced UV Inactivation of Adenoviruses under Polychromatic UV Lamps

Adenovirus is recognized as the most UV-resistant waterborne pathogen of concern to public health microbiologists. The U.S. EPA has stipulated that a UV fluence (dose) of 186 mJ cm⁻² is required for 4-log inactivation credit in water treatment. However, all adenovirus inactivation data to date published in the peer-reviewed literature have been based on UV disinfection experiments using UV irradiation at 253.7 nm produced from a conventional low-pressure UV source. The work reported here presents inactivation data for adenovirus based on polychromatic UV sources and details the significant enhancement in inactivation achieved using these polychromatic sources. When full-spectrum, medium-pressure UV lamps were used, 4-log inactivation of adenovirus type 40 is achieved at a UV fluence of less than 60 mJ cm⁻² and a surface discharge pulsed UV source required a UV fluence of less than 40 mJ cm⁻². The action spectrum for adenovirus type 2 was also developed and partially explains the improved inactivation based on enhancements at wavelengths below 230 nm. Implications for water treatment, public health, and the future of UV regulations for virus disinfection are discussed.     

Application of machine learning methods to pathogen safety evaluation in biological manufacturing processes

The production of recombinant therapeutic proteins from animal or human cell lines entails the risk of endogenous viral contamination from cell substrates and adventitious agents from raw materials and environment. One of the approaches to control such potential viral contamination is to ensure the manufacturing process can adequately clear the potential viral contaminants. Viral clearance for production of human monoclonal antibodies is achieved by dedicated unit operations, such as low pH inactivation, viral filtration, and chromatographic separation. The process development of each viral clearance step for a new antibody production requires significant effort and resources invested in wet laboratory experiments for process characterization studies. Machine learning methods have the potential to help streamline the development and optimization of viral clearance unit operations for new therapeutic antibodies. The current work focuses on evaluating the usefulness of machine learning methods for process understanding and predictive modeling for viral clearance via a case study on low pH viral inactivation.     

Predicted inactivation of viruses of relevance to biodefense by solar radiation

UV radiation from the sun is the primary germicide in the environment. The goal of this study was to estimate inactivation of viruses by solar exposure. We reviewed published reports on 254-nm UV inactivation and tabulated the sensitivities of a wide variety of viruses, including those with double-stranded DNA, single-stranded DNA, double-stranded RNA, or single-stranded RNA genomes. We calculated D(37) values (fluence producing on average one lethal hit per virion and reducing viable virus to 37%) from all available data. We defined "size-normalized sensitivity" (SnS) by multiplying UV(254) sensitivities (D(37) values) by the genome size, and SnS values were relatively constant for viruses with similar genetic composition. In addition, SnS values were similar for complete virions and their defective particles, even when the corresponding D(37) values were significantly different. We used SnS to estimate the UV(254) sensitivities of viruses for which the genome composition and size were known but no UV inactivation data were available, including smallpox virus, Ebola, Marburg, Crimean-Congo, Junin, and other hemorrhagic viruses, and Venezuelan equine encephalitis and other encephalitis viruses. We compiled available data on virus inactivation as a function of wavelength and calculated a composite action spectrum that allowed extrapolation from the 254-nm data to solar UV. We combined our estimates of virus sensitivity with solar measurements at different geographical locations to predict virus inactivation. Our predictions agreed with the available experimental data. This work should be a useful step to understanding and eventually predicting the survival of viruses after their release in the environment.     

Viral adventitious agent detection using laser force cytology: Intrinsic cell property changes with infection and comparison to in vitro testing

Adventitious agent testing in biomanufacturing requires assays of broad detection capability to screen for as many infectious agents as possible. The current gold standard for general infectious adventitious virus screening is the in vitro assay in which test articles are cultured onto a panel of different cell lines and observed for cytopathic effect (CPE). However, this assay is inherently subjective due to the nature of visual observation of cell morphology and labor and time intensive, requiring highly trained personnel to identify CPE. Laser force cytology (LFC) is an alternative, automated analytical method that uses a combination of optical and fluidic forces along with imaging to objectively and quantitatively assess CPE in cell culture. Importantly, because LFC uses no labels or antibodies, the assay is appropriate for general adventitious agent testing. Using LFC, changes in cellular features associated with virally infected cells were identified using principal component analysis. Using these features of infected cells, the sensitivity and earliness of detection with LFC was directly compared with the in vitro assay for a diverse panel of viruses incubated with chinese hamster ovary (CHO), Vero, and Medical Research Council cell strain 5 (MRC-5) cells. LFC detected viral infection with a sensitivity equal to the in vitro assay on average, but in certain virus and cell combinations including mouse minute virus (MMV) and reovirus 3 in CHO cells, detection was 4 days earlier and for MMV, the limit of detection was 10-fold lower. Overall, these results demonstrate the ability of LFC to serve as a biopharmaceutical adventitious agent testing methodology with sensitivity equivalent to the in vitro assay, but in an objective and automated manner.     

Identification and quantitation of vesivirus 2117 particles in bioreactor fluids from infected Chinese hamster ovary cell cultures

The prevention of adventitious agent contamination is a top priority throughout the entire biopharmaceutical production process. For example, although viral contamination of cell banks or cell cultures is rare, it can result in serious consequences (e.g., shutdown and decontamination of manufacturing facilities). To ensure virus free production, numerous in vivo and in vitro adventitious agent assays and biophysical characterizations such as electron microscopy are conducted on cell banks, raw materials, process materials, and drug substances throughout the manufacturing process. Molecular assays such as PCR and other nucleotide‐based techniques are also routinely used for screening and identification of any viral agents. However, modern techniques in protein identification of complex protein mixtures have not yet been effectively integrated throughout the industry into current viral testing strategies. Here, we report the identification and quantitation of Vesivirus 2117 particles in bioreactor fluid from infected Chinese hamster ovary cell cultures by global protein sequencing using mass spectrometry in combination with multi‐dimensional liquid‐chromatography. Following mass spectrometric data acquisition and rigorous data analysis, six virus specific peptides were identified. These peptides were fragments of two structural proteins, capsid protein pre‐cursor (four unique peptides) and small structural protein (two unique peptides), from the same species: Vesivirus 2117. Using stable heavy isotope‐labeled peptides as internal standards, we also determined the absolute concentration of Vesivirus particles in the bioreactor fluid and the ratio of two capsid proteins (VP1:VP2) in the particles as approximately 9:1. The positive identification of Vesivirus 2117 was subsequently confirmed by RT‐PCR. Biotechnol. Bioeng. 2013; 110: 1342–1353. © 2012 Wiley Periodicals, Inc.     

Photocatalytic disinfection of Giardia intestinalis and Acanthamoeba castellani cysts in water

In this study, disinfection of water containing Giardia intestinalis and Acanthamoeba castellani cysts with TiO2 and modified catalyst silver loaded TiO2 (Ag-TiO2) was investigated. Destruction of the parasites was evaluated after UV illumination of the suspension consisting 5 x 10(8)-13.5 x 10(8)cysts/mL in the presence of 2g/L neat or modified TiO2 at neutral pH. In the initial stage, the solid photocatalyst particles penetrated the cyst wall and then oxidant species produced by TiO2/UV destroyed both cell wall and intracellular structure. In the case of G. intestinalis inactivation (disinfection) performance of TiO2/UV system reached 52.5% only after 25 min illumination and total parasite disinfection was achieved after 30 min illumination. However, silver loaded TiO2 seemed to be more effective as this loading provided better catalytic action as well as additional antimicrobial properties. Cell viability tests showed that parasite cysts, their walls in particular, were irreversibly damaged and cysts did not re-grow. Nevertheless the studied system seemed to be ineffective for the inactivation of A. castellani. Inactivation percentages of TiO2/UV and Ag-TiO2/UV systems were far lower than that of UV alone, being 50.1% and 46.1%, respectively.     

UV Disinfection of Adenoviruses: Molecular Indications of DNA Damage Efficiency

Adenovirus is a focus of the water treatment community because of its resistance to standard, monochromatic low-pressure (LP) UV irradiation. Recent research has shown that polychromatic, medium-pressure (MP) UV sources are more effective than LP UV for disinfection of adenovirus when viral inactivation is measured using cell culture infectivity assays; however, UV-induced DNA damage may be repaired during cell culture infectivity assays, and this confounds interpretation of these results. Objectives of this work were to study adenoviral response to both LP and MP UV using (i) standard cell culture infectivity assays and (ii) a PCR assay to directly assess damage to the adenoviral genome without introducing the virus into cell culture. LP and MP UV dose response curves were determined for (i) log inactivation of the virus in cell culture and (ii) UV-induced lesions per kilobase of viral DNA as measured by the PCR assay. Results show that LP and MP UV are equally effective at damaging the genome; MP UV is more effective at inactivating adenovirus in cell culture. This work suggests that the higher disinfection efficacy of MP UV cannot be attributed to a difference in DNA damage induction. These results enhance our understanding of the fundamental mechanisms of UV disinfection of viruses—especially double-stranded DNA viruses that infect humans—and improve the ability of the water treatment community to protect public health.     

Effect of exposure to UV-C irradiation and monochloramine on adenovirus serotype 2 early protein expression and DNA replication

The mechanisms of adenovirus serotype 2 inactivation with either UV light (with a narrow emission spectrum centered at 254 nm) or monochloramine were investigated by assessing the potential inhibition of two key steps of the adenovirus life cycle, namely, E1A protein synthesis and viral genomic replication. E1A early protein synthesis was assayed by using immunoblotting, while the replication of viral DNA was analyzed by using slot blotting. Disinfection experiments were performed in phosphate buffer solutions at pH 8 and room temperature (UV) or 20 degrees C (monochloramine). Experimental results revealed that normalized E1A levels at 12 h postinfection (p.i.) were statistically the same as the corresponding decrease in survival ratio for both UV and monochloramine disinfection. Normalized DNA levels at 24 h p.i. were also found to be statistically the same as the corresponding decrease in survival ratio for monochloramine disinfection. In contrast, for UV disinfection, genomic DNA levels were much lower than E1A or survival ratios, possibly as a result of a delay in DNA replication for UV-treated virions compared to that for controls. Future efforts will determine the pre-E1A synthesis step in the adenovirus life cycle affected by exposure to UV and monochloramine, with the goal of identifying the viral molecular target of these two disinfectants.     

Role of housing modalities on management and surveillance strategies for adventitious agents of rodents

Specific pathogen-free (SPF) rodents for modern biomedical research need to be free of pathogens and other infectious agents that may not produce disease but nevertheless cause research interference. To meet this need, rodents have been rederived to eliminate adventitious agents and then housed in room- to cage-level barrier systems to exclude microbial contaminants. Because barriers can and do fail, routine health monitoring (HM) is necessary to verify the SPF status of colonies. Testing without strict adherence to biosecurity practices, however, can lead to the inadvertent transfer of unrecognized, inapparent agents among institutions and colonies. Microisolation caging systems have become popular for housing SPF rodents because they are versatile and provide a highly effective cage-level barrier to the entry and spread of adventitious agents. But when a microisolation-caged colony is contaminated, the cage-level barrier impedes the spread of infection and so the prevalence of infection is often low, which increases the chance of missing a contamination and complicates the corroboration of unexpected positive findings. The expanding production of genetically engineered mutant (GEM) rodent strains at research institutions, where biosecurity practices vary and the risk of microbial contamination can be high, underscores the importance of accurate HM results in mitigating the risk of the introduction and spread of microbial contaminants with the exchange of mutant rodent strains among investigators and institutions.     

High-intensity narrow-spectrum light inactivation and wavelength sensitivity of Staphylococcus aureus

This study was conducted to investigate the bactericidal effects of visible light on methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MRSA), and subsequently identify the wavelength sensitivity of S. aureus, in order to establish the wavelengths inducing maximum inactivation. Staphylococcus aureus, including MRSA strains, were shown to be inactivated by exposure to high-intensity visible light, and, more specifically, through a series of studies using a xenon broadband white-light source in conjunction with a selection of optical filters, it was found that inactivation of S. aureus occurs upon exposure to blue light of wavelengths between 400 and 420 nm, with maximum inactivation occurring at 405+/-5 nm. This visible-light inactivation was achieved without the addition of exogenous photosensitisers. The significant safety benefit of these blue-light wavelengths over UV light, in addition to their ability to inactivate medically important microorganisms such as MRSA, emphasises the potential of exploiting these non-UV wavelengths for disinfection applications.     

国内外关于人用疫苗病毒安全性检测的研究

人用疫苗产品安全性检测的重点是确定生产基质及原辅料有无潜在病毒污染。按照2010版中国药典(以下简称CP)病毒外源因子检查,并与欧洲药典7.0版(以下简称EP7.0)和美国药典(以下简称USP34/NF29)进行比较,发现2010版CP在细胞基质病毒外源因子检测要求上比2005版CP有较大的提高,并对检测结果有效性的控制进一步加强。USP34/NF29规定检测的病毒种类繁多,并且根据种属来源的特异性及病毒的亲嗜性,列出了敏感指示细胞系,而2010版CP对上述部分内容未作规定或欠详细表述。因此在今后药典的标准提高方面,应结合国内外相关病毒外源因子检测的实际情况,加强疫苗安全性;以EP7.0或USP34/NF29标准为依据进行药品研发及药品审评工作的人员应注意其中的差别,勿混淆套用。