Conference report WHO informal consultation on the draft WHO Guideline on the phasing out of animal tests for the quality control of biological products

The selection of suitable cell cultures for use in the biological industry as well as research and diagnostic studies is critical. One of the factors affecting cell culture that can affect the results of studies is the contamination with viral agents. Therefore, efforts to preserve the health of cell cultures from contamination sound logical, and the use of virus-free cells is vital in research and diagnostic studies as well as in the manufacturing industries. For this purpose, it is crucial to use fast and correct diagnostic methods to detect the presence of critical viral contaminants in cells. Moreover, the use of susceptible diagnostic methods is also doubly important, especially in the case of contaminants that may remain hidden. Therefore, in this study, the BHK-21C5 cell line, one of the most widely used cells in the production and quality control of biological products and virological studies, was examined in terms of contamination with the most important viruses such as BVD and BLV. Detection of possible contaminants by using two-step RT-PCR to detect the 5' UTR portion of the BVD virus. Moreover, Nested PCR was carried out to detect the BLV virus using the gp51 env gene region. Also, an Flk cell line and Hela cell line that were consistently contaminated with the BLV virus were used as positive controls in Nested PCR. Due to the absence of bands in the BHK-21C5 cell column and the bandwidth observed in the positive control column (BVDV) in the range of 283 bp, non-contaminating of the cell clone with the BVD virus was proved. Also, no band was observed in well related to BHK-21C5 cell, and no cell clone contamination with BLV was confirmed, and in wells related to the positive controls (Flk and Hela cells), the bands have seen in the range 444bp. So, the results showed that no obvious or covert viral contamination effect was observed in the cell clone studied. Hence, the use of this cell line seems unobstructed in the quality control and production of biological products and research and diagnostic studies.

According to the latest requirements of World Health Organization and U.S.Food and Drug Administration for detecting mycobacteria in biological products,mycobacteria detection is first added to quality control of live attenuated Japanese encephalitis vaccine.This article reviews classification and characteristics of mycobacteria,pathogenicity of different kinds of mycobacteria and common technology for detecting mycobacteria,and introduces different methods for detecting mycobacteria in quality control of Japanese encephalitis vaccine.Nucleic acid amplification assay for detecting mycobacteria is first applied to quality control of biological products in our country. Key words: Mycobacterium ; Biological products ; Quality control ; Japanese encephalitis vaccines ;

There is always a concern about the quality of cell-based products in terms of the contamination of the cells and their lack of efficiency. Therefore, it is of prime importance to ensure these cells' identity, purity, efficacy, and suitability for the production of biological products and diagnostic uses. Hence, cells must be identified, evaluated, documented, and stored to be used consistently and efficiently. With these conditions, vaccine manufacturers have a suitable reserve of efficient and valuable cells for the production and quality control of biological products. In this review article, a strategic plan was drawn for cell-substrate well-characterization and identification according to scientific principles, the author's work experience, and regulatory guidance for the optimal use of that cell in research and diagnostic studies especially for the biological product production process. For this purpose, all aspects of cell identification, cell evaluation, and cell characterization are discussed. Because of the importance of cell identity in the competence of a cell substrate, in the cell identification section, all aspects of cell identification, including general cell information and specific cell characteristics, especially in terms of cell passage history, cell storage conditions, and cell coding and labeling, were studied. In the part of cell evaluation and determination of cell characteristics, all required tests to determine cell characteristics from various aspects, including determination of cell identity, cell growth conditions, cell quality, efficiency, and the possibility of cell contamination with adventitious agents, including cellular, viral, bacterial, mycoplasma, and mycobacterial agents were introduced. Due to the importance of endogenous virus contamination, this topic is specially discussed. In addition, the stability of the cell both from the aspect of genetic stability and from the aspect of stability of cell efficiency, were discussed. In the end, while reviewing the necessary documents to be under the control of the cell for use in the laboratory, based on the studies conducted, the certificate of the cell has been compiled. Therefore, on this basis, the studied cell can be used for research and diagnostic studies of virology, especially for the production and quality control of biological products.

INTRODUCTION. Many countries are implementing an increasing number of alternatives to animal testing in the research, development, and quality control of medicines under the concept of 3Rs (replacement, reduction, and refinement). However, the implementation of in vitro alternatives to in vivo methods into batch release testing of biologicals, primarily vaccines, is a challenging process. The introduction of the 3Rs principles into regulatory and industry standards requires a comprehensive analysis, including, among other things, a study of specific considerations for national frameworks.AIM. This study aimed to analyse the degree of global integration of the 3Rs principles in the quality control of biological medicinal products and to assess existing challenges and opportunities for a successful transition to non-animal quality control methods in the Russian Federation.DISCUSSION. The key advantages of in vitro biological methods over in vivo approaches for quality control of medicinal products include reduced variability, higher specificity, and shorter testing timelines. Analysis of assessments by regulatory authorities and pharmaceutical manufacturers regarding the opportunities and challenges in implementing the 3Rs principles has identified the following main arguments for replacing in vivo methods: ethical considerations, relevance of reducing testing time, decreased variability. The integration of in vitro methods is hindered by insufficient information on alternative approaches and the lack of harmonised regulatory frameworks for adopting 3Rs principles. For instance, the European Pharmacopoeia has eliminated all general safety testing of biological medicinal products in animals as part of its 3Rs implementation. Furthermore, the World Health Organization has developed guidelines for the phased discontinuation of animal testing in the quality control of biological products.CONCLUSIONS. The main challenges associated with 3Rs implementation include the difficulty of confirming the in vitro – in vivo correlation (particularly, by comparing in vivo and in vitro methods), the lack of regulatory harmonisation, and the insufficient regulatory support for manufacturers in adopting alternative methods. The international harmonisation of regulatory requirements is necessary to effectively address these issues and successfully implement the 3Rs principles in the quality control procedures for biologicals released in different countries, including the Russian Federation.

Development and application of potency assays based on genetically modified cells for biological products

Mycoplasma is the smallest self-replicating bacteria, figuring as common contaminant of eukaryotic cell cultures. Production inputs and operator's manipulation seem to be the main sources of such contamination. Many analytical approaches have been applied for mycoplasma detection in cell cultures and also in biological products. However, unless they were validated, only indicator cell culture and bacteriological culture are considered as compendial methods for quality control of biological products. Nano-flow cytometry has been pointed out as an alternative technique for addressing prokaryotic and eukaryotic cell viability being a substantial tool for reference material production. In this study, a viability-flow-cytometry assay was standardized for M. gallisepticum and then applied to other cell-culture-contaminant mycoplasmas. For this, M. galliseticum's growth rate was observed and different treatments were evaluated to establish low viability cultures (cell death-induced control). Distinct viability markers and their ideal concentrations (titration) were appraised. Ethanol treatment showed to be the best death-inducing control. CFDA and TOPRO markers revealed to be the best choice for detecting live and dead mycoplasma frequencies, respectively. The standardized methodology was applied to Mycoplasma arginini, M. hyorhinis, M. orale, Spiroplasma citri and Acholeplasma laidlawii. Significant statistical difference was observed in the percentage of viable cells in comparison to ethanol treatment for A. laidlawii in CFDA and in both markers for M. gallisepticum, M. hyorhinis and S. citri. In summary, we standardized a flow cytometry assay for assessing M. gallisepticum - and potentially other species - viability and ultimately applied for reference material production improving the quality control of biological products.

Mycobacteria can be one of the main contaminants of biological products, and their presence can have serious consequences on patients' health. For this reason, the European Pharmacopoeia mandates the specific testing of biological products for mycobacteria, a critical regulatory requirement aimed at ensuring the safety of these products before they are released to the market. The current pharmacopeial reference, i.e., microbial culture method, cannot ensure an exhaustive detection of mycobacteria due to their growth characteristics. Additionally, the method is time consuming and requires a continuous supply of culture media, posing logistical challenges. Thus, to overcome these issues, pharmaceutical industries need to consider alternative non-microbiological techniques to detect these fastidious, slow-growing contaminating agents. This review provides an overview of alternative methods, which could be applied within a quality control environment for biological products and underlines their advantages and limitations. Nucleic acid amplification techniques or direct measurement of mycobacteria stand out as the most suitable alternatives for mycobacterial testing in biological products.

The impact of Microwave Temperature Sounder (MWTS) radiances on the prediction of the Chinese Numerical Weather prediction (NWP) system-GRAPES (Global and Regional Assimilation and PrEdiction System) with comparison of two Quality Control (QC) schemes was researched. The main differences between the two schemes are cloud detection, O-B (brightness temperature difference between observation and model simulation) check and thinning. To evaluate the impact of the two QC schemes on GRAPES, a typhoon case study and cycle experiments were conducted. In the typhoon case study, two experiments were conducted using both the new and old QC schemes. The results show that outliers are removed in the new QC while they exist in the old QC. The analysis and the model forecast are subsequently generated after assimilating data from the two QC schemes. The model-predicted steering flows more southward with the new QC scheme, and as a result, the forecast track in the experiments is more southward, i.e., closer to the best track than the old scheme. In addition to the case study, four impact cycle experiments were conducted for 25-day periods. The results show that the new QC scheme removed nearly all the biases whereas the old scheme could not. Furthermore, the mean and standard deviation of analysis increments with the new scheme is much smaller than those of O-B. In contrast, the old scheme values are either slightly smaller or the same. Verifications indicate that forecast skill is improved after applying the new scheme. The largest improvements are found in the Southern Hemisphere. According to the results above, MWTS with the new QC scheme can improve the GRAPES forecast.

rIntroduction: Recombinant human erythropoietin (rhEPO) has been produced in different conditions and cell lines. Identification, isolation, and purification of this protein from various sources have pivotal role in clinical applications. Hence, Clinical trials should be carried out for the identification of purity and aggregation of biological EPO. Moreover, the purification of EPO cooperates with various recommended processes has been manifested, but the strategies schemes (i.e., liquid chromatography (HPLC) and ion exchange chromatography (IEX) are often used in combination. In the current study, the quality of purification, biological modifications and the stability of rhEPO using various chromatography methods including HPLC and IEX have been assessed.Materials and Methods: rhEPO was expressed in the Chinese hamster ovary (CHO) cells and purified by the general requirements for the quality control of biological products. For the assessment of the influence of IEX in the purity pattern of rhEPO, HPLC and biological analysis were performed for 3 samples.Results: Our results revealed that the combination of 4 strategies represent confident methods for evaluating the quality of this biological medicinal product; moreover, purity and biochemical applications will yield to a relatively pure protein preparation. The activity of EPO was presented by monomeric isoform and high acid sialic purification in final product. Moreover, the determination of the biochemical reactions rate and their relationship tests were obtained by change in electrical conductivity with pH being 31.5 and 5, respectively.Conclusion: Taken together, our results indicated that different purification process based on our results can increase the accuracy of rhEPO purification.

673. Establishment of a National Reference Standard for Reverse Transcriptase Activity Assay and Its Applicability in Quality Control of Gene therapy Products

Biopotency Assays: an Integrated Application to Quality Control of Chinese Materia Medica

WHO informal consultation on development of guidelines on procedures and data requirements for changes to approved biotherapeutic products, Seoul, Republic of Korea, 27–28 April 2017

A biological product is defined as “a virus, therapeutic serum, toxin, antitoxin, vaccine, blood, blood component or derivative, allergenic product, or analogous product, or any other trivalent organic arsenic compound, applicable to the prevention, treatment or cure of a disease or condition of human beings”. Throughout the 20th century, the world witnessed great discoveries in the biological sciences. One of the earliest biological products introduced to the U.S. marketplace was a blood protein called Factor VIII first sold in 1966. The earliest FDA approval for a modern biotech product designed for human therapeutic use was given to human insulin in 1982, approval was given in 1985 to a human growth hormone (HGH) for the treatment of dwarfism. In the 1990s FDA granted approvals for vaccines against rabies, tetanum toxoids, and pertussis. The manufacturing process for a biological product usually different from the process for drugs. The manufacture of biological medicinal products involves certain specific considerations arising from the nature of the products and the processes. Persons responsible for production and quality control should have an adequate background in relevant scientific disciplines, such as bacteriology, biology, biometry, chemistry, medicine, pharmacy, pharmacology, virology, immunology and veterinary medicine. The degree of environmental control of particulate and microbial contamination of the production premises should be adapted to the product and the production step. Animals are used for the manufacture of a number of biological products, in addition, animals may also be used in the quality control of most sera, antibiotics and vaccines. All biological products should be clearly identified by labels which should be approved by the national control authority. The evaluation of stability may necessitate complex analytical methodologies. Assays for biological activity, where applicable, should be part of the pivotal stability studies.

The aim of the present work was to evaluate an environmental monitoring program for clean rooms, or classified environments, involved in the filling and quality control of biological products produced by Butantan Institute, Sao Paulo, Brazil. This monitoring established the quantification, characterization and seasonality of the microorganisms in air and operators and, moreover, determined the alert and action limits. The total detectable microbial number showed some contrasts in installed air purification systems and in the operational impact on adopted procedures. The typical microbial population consisted of Staphylococcus sp, Micrococcus sp, Bacillus sp and Penicillium sp. The highest microorganism concentration occurred during summer and springtime. The established internal alert and action limits supported the operational procedures. Therefore, the environmental monitoring program is recommended for other laboratories involved in the production of vaccines, hyperimmune sera and biopharmaceuticals.

Monoclonal Antibodies (mAbs) are used for biomedical research, diagnosis, treatment, production, and the quality control of biological products. mAbs are also very helpful in poliovirus research studies because it is still one of the major public health problems in developing countries. The main objective of this study was the production of mAbs against Poliovirus Type 2 (PV2) to be prepared and respond to the re-emergence of this virus. After fusion of immunized B cells prepared from mice with myeloma tumor cells and screening of about 250 hybridoma colonies, 22 with the highest antibody titer and without cross-reaction with others types were selected and cloned by limiting dilution. In the end, two colonies capable of secreting mAbs against epitopes of PV2 were used to produce mAbs. The mAbs were characterized by antibody assays, isotyping, and epitopes analysis using western blotting test, the crossreactivity with other types, as well as stability, sterility, and mycoplasma tests. The results indicated that the produced mAbs had high specificity, sensitivity and stability against PV2 without any cross-reactivity and were of IgG1 Kappa chain with similar bands at 26 kDa during electrophoresis associated with viral protein VP3 neutralization. These mAbs were specific in serum neutralization tests for PV2 vaccine strain, and therefore, they are potentially valuable for routine polio research, diagnosis, isolation, production, and control of poliovirus vaccines.