Abstract: Validation of process is an important guarantee of drug quality. Due to biological products derived from the cell matrix, the preparation process is complex, with incomplete characterization of the structure, biological products, process validation needs special consideration. In this paper, according to the relevant guiding principles at home and abroad, combined with the practice of drug review and evaluation, the author summarizes the biological characteristics of the process of cell substrate, fermentation and purification process, virus safety, chromatography medium life and disposable reactor consumables, and intermediate stability Sexual and other general requirements. At the same time, the current common problems in the registration of biological products are discussed.
Key words: biological products; process validation; process development; process characterization; process validation
The general points to consider and discussion of biologics process validation
Due to derived from living subsubstrate, complexity of manufacturing process and not-well characterization of molecular structure, some specific points to consider for process validation of biologics were required. In this review, the general issues related to biologics process validation was summarized, including cell substrate, viralsafety, fermentation and purify process, single used bioreactor, chromatographylife, and stability and holding time of intermediate. Moreover, the common problems involved in the regulatory submission of process validation data were also discussed.
Key words: Biologics; Process validation ; Process development ; Process characterization; Process verify;
Foreword
Process validation is the process of demonstrating that a manufacturing process consistently and consistently produces a product that meets its intended use and registration requirements in accordance with the specified manufacturing parameters [1] . The clinical effectiveness and safety of drugs depend on the quality attributes of their products. According to the concept of Quality by Design ( QbD ), end product quality is not only dependent on quality standards and product inspection, but also on process design and process verification [2] . Process design defective drugs, even through the terminal inspection can not guarantee the process continued to produce qualified products. Based on the above advanced concepts, the focus of pharmaceutical research and development and evaluation has been gradually shifted from the research of quality standards many years ago to the research and evaluation of process [3] .
Biological products, including human vaccines, recombinant DNA proteins, recombinant monoclonal antibodies, blood products and tissue extracts and so on. Compared with small molecule chemical drugs, the biological substrate is composed of cells, microorganisms, blood, tissues and body fluids. The production and preparation process of the biological products is complicated. According to the current analysis methods, the molecular structure of the biological products can not be fully characterized. Therefore, in order to ensure the consistency of the quality of biological products, the design, verification and verification of the production process are particularly important. This paper first introduces the development of the concept of process validation and the latest requirements, combined with its own characteristics of biological products, focusing on biological products process validation special consideration. Finally, from the drug review point of view, we discuss the common problems in the current registration of biological products.
First, the concept and implementation of process validation
1.1 process validation concept development and related requirements
In 1987 , the United States FDA was the first to promulgate the "Guide for Process Validation". It first proposed the concept and implementation of process validation. Since then, with the development of industry technology, risk control and the application of advanced concepts such as QbD , the United StatesFDA released a new Guide to Process Validation in 2011 , The concept of "product life cycle" was introduced to emphasize the scientific, systematic, and sustainable nature of process validation. And process verification is divided into three phases: Process Design , Process Qualification and Continued Process Verification [2] . Since then, the EU EMEA issued the "Guide for Process Validation" in 2012 , pointing out that the process validation method can be implemented by traditional process verification, continuous process verification or a combination of both [4] . In 2016 , the EU EMEA proposed the content and specific requirements for the verification of the biological raw material drug process in response to the registration requirements of the biological products listing and the process change. Which the upstream process, downstream technology, multi-factory production, rework and other processes involved in the certification made specific requirements. [5]
China's 2010 edition of "Good Manufacturing Practices for Pharmaceutical Production" in the relevant requirements of the process validation focused on the seventh chapter (confirmation and verification), including a clear process validation should include the first verification, affecting the quality of the product after a major change to verify the necessary Revalidation and continuous process validation during the product life cycle to ensure that the process is always validated. Specific provisions such as: "process validation should prove that a production process in accordance with the provisions of the process parameters can continue to produce the intended use and registration requirements of the product"; "The use of new production prescription or production process, should verify the suitability of its conventional production "Confirmation and verification is not a one-time action. After the first confirmation or verification, it should be re-confirmed or re-verified according to the product quality review and analysis. The key production processes and operating procedures should be re-verified periodically to ensure that they can achieve the expected Results "and so on [1] .
In addition, according to the current Measures for the Administration of Drug Registration, on the first registration of manufactured drugs, after the adoption of the technical review, on-site inspections of commercial-scale production processes should also be carried out and samples taken for testing to confirm the feasibility of the production process . Drug trial center will combine the technical review comments, sample production site inspection reports and sample test results comprehensive evaluation [6] .Therefore, the scale of the proposed commercial production process verification is also one of the necessary conditions to decide whether the drug can be listed.
1.2 process validation and methods of implementation
The implementation of process verification should focus on the understanding of production technology and products. In accordance with the latest "Process Validation Guide" promulgated by FDA , it can be implemented in stages according to process design, process confirmation and continuous process verification [2] . The process design stage should be based on the knowledge acquired during development and pilot production to establish the production process design space and control strategy; process validation is the process design assessment to determine if the process has the ability to be re-commercialized; the process validation is for To ensure that the commercialization of chemicals continues to be controllable and to evaluate process changes by collecting and evaluating information and data on process performance after the product is launched.
Process design order In order to examine the relationship between process parameters (input) and product quality (output), to provide a scientific basis for the establishment of design space and production control strategy, the use of small equipment DOE ( Designof Experiments ) experimental design can be used to investigate. Such as: culture process development using shake flask, spin-off ( Spintube ) or small fermentor while a variety of fermentation parameters (medium, temperature, pH and dissolved oxygen, etc.) to optimize [7,8] ; purification process development process In a 96- well plate ( PreDictor plate96 ) high-throughput screening of chromatography media or buffer [9] ;
Process validation phase is to confirm the commercialization of cGMP process conditions, to prove that the product can be stable and reproducible after the listing to produce a consistent quality of the product. Commercial production process validation should include two elements, namely, plant design, equipment and facilities validation, and process performance qualification ( PPQ ), which are performed using certified plant, equipment and facilities and trained by trained personnel In accordance with the commercial production process, control procedures for commercial products, such as the production process. Therefore, the bio-products registered clinical trials, the trial of three batches of clinical trials of the production can not yet be a rigorous process to confirm. Process validation refers to biological products reported before the pre- cGMP conditions required for three batches to be listed on the scale of production. The robustness of the commercial process can be finally confirmed through the process parameters of this stage, the central control indicator and the quality of the product .
Continuous verification of the process refers to the continuous monitoring of the product after it is put on the market in order to ensure that the process is always in control. After the biological products are registered and produced, the batch commercial production data should be continuously collected and analyzed. This will not only ensure the controllability of the quality of the listed products, but also provide "comparable" research data support for subsequent technological changes and product quality improvement.
Second, biological products process validation special consideration
Compared with the traditional small molecule drugs, the production process of biological products possesses the following characteristics: ( 1 ) the products are derived from living cell matrix or bio-derived substances; ( 2 ) the production process involves complicated multi-process steps; ( 3 ) Product molecular structure is complex, structural confirmation has some incomplete, at the same time emphasize the importance of biological activity. Such as: therapeutic recombinant monoclonal antibody generally used mammalian cells expressed by animal cell culture, multi-step chromatography purification made of lyophilized or liquid preparation . There are theoretically at least 108 variants of IgG -type antibodies[9] , whereas prophylactic vaccines are more complex, usually complete pathogen or pathogen components, combined with adjuvant compatibility and interaction. At present, most of the preventive vaccines can not provide complete structural confirmation [10] .
Therefore, the validation of biological products should be combined with the complexity of biological products and product variability for special consideration. For example, in the production process involving a cellular matrix, body fluid, tissue or blood at risk of virus safety, virus inactivation of the cell matrix should be considered in process validation; for recombinant proteins expressed using continuous passage engineering cells, process validation should consider engineering cell lines Of the pass-through stability; for the purification process using multiple steps of biological products, the downstream process validation should consider the product-related impurities and process-related residues of impurities or removal; for end products containing multiple forms of biological products, process validation Use advanced characterization tools to provide structural confirmation information as much as possible.
2.1 on the production of cell matrix validation
Most of the prevention or treatment of biological products, products derived from the living cell matrix. Such as: the production of the vaccine matrix mainly using primary cells (mouse kidney cells, monkey kidney cells, chicken embryo cells, etc.), diploid cells and passage cells ( Vero ) [10] . Therapeutic recombinant proteins are expressed heterologously by mammalian cells ( CHO , SP2 / 0 , NS0, etc.) [11] ; therapeutic cells are derived from the patient's autologous cells. Therefore, in order to ensure product safety and quality consistency, biological substrates should be fully verified.
According to the requirements of 2015 Chinese Pharmacopoeia, production cells should be managed strictly according to the multi-level seed bank (master cell bank, working cell bank) and a comprehensive test should be performed on the master cell bank, working cell bank, and production end-stage cells ( EPCs) , Purity, Internal / External Factors, Tumorigenic / Oncogenic Types, Karyotypes, etc.). General master cell bank should conduct a comprehensive test; working cell bank exogenous factor detection, should focus on the main cell bank to the working cell bank may be introduced during the passage of the virus. For example, animal origin material (porcine trypsin or bovine serum) is used in the passage or construction of cell bank. The cell bank should also detect porcine / bovine-derived virus. For passaging cells that have been shown to be neoplastic ( CHO , NSO , HEK293 , BHK21, etc.) for the production of therapeutic biologicals, cytotoxicity assays may no longer be performed. Domestic reporting Clinical trials generally require the identification of terminal production cells ( EPCs ). If the culture process changes occur during the clinical trial phase, the EPC cell test needs to be re-performed [12] .
In addition, in order to ensure the consistency of the end product quality, the engineering cells should be limited generations in the production of the passage, the number of passages should be verified by genetic stability experiments. That is, the genetic stability studies should cover more generations than the largest number of passages in commercial-scale production. General working cells in the simulation of real production technology, the use of small bioreactors for continuous subculture.Stability indicators should also include the production of genotypes (gene sequence of interest, gene copy number, integration sites, etc.) and phenotype (viability, metabolism, target product titer, etc.)[13] .
2.2 on fermentation and purification process validation
Biological products of liquid production process is generally divided into cell-based fermentation upstream process, and multi-step purification process based downstream process. For the study of the process characterization of the stock solution, the rationality of operating parameters of each process operation should be indicated and the necessary acceptance standards for the central control system should be established. Research and validation of cell culture processes should focus on the effects of operating conditions on cell growth properties, metabolic status, and the product of interest. And, should be based on the maximum passage generation, microbial contamination or product quality indicators of the establishment of fermentation broth, such as sterile, mycoplasma, specific virus, the desired product yield.
Downstream purification process research and validation should focus on the key processes for product-related impurities (aggregates, degradation products, charge isomers, hydrophobic variants, etc.), process-related impurities (host protein, host DNA , , Antibiotics, etc.) can be effectively removed or residual levels [14] . For chemically coupled modified products (chemically coupled antibody drugs, chemically modified peptide drugs, etc.), process validation should also focus on process control of free modifying groups, unconjugated protein ratios, and the like. For downstream vaccines, processes should focus on validated antigen extraction and impurity removal [15] .
2.3 Process Verification of Virus Security
There is a risk of virus contamination in biotech products, cell matrices and some of the raw materials produced (trypsin, serum). Furthermore, since retrovirus particles are contained in cell genomes such as CHO and SP2 / 0 , verification of the virus safety of biological products requires attention not only for the source of the cell matrix and the raw materials, but also for the removal / inactivation ofviruses in the purification process Ability [16] . The current domestic general requirements, biological products into the clinical trial before the completion of the key processes of virus removal and inactivation verification, reporting stage of production will be combined with the service life of chromatography medium to complete the process of virus inactivation to verify.
Validation of virus inactivation / removal process Validation of the inactivation / removal efficiencyof the main processes (low pH incubation, S / D treatment, nanofiltration, etc.) is mainly carried out by using a scale-down model to simulate the actual process conditions . The virus used in the validation process should be representative. For example, enveloped viruses ( MuLV , VSV or PRV ) can be used as indicator viruses for low pH incubation ; parvovirus ( PPV or MMV ) can be used as the indicator virus for membrane filtration . The general requirement for virus inactivation is that at least two steps result inmore than 4 logs of virus removal / inactivation [16] [17] .
It is noteworthy that, for virus inactivation process validation, it is necessary to ensure that the efficiency of inactivation of the model virus, but also pay attention to product quality changes. It has now been found that IgG4 -type antibodies (eg Nivolumab ) are more sensitive to pH during acid incubation[18] and therefore their process validation should also examine the effect of virus inactivation on product quality (antibody purity and activity, or vaccine antigen content And potency, etc.). For traditional vaccines such as inactivated vaccines, since the product itself is a virus and the viral load is much higher than the potential theoretical value of biotech products, its virus inactivation verification is required in addition to the conventional inactivation test for the inactivation endpoint. In addition, Inactivation process sampling for virus inactivation kinetics curve; at the same time it needs to be confirmed by the study, inactivated antigen still has sufficient immunogenicity.
2.4 on the chromatographic medium, one-time validation of the applicability of the reactor
Chromatographic procedures (affinity, hydrophobic, ion exchange, etc.) as biological products to remove the main impurities and virus inactivation process, its processing power and purification effect depends on the service life of the chromatography medium. Therefore, the general biological products in the registered production, should be used to reduce the size of the model to simulate the actual process conditions, according to process performance (protein loading, recovery) and product quality (purity, process impurities remaining) to confirm its service life Number) [14] .
In recent years, as a one-time reactor to avoid cleaning, sterilization and other validation costs, large-scale cell culture has been widely used. Such as: the use of disposable bioreactor flow culture animal cells to produce recombinant antibodies [7] , the use of disposable reactor culture CHO-C 28 cells to produce HbSAg antigen [19] . However, taking into account the applicability and potential risks of disposable bioreactor materials, process validation studies such as Extractable and Leachables should beconducted . EMEA 's process validation document also states that the process development phase should be fully disposable consumables to consider the risk assessment of leachables and extracts, to take supplier documentation (eluate data) or to study using simulation conditions. Process validation phase, should explain the one-time consumables for the production performance (such as cell viability), product quality[5] .
2.5 intermediate products, liquid and preparation preservation and transport verification
Due to the instability of biological macromolecules, the general stock solution of biological products is frozen, and the preparation is stored and transported in a refrigerated manner. According to the newly promulgated Guiding Principles for the Stability of Biological Products, the selection of stock solutions, storage conditions, expiration date and transportation conditions need to be verified through stability studies (influencing factors, compulsive degradation, acceleration and long-term). In addition, the production process of some biological products is limited by the site and the process conditions. Some of the intermediates may be held at a certain stage of the process for a short time . For example, the affinity chromatography may be stored for a short time during antibody production Or refrigerated before further processing; inclusion of insulin in the production process after harvesting and other processing. At this point you need to verify the conditions for the storage of intermediate goods, valid period and shipping conditions. [20]
Third, the common problems in drug registration management
In the biopharmaceutical evaluation, the process validation data is the most direct proof of the scientific, rational and reproducible production process. At present, China's pharmaceutical manufacturing enterprises in the registration information registration process, the lack of process validation data is the more common defects in pharmaceutical research. Registration information often appear "there is no technology research and development process validation," "process validation process without research and development," and even "neither process research and development, there is no process validation" and other issues. All of the above conditions make it impossible to demonstrate process robustness and product consistency through process validation data. The following will be combined with the review of practice, the current domestic drug registration management process validation related issues and the underlying causes are discussed.
3.1 , process research is not sufficient to verify the batch / size is not representative
As the process validation research needs pharmaceutical manufacturers from many aspects such as raw materials, personnel, equipment and facilities to be guaranteed, the entire process of verification process of capital investment, high time and cost. In order to convert R & D results into production, applicants often try to reduce the number of process verification batches, compress the process verification time, resulting in inadequate process studies and unidentified batches / sizes. In addition, since the research and development of biological products in China have long followed the imitation of original research products from abroad, the production process routes can learn from the original research products or adopt the platform preparation technology, which is also likely to cause the applicants to lack sufficient research and process qualification Pay attention.
A comprehensive and in-depth study of the production process at the stage of process research will help to understand the influence of process parameters on product quality and thus reserve enough design space for the adjustment of process parameters. It can be said that the more full pre-technology research, product approval after the listing of the possibility of changing the process will be smaller. On the contrary, will result in the future product can not be manufactured in accordance with manufacturing procedures or a major process change after listing.
Process validation process validation phase should be consistent with cGMP specifications for research, and as far as possible the worst-case process conditions to confirm the process to be listed on the robustness. At present, the batch verification of the domestic production of listed varieties lacks a sound and comprehensive design. In addition to the routine release inspection and process control test data, the following factors are not sufficiently considered: the representative of the production process parameters and the consideration of the challenging test; the column And other equipment life research; production scale of the terminal generation cells and / or strains of sequencing research.
3.2 , the lack of product " sustainable " process validation
At present, the requirements of process verification both at home and abroad point out that confirmation and verification are not transient behaviors. After the first confirmation or verification, they should be re-confirmed or re-verified according to the product quality review and analysis. However, the understanding of process validation by some drug manufacturers is verification of the "point" of reporting time. That is, only three batches of clinical trials or registered production meet the proposed quality standards, lack of consideration of the whole life cycle of process validation, The lack of comparability of process and product quality at all stages, especially the comparability of listed and clinical samples.
For clinical studies or process changes that occur after the product is launched, the concept of process validation at this time should be combined or extended to comparability studies. That is, by examining batch release, structural verification and stability studies (especially accelerated or forced degradation) of multiple batches of products before and after the change, it is confirmed that product quality is consistent before and after the process is changed. If there are quality differences or pharmacies alone can not explain the comparability of product quality, non-clinical, clinical and other comparable research should continue [21] .
3.3 , the concept of process validation lags behind the lack of relevant guiding principles
The number of process validations is based on the complexity of the process, the level of variation in the process, and the level of knowledge about the process. Process validation of biological products should encourage the use of multiple batches of data at different stages [22] . Part of the applicants' narrow understanding of process validation as three trial-to-market-scale pre-production in three batches of pilot-scale production and registration during the registration clinical phase actually stems from a one-sided understanding of the concept of process validation in the 1980s Has contradicted the "drug-based life cycle" process validation philosophy currently advocated. Even in the practice of drug registration management, there are still some problems such as the submission of research materials on pilot production process during the application stage for listing and the verification of the commercial scale have not yet been completed. This further exposes drug developers lacking the necessary knowledge and understanding of the concept of process validation.
In our country, the related requirements of the validation of biological products are scattered in the "Good Manufacturing Practices for Pharmaceutical Production", "General Introduction to Chinese Pharmacopoeia" and the guiding principles of quality control of related products (recombinant DNA products, human monoclonal antibodies, conjugate vaccines, Peptide vaccine, vaccine production process changes, blood products virus inactivation) and other documents. There is no specific guiding principle system to verify the requirements of biological products process, but also cause the industry for biological products process verification lack of complete, in-depth understanding of the important reasons. In this regard, the EU 's "Requirements for Process Validation and Reporting of Biotechnological Source Products" issued in 2016 covers the verification requirements of upstream processes, downstream processes, processing of multiple products and rework of biological products. For the verification of domestic biological products processes, A certain degree of practical significance.
Conclusion
Process validation is to ensure that biological products after the listing of safe, effective, quality control of prerequisites. Advocacy and implementation of advanced technology validation concept is conducive to the long-term development of China's bio-pharmaceutical industry. The author believes that the industry should fully understand the process of verification brought about the initial investment and future earnings. Although process validation can increase enterprise R & D costs and delay product launches. However, by verifying the robustness and flexibility of the manufacturing process, process validation actually reduces the risk of product changes in finished products and processes. From the life cycle of pharmaceuticals, good process verification can bring sustained economic returns for manufacturing enterprises; for regulatory authorities, advanced process verification concepts should be advocated to standardize and guide the scientific research on process validation in an efficient and scientific manner . In recent years, the United States FDA and the European EMEA have all advocated for modular virus verification [17] . That is: different mAb using the same purification process, for a module of virus validation can be extrapolated to other antibodies. In accordance with the concept of clinical application of foreign platform for verification. At the stage of re-production of product-specific virus removal and clearance validation [9] . In addition, the scale of the proposed commercial scale batch of foreign products can be marketed. In our country, based on the current domestic laws and regulations, we can not go on the market to verify batch products of process verification at the stage of delivery. This not only reduces the applicant's enthusiasm for process validation but also limits the batch, size and verification of process validation. Therefore, based on the current development of the domestic biological industry and drawing on the advanced international concepts and experiences, it is of great scientific and effective significance to carry out the verification of the biological products in a scientific and efficient way, which deserves to be discussed by the industry and the regulatory community.
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"Chinese Journal of Biological Products" 2017 the sixth period
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