Lot Release Research Articles

국내 혈장분획제제 안전관리 정책과 특성에 대한 고찰

Plasma-derived medicinal products (PDMPs) are pharmaceuticals prepared using proteins in plasma, a liquid component of blood. Regarding the use of human blood as a raw material in PDMP manufacturing, it has unique characteristics unlike other pharmaceuticals (for example, limitations of scientific identification, finiteness of blood, and risk of infection through disease pathogens, such as the human immunodeficiency virus, hepatitis B virus, and hepatitis C virus in the blood). The safety management policy of PDMPs in Korea started with the enactment of the Blood Management Act in 1970. After that, the current safety management system was established through product approval management according to the Pharmaceutical Affairs Act in 2006, higher standards of safety management for imported source plasma in 2009, and expansion of management standards for source plasma in 2012. The safety management of PDMPs involves several important policies. Public management has been carried out by the Korean Red Cross and national government, and domestic plasma should be used first, while imported plasma may be used only if there is not enough. Additionally, to ensure blood products safety, plasma testing, inventory hold, and plasma master file reporting obligations are being implemented. In addition, it is managed as a subject of national lot release, which should also be quality-confirmed by the country. Because the supply of domestic source plasma, which is the raw material of PDMPs, has shown a decreasing trend in recent years, continuous monitoring and preparation is required. In this study, we describe the history of safety management policies, including the definition and classification of PDMPs, product status, usage status of source plasma, and the related policy guidelines and characteristics.

Target cell line characterization reveals changes in expression of a key antigen that impacts T cell dependent cellular cytotoxicity assay performance

Cell lines are important tools regularly used for in-vitro potency assays employed in Good Manufacturing Process (GMP) lot release and stability testing of different therapeutic modalities. Characterization of these cell lines is key to understanding their performance.Bispecific T cell engager (BiTE®) molecules are an exciting modality in Amgen's therapeutic product portfolio. BiTE® molecules are engineered as two linked, single chain antibody domains. One domain targets the cluster of differentiation 3 (CD3) domain of T cells, and the other domain targets a specific antigen. The mechanism of action of the BiTE® molecule is to bring T cells into close proximity of tumor cells to facilitate tumor cell killing. One BiTE® molecule in development, AMG 757, is directed against delta-like ligand 3 (DLL3), which is expressed in small cell lung cancer tumor cells. AMG 757 is a half-life extended bispecific T cell engager (HLE BiTE®) construct. The bioassay employed to demonstrate the mechanism of action of AMG 757 is a T cell dependent cellular cytotoxicity (TDCC) cell-based assay requiring two cell lines, the effector T cell line HuT-78, and engineered tumor target cells, SHP-77-Luc. During the course of development of this bioassay, characterization of the SHP-77-Luc line showed an increase in the luminescence assay signal and Maximum-to-Minimum (Max-to-Min) ratio of the dose response curve as the passage number of the cells increased. Our research revealed an increase in not only luciferase expression but also an increase in the cell surface and intracellular expression levels of DLL3 over time in culture, which ultimately resulted in the increased assay signal window.

Bayesian statistical approaches to drug product variability assessment and release

The determination of the variability of critical dosage form attributes has been a challenge in establishing the quality of pharmaceutical products. During the development process knowledge is minimal. Consequently, ad hoc statistical tools such as hypothesis or significance tests, with calibrated decision error rates are often used in an effort to vet CQAs (Critical Quality Attributes) and keep their levels “between the curbs”. As progress moves towards product launch, process and mechanistic understanding grows considerably and there are opportunities to leverage that knowledge for predictive modeling. Bayesian models offer a coherent strategy for integrating prior knowledge into both experimental design as well as predictive analysis for optimal risk-based decision making. This is because the Bayesian paradigm, unlike the frequentist paradigm, can assign probabilities to underlying states of nature that directly impact safety and efficacy such as the population distribution of tablet potencies or dissolution profiles in a batch. However, there are challenges and reluctance in switching to a predictive modeling quality framework once regulatory approval has been attained. This paper offers encouragement to make this switch.In this paper, we review a joint Long Island University - Purdue University (LIU-PU) FDA funded project whose purpose was to further integrate the concepts of this adaptive approach to lot release with the rationale and methods for data generation and curation and to extend the testing of this approach. We discuss the utility of the approach in product development. We consider the regulatory compliance implications, with examples, and establish a potential way forward toward implementation of this approach for both industry and regulatory stake-holders.

Driving CAR-T from early-stage development to clinical filing and lot release

Driving CAR-T from early-stage development to clinical filing and lot release Approach to assess the efficacy, potency, persistence, and safety of your CAR-T cell therapy in vitroEvaluating the efficacy and safety profile of CAR T in vivo and lessons learnedShare regulatory expectations (guidelines and how to interpret them) and challenges with selecting sustainable CAR-T cell potency assays for development that reflect the MoA and are able to meet method validation requirements.   Discuss biological activity assays at play for CAR-T and inform on how to approach determination for lot release of CAR-T cells will be presented as it plays a key role in defining the quality of the product.Dr Ulrike Herbrand joined Charles River Laboratories in 2007. She is Scientific Director Global in vitro Bioassays and Head of the Bioassay Research & Development team at Charles River Laboratories’ site in Erkrath, Germany. She gained a PhD in biological sciences during her time at the Max-Planck-Institute for Molecular Physiology in Dortmund (Germany) and worked five years at post-doctoral positions at medical research centers in the field of cancer research. She is an expert in mechanism of action-reflecting bioassays.Dr Julia Schüler studied veterinary medicine in Switzerland and Germany where she received her DVM. Afterwards, Julia joined the lab of Heiner Fiebig, at Oncotest GmbH, in Freiburg, Germany receiving her PhD on orthotopic implantation of solid cancer PDX. After a post-doc in innate immunity at the Max-Planck institute she re-joined Oncotest in 2002. Since then, she is driving the oncology model development strategy within Oncotest and since 2015 within Charles River. She currently holds the position of a research director at Charles River.Sophie Vermond is a Senior Scientist at Charles River Laboratories in Leiden, the Netherlands. Her research focuses on efficacy and toxicity testing of novel drug therapies in human primary cell-based models. Her main focus is T cell therapy safety studies. Furthermore, she is head of the flow cytometry group.

Long-term monitoring of antibody titers against microorganisms in human intravenous immunoglobulin lots derived from healthy donors in Japan manufactured during 1998-2018.

Studies evaluating long-term trends of intravenous immunoglobulin (IVIG) titers against microorganisms such as bacteria and fungi have not been conducted. We herein evaluated long-term trends of titers of IVIG lots manufactured from 1998 to 2018, derived from donors in Japan, against microorganisms affecting patients with primary or secondary immunodeficiency diseases. Titers against four strains of Pseudomonas aeruginosa, three strains of Staphylococcus aureus, four strains of Haemophilus influenzae, two strains of Klebsiella pneumoniae, one strain of Streptococcus pneumoniae, Enterococcus faecium, Bordetella pertussis, Serratia marcescens, and Candida albicans, were tested immediately after lot release in a commercial clinical testing facility in 1998. The long-term (whole period) and short-term (first and second half of the period) trends of titers were evaluated using regression analysis. The IVIG lots indicated meaningful titers against all microorganisms that were stable in the short term. The long-term trends could be categorized into stable, slightly decreasing, or decreasing trends, except for the metallo-β-lactamase-producing K. pneumoniae, which indicated a slight increase. Notably, three strains of P. aeruginosa showed remarkable decreasing long-term trends in the titer. Some titers indicate decreasing trends against microorganisms over the long-term, however, is not clear whether the phenomenon diminishes the performance of IVIG. The titers of the IVIG lots could provide helpful information to optimize replacement therapy, such as considering trough values based on the titers in the patient plasma. Therefore, continuous monitoring of IVIG titers against microorganisms is important to understand titer fluctuation and epidemiological background.

Accelerated national lot release on COVID-19 vaccines in Republic of Korea

Independent quality testing of samples from vaccine lots is part of quality assurance, especially to ensure the consistency of production lot by lot. Effective national lot release system that ensures the quality of each lot of vaccine before it is on the market is important because vaccines are intended to healthy people. In order to respond more quickly to public health crises such as the COVID-19 pandemic, the MFDS implements accelerated national lot release for rapid vaccination in Republic of Korea.For the accelerated system, improvement has been made in terms of timing of application for lot release and required documents. In addition, the processing period has been shortened and sampling method and test items have been streamlined. A thorough preparation for accelerated lot release has been developed by establishing test methods for a new platform in advance. As a result, a total of 43.88 million doses have been released within eight days on average. The accelerated lot release system has contributed significantly to rapid COVID-19 vaccination in Korea.

Considerations for use of hematopoietic stem cells in allogeneic therapies

This article reviews some of the considerations and unforeseen challenges with sourcing and preparing regulatory filings using donor supplied Hematopoietic stem cells (HSCS) in novel allogeneic cell and gene therapies. There are unique complications not seen with autologous therapies as developers must understand the regulatory requirements for donor sourcing, testing and isolation of CD34+ cells for IND submissions. Finding the right partner for sourcing as well as lot release testing is discussed. Understanding the procurement requirements for the starting cells and the manufacturing process used is reviewed. This article covers some of the areas that organizations may miss when developing these types of cell and gene therapy products for the first time.

Continuous Method Validation: Beyond One-Time Studies to Characterize Analytical Methods

A validated method is used to measure quality attributes of manufactured lots to support the production of active ingredient or drug product for a regulatory filing or product release. Various regulatory guidelines provide expectations on how to validate a method, including accuracy and precision. Validation studies are not typically designed to have the size or scope needed to fully characterize a method. We can and should, however, capitalize on the historical data that is generated from method development, transfer, and clinical trial lot release to facilitate the characterization. Using prior information from these sources affords a more confident characterization of the method’s accuracy and precision. This article focuses on Bayesian methods for using the stream of available method data. Considerations are given for prior distributions, including methods to control the influence of prior data. Two case studies are presented to illustrate the continuous validation approach. This is a joint effort of the DIA Bayesian Scientific Working Group and the American Statistical Association Biopharmaceutical Nonclinical Working Group.

Points to consider for COVID-19 vaccine quality control and national lot release in Republic of Korea: focus on a viral vector platform.

Due to the global public health crisis caused by the coronavirus disease 2019 (COVID-19) pandemic, the importance of vaccine development has increased. In particular, a rapid supply of vaccines and prompt deployment of vaccination programs are essential to prevent and overcome the spread of COVID-19. As a part of the vaccine regulations, national lot release is regulated by the responsible authorities, and this process involves the assessment of the lot before a vaccine is marketed. A lot can be released for use when both summary protocol (SP) review and quality control testing are complete. Accelerated lot release is required to distribute COVID-19 vaccines in a timely manner. In order to expedite the process by simultaneously undertaking the verification of quality assessment and application for approval, it is necessary to prepare the test methods before marketing authorization. With the prolonged pandemic and controversies regarding the effectiveness of the COVID-19 vaccine against new variants, public interest for the development of a new vaccine are increasing. Domestic developers have raised the need to establish standard guidance on the requirements for developing COVID-19 vaccine. This paper presents considerations for quality control in the manufacturing process, test items, and SP content of viral vector vaccines.

A Systematic Approach for the Evaluation, Validation, and Implementation of Automated Colony Counting Systems.

For several years, automated colony counting systems have been available with varying degrees of automation. Ever more sophisticated instruments are now increasingly used in microbiological laboratories of pharmaceutical quality control. In addition to the colony counting device, the instruments are now also equipped with robotic systems performing the entire handling of the petri dishes, e.g., automated internal transportation of petri dishes from the incubator chamber to the instrument's enumeration device and back. Moreover, the subjective evaluation of microbial enumeration tests by analysts is replaced with a more accurate and precise process. This leads to significant improvements to data integrity compliance. Automated colony counting systems also often enable cost reduction in the microbiological laboratory, e.g., by not requiring a contemporaneous verification by a second analyst. They also enable direct integration of count data into an existing laboratory information management system, reducing the hands-on time, costs per test and also preventing human errors caused by manual transcription. Altogether, these instruments will lead to improved monitoring and assurance of control of biopharmaceutical processes and manufacturing environments, as well as shortened cycle times in the supply chain. Regulators are encouraging the biopharmaceutical industry to adopt these innovative systems. For example, this year a BioPhorum member company received the first health authority approvals from EU, US, CH, Canada, Australia, and China for the use of automated colony counting systems for in-process bioburden testing and the release of drug substance lots, with an incubation time reduced by about 50%. Although these approvals are for release testing of drug substance lots, the instruments can also be used for environmental monitoring, testing of water samples, etc. This article describes a systematic 9-step approach to the evaluation, equipment qualification, and deployment of automated colony counting systems, which can be applied by biopharmaceutical companies wanting to take advantage of their numerous benefits.

Characteristics of SARS-CoV-2 mRNA/LNP vaccines and their official verification

SARS-CoV-2-causing COVID-19, of which outbreak was initially reported from China at the end of 2019, became a large-scale global epidemic in 2020. To cope with COVID-19, a novel type of vaccines has been developed with unprecedented speed and has been very useful in many countries, including Japan, for controlling the severity of this infectious disease. Two items of the coronavirus-modified uridine RNA vaccine, in which mRNA encoding the SARS-CoV-2 spike protein is embedded in lipid nanoparticles (LNP), are currently being marketed in Japan. Each of them has very high efficacy and almost no serious side effects, although fever and fatigue that subside in about 2-3 days are observed. In this symposium, I would like to talk about the characteristics of the mRNA/LNP vaccine, which was generated by combining the best of various sciences and technologies, based on publicly available information. Unlike conventional virus vaccines, the above mRNA/LNP vaccine does not contain any components directly derived from living organisms and is more like a chemical medicine. However, the vaccine is still subject to official verification at the time of lot release. The global standard that must be implemented at the time of lot release of a vaccine is the review of the Summary Lot Protocol (SLP),which summarizes the manufacturing and quality testing records for each lot, to determine whether it conforms to the approved content of the product. The SLP review is also conducted for the mRNA/LNP vaccines by the National Institute of Infectious Diseases.

Rapid, high throughput protein estimation method for saponin and alhydrogel adjuvanted R21 VLP Malaria vaccine based on intrinsic fluorescence

Protein content estimation of recombinant vaccines at drug product (DP) stage is a crucial lot release and stability indicating assay in biopharmaceutical industries. Regulatory bodies such as US-FDA and WHO necessitates the quantitation of protein content to assess process parameters as well as formulation losses. Estimation of protein content at DP stage in presence of adjuvants (e.g AlOOH, AlPO4, saponin and squalene) is quite challenging, and the challenge intensifies when the target protein is in Virus like particles (VLP) form, owing to its size and structural complexity. Methods available for protein estimation of adjuvanted vaccines mostly suffer from inaccuracy at lower protein concentrations and in most cases require antigen desorption before analysis. Present research work is based on the development of a rapid plate-based method for protein estimation through intrinsic fluorescence by using Malaria vaccine R21 VLP as a model protein. Present method exhibited linearity for protein estimation of R21, in the range of 5–30 µg/mL in Alhydrogel and 4–20 µg/mL for Matrix M adjuvant. The method was validated as per ICH guidelines. The limit of quantification was found to be 0.94 µg/mL for both Alhydrogel and Matrix M adjuvanted R21. The method was found specific, precise and repeatable. This method is superior in terms of less sample quantity requirement, multiple sample analysis, short turnaround time and is non-invasive. This method was found to be stability indicating, works for other proteins containing tryptophan residues and operates well even in presence of host cell proteins. Based on the study, present method can be used in vaccine industries for routine in-process sample analysis (both inline and offline), lot release of VLP based drug products in presence of Alhydrogel and saponin based adjuvant systems.

The Regulatory Evaluation of Vaccines for Human Use.

A vaccine is an immunogen, the administration of which is intended to stimulate the immune system to prevent, ameliorate, or treat a disease or infection. A vaccine may be a live attenuated preparation of microorganisms, inactivated (killed) whole organisms, living irradiated cells, crude fractions, or purified immunogens, including those derived from recombinant DNA in a host cell, conjugates formed by covalent linkage of components, synthetic antigens, polynucleotides (such as the plasmid DNA vaccines), mRNA, living vectored cells expressing specific heterologous immunogens, or cells pulsed with immunogen. Vaccines are highly complex products that differ from small molecule drugs because of the biological nature of the source materials such as those derived from microorganisms as well as the various cell substrates from which some are derived. Regardless of the technology used, because of their complexities, vaccines must undergo extensive testing and characterization. Special expertise and procedures are required for the manufacture, control, and regulation of vaccines. Throughout their life cycle from preclinical evaluation to post-licensure lot release testing, vaccines are subject to rigorous testing and oversight by manufacturers and national regulatory authorities. In this chapter, an overview of the regulatory evaluation and testing requirements for vaccines is presented.

Development and validation of a reporter gene assay to determine the bioactivity of anti-CTLA-4 monoclonal antibodies

CTLA-4 is an important immune checkpoint for the regulation of T cell activation, and anti-CTLA-4 monoclonal antibodies (mAbs) are being developed as mono- or combination therapy for various tumors with reliable clinical efficacy. Ipilimumab is the first approved inhibitor of immune checkpoint, and many other anti-CTLA-4 mAbs, including ipilimumab biosimilars, are in different stages of clinical trials. However, due to the immunomodulating nature of the mAbs targeting CTLA-4, mode of action (MoA) and cell-based bioassay to determine their bioactivities as the lot release or stability test has been a great challenge to quality control laboratories. In this study, we have developed and validated a reporter gene assay (RGA), in which two kinds of cell lines were engineered to measure the bioactivity of anti-CTLA-4 mAbs. Raji cells were stably transfected with the membrane-anchored anti-CD3 single chain antibody fragment (scFv) as antigen-presenting cells (APCs, Raji-CD3scFv cells), while Jurkat cells were stably transfected with CTLA-4 with Y201V mutation and NFAT controlled luciferase as the effector cells (Jurkat-CTLA-4-NFAT-luc cells). The ligation of CD80/CD86 on the APCs with CTLA-4 could reduce the luciferase expression accompanied with the activation of effector cells, while the anti-CTLA-4 mAb could reverse the reduction, which resulted in good dose response curve to determine its bioactivity. After optimizing various assay conditions, we performed full validation according to ICH-Q2 (R1), which demonstrated the excellent specificity, accuracy, precision, linearity, and the cell passage stability. The satisfied performance characteristics render the RGA a good bioassay in the bioactivity determination of anti-CTLA-4 mAbs, as applied in characterization, batch release control, stability study, and biosimilar assessment.

Adoptive Therapy with Allogeneic Cord Blood T Regulatory Cells Improves Transfusion Requirement in Bone Marrow Failure Syndromes

Adoptive Therapy with Allogeneic Cord Blood T Regulatory Cells Improves Transfusion Requirement in Bone Marrow Failure Syndromes

Aggregation and Size Attributes Analysis of Unadsorbed and Adjuvant-adsorbed Antigens using a Multispectral Imaging Flow Cytometer Platform

Various vaccine quality attributes should be monitored to ensure consistency, potency, purity, and safety of vaccine products prior to lot release. Vaccine particle size and protein antigen aggregation are two important considerations for particle-adsorbed vaccines. In this study, we evaluated the use of imaging flow cytometry as a potential all-in-one platform to measure adjuvant particle size and to detect protein aggregates through a combination of brightfield microscopy, side scatter detection, and fluorescence microscopy. An aluminum phosphate adjuvant was analyzed for size using the brightfield function, and the size measurement was compared against laser diffraction. Heat-induced protein aggregates of either unadsorbed antigens or aluminum phosphate adjuvant-adsorbed antigens were stained with the fluorescent ProteoStat aggregation dye, followed by detection and analysis using a combination of the brightfield and fluorescence microscopy functions. The change in aggregation of unadsorbed antigens was confirmed using dynamic light scattering. These results demonstrate the versatility of the imaging flow cytometry platform for the evaluation of multiple vaccine quality characteristics.

A reporter gene assay for determining the biological activity of therapeutic antibodies targeting TIGIT

T cell immunoglobulin and ITIM domain (TIGIT) is a novel immune checkpoint that has been considered as a target in cancer immunotherapy. Current available bioassays for measuring the biological activity of therapeutic antibodies targeting TIGIT are restricted to mechanistic investigations because donor primary T cells are highly variable. Here, we designed a reporter gene assay comprising two cell lines, namely, CHO-CD112-CD3 scFv, which stably expresses CD112 (PVRL2, nectin-2) and a membrane-bound anti-CD3 single-chain fragment variable (scFv) as the target cell, and Jurkat-NFAT-TIGIT, which stably expresses TIGIT as well as the nuclear factor of activated T-cells (NFAT) response element-controlled luciferase gene, as the effector cell. The anti-CD3 scFv situated on the target cells activates Jurkat-NFAT-TIGIT cells through binding and crosslinking CD3 molecules of the effector cell, whereas interactions between CD112 and TIGIT prevent activation. The presence of anti-TIGIT mAbs disrupts their interaction, which in turn reverses the inactivation and luciferase expression. Optimization and validation studies have demonstrated that this assay is superior in terms of specificity, accuracy, linearity, and precision. In summary, this reliable and effective reporter gene assay may potentially be utilized in lot release control, stability assays, screening, and development of novel TIGIT-targeted therapeutic antibodies.

Development of a novel reporter gene assay to evaluate antibody-dependent cellular phagocytosis for anti-CD20 therapeutic antibodies

More than 100 monoclonal antibodies (mAbs) have been approved by FDA. The mechanism of action (MoA) involves in neutralization of a specific target via the Fab region and Fc effector functions through Fc region, while the latter include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). ADCP has been recognized one of the most important MoAs, especially for anti-cancer mAbs in recent years. However, traditional bioassays measuring ADCP always introduced primary macrophages and flow cytometry, which are difficult to handle and highly variable. In this study, we engineered a monoclonal Jurkat/NFAT/CD32a-FcεRIγ effector cell line that stably expresses CD32a-FcεRIγ chimeric receptor and NFAT-controlled luciferase. The corresponding mAb could bind with the membrane antigens on the target cells with its Fab fragment and CD32a-FcεRIγ on the effector cells with its Fc fragment, leading to the crosslinking of CD32a-FcεRIγ and the resultant expression of subsequent NFAT-controlled luciferase, which represents the bioactivity of ADCP based on the MoA of the mAb. With rituximab as the model mAb, Raji cells as the target cells, and Jurkat/NFAT/CD32a-FcεRIγ cells as the effector cells, we adopted the strategy of Design of Experiment (DoE) to optimize the bioassay. Then we fully validated the established bioassay according to ICH-Q2(R1), which proved the good assay performance characteristics of the bioassay, including specificity, accuracy, precision, linearity, stability and robustness. This RGA can be applied to evaluate the -ADCP bioactivity for anti-CD20 mAbs in lot release, stability testing as well as biosimilar comparability. The engineered cells may also potentially be used to evaluate the ADCP bioactivity of mAbs with other targets.

A practical approach for gmp-compliant validation of real-time PCR method for mycoplasma detection in human mesenchymal stromal cells as advanced therapy medicinal product

BackgroundManufacturing of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP) for clinical use involves an ex vivo expansion, which leads to a risk of contamination by microbiological agents. Even if manufacturing under Good Manufacturing Practice (GMP) license minimizes this risk, contamination of cell cultures by mycoplasmas still represents a widespread problem. Furthermore, the absence of mycoplasma contamination represents one of ATMPs release criteria. Since July 2007, European Pharmacopoeia (EuPh) offers the possibility to replace official mycoplasma detection methods with Nucleic Acid Amplification techniques, after suitable validation. As an Italian authorized Cell Factory, we developed an in-house GMP-compliant validation of real-time PCR method for mycoplasma detection in human Mesenchymal Stromal Cells, according to EuPh sec. 2.6.7 and International Conference on Harmonization Q2. Materials and methodsThe study was performed in compliance with GMP international requirements with MycoSEQ™ Mycoplasma Detection Assay (Thermofisher) on QuantStudio5 real-Time PCR (Applied Biosystems). Assay validation was developed to evaluate sensitivity, interferences matrix-related, specificity and robustness. ResultsMycoSEQ™ Mycoplasma Detection Assay has been successfully validated on human Mesenchymal Stromal Cells as results comply with validation protocol acceptance criteria. ConclusionsMycoSEQ™ Mycoplasma Detection Assay is a fast, sensitive and specific PCR-based Nucleic Acid Test assay that can be used as an alternative to official mycoplasma test methods for lot release of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP). Moreover, our study underlines the presence of interference on real-time PCR reaction due to matrix composition, pointing out a practical approach for method validation (i.e interference removal).

Development and validation of a novel reporter gene assay for determination of recombinant human thrombopoietin

Recombinant human thrombopoietin (rhTPO) was approved by the National Medical Products Administration in 2010 for the treatment of thrombocytopenia in patients with immune thrombocytopenic purpura and chemotherapy-induced thrombocytopenia. Nevertheless, no method for determining rhTPO bioactivity has been recorded in different national/regional pharmacopoeia. Novel methods for lot release and stability testing are needed that are simpler, quicker, and more accurate. Here, we developed a novel reporter gene assay (RGA) for rhTPO bioassay with Ba/F3 cell lines that stably expressed human TPO receptor and luciferase reporter driven by sis-inducible element, gamma response region, and gamma-interferon activated sequence. During careful optimization, the RGA method demonstrated high performance characteristics. According to the International Council for Harmonization Q2 (R1) guidelines and the Chinese Pharmacopoeia 2020 edition, the validation results demonstrated that this method is highly time-saving, sensitive, and robust for research, development, manufacture, and quality control of rhTPO.