About print and distribute stem cell preparations before the quality control and clinical research guidelines (trial)

The national health and family planning commission of the People's Republic of China

The 2015-08-21

Countries who do science hair [2015] no. 46

Provinces, autonomous regions and municipalities directly under the central government health family planning, food and drug supervision bureau, the xinjiang production and construction corps health, food and drug supervision bureau, national health related units directly under state family planning commission, the food and drug supervision bureau directly under the units:

To strengthen quality management of stem cells in the preparation and clinical research in China, the state health and family planning commission and food and drug supervision administration according to the "stem cell clinical research management approach (trial)", jointly formulate the "stem cell preparations before the quality control and clinical research guiding principles (try out)" (available from the national health and family planning commission or the food and drug supervision administration website). Hereby printed and distributed to you, please comply with them.

General office of national health and family planning commission

The food and drug supervision administration office

On July 31, 2015

Stem cell preparations before the quality control and clinical research guiding principle

(trial)

One, foreword

Second, the quality control of stem cell preparations

(a) stem cell collection, separation and the establishment of the stem cells (department)

(2) the preparation of stem cell preparations

(3) stem cells in the preparation of inspection

(4) the quality of the stem cell preparation research

Third, stem cell preparations of preclinical studies

(a) safety evaluation

(2) the effectiveness evaluation

Noun explanation

reference

A preface.

Stem cells is a kind of with different differentiation potential, and in the condition of undifferentiated cell self-renewal. Sources of stem cell therapy refers to the application of autologous or allogeneic stem cells after in vitro operation input (or into) the human body, used for disease treatment process. Such operations include stem cells in vitro separation, purification, amplification, modifications, the establishment of the stem cells (department), differentiation, frozen storage and recovery after frozen storage process. Used for cell therapy of stem cells including adult stem cells, embryonic stem cells and induced pluripotent stem cells (iPSC). Adult stem cells including autograft or allograft, differentiation of fetal or adult different organizations, as well as the development accompanied by organization (e.g., umbilical cord, amniotic membrane, placenta, etc.) between the source of hematopoietic stem cells, mesenchymal stem cells, various types of progenitor cells or progenitor cells, etc.

At home and abroad have carried out a number of stem cells (hematopoietic stem cells) clinical application research, involving a variety of types of stem cells and various types of disease. The main disease types including joint disease, liver cirrhosis, graft rejection (GVHD), spinal cord injury (sci), host and degenerative neurological diseases and diabetes, etc. Many types of stem cells from bone marrow, adipose tissue, umbilical cord blood and umbilical cord or placenta tissue between the source of mesenchymal stem cells, they have certain multi-directional differentiation potential and anti-inflammatory and immune regulation ability, etc.

For stem cells treatment preparation technology and treatment, diversity, complexity and particularity. But as a new biological treatment products, all stem cell preparations can follow a common research and development process, that is, from the preparation of stem cell preparation, in vitro and in vivo animal experiments, to into the process of clinical research and clinical treatment. Every stage of the process, must be for the use of stem cell preparations in terms of cell quality, safety and biological effect research and quality control.

The guiding principle proposed is applicable to all kinds of possible application to clinical stem cells (in addition to the existing provisions of hematopoietic stem cell transplantation) in the preparation and the basic principles of pre-clinical stage. Each specific stem cell preparation preparation and use process, must have a strict standard operating procedures and according to its implementation, to ensure that the stem cells in the preparation of quality control and safety and efficacy of treatment. Each research project specific stem cells involved in the preparation, should be based on the basic requirements of the guiding principles for different stages, combined with their stem cell preparations and the particularity of indications, prepare and implement related preclinical study of stem cells.

2. The quality control of stem cell preparations

(a) stem cell collection, separation and the establishment of the stem cells (department)

1. The demand for stem cell donor

Each stem cell preparations must have, including donor information, clear cell preparation and biological character information. As one of the important contents of the cells in the preparation of information, need to provide access method and way of stem cells as well as relevant clinical data, including donor general information, medical history, family history, etc. Past medical history and family history of hereditary disease (monogenic and polygenic diseases, including cardiovascular disease and cancer, etc.) related information in detail. Clinical research for allogeneic stem cell donor, must pass a test screening proved important specific viruses, including HIV, HBV, HCV, HTLV, EBV, CMV, etc.) of the infection, no treponema pallidum infection. When it is necessary to collect the donor ABO blood group, HLA class I and class II classification data, in order to have traceability queries. Surplus embryos produced by the technique such as using in vitro fertilization (ivf) as the main source of established human embryonic stem cell lines, must be able to trace the donor gametes, and accept the screening and detection. Shall not use previous history of suffering from severe infectious disease and family history of a specific genetic disease as a source of allogeneic stem cell donor.

Source of autologous stem cell donor, according to the characteristics of stem cells in the preparation, the source of tissue or organs, as well as the clinical indications, can be asked about the quality of the donor and screening criteria and project to adjust.

2. Stem cell collection, separation and stem cells (department) establishes the basic stage quality control requirements

Should set collection, stem cells and stem cells (department) to establish the standard operation and management of program, and in accord with "drug production quality management norms" (GMP) requirements on the basis of strictly implemented. Standard operating procedures should include the operating personnel training; Materials, the use and management of instruments and equipment; Stem cell collection, separation, purification and amplification and cells (system) to establish; Cell storage, transportation and relevant safeguard measures, and clean environment standard and regular maintenance and testing, etc.

To minimize the variability of different batch cells in the research process, the researchers in the preparation stage of stem Cell preparations to source rich with a particular generation time of cells establish a multi-stage library, such as the Master Cell Bank (the Master Cell Bank) and Working Cell Bank (Working Cell Bank). Cells in the basic quality requirements, it is need to have a clear cell identification features, no foreign microbial pollution.

In stem cell collection, separation and stem cell stage of establishing a (department), shall be of autologous sources, without the complex operation of stem cells in vitro, activity identification, the survival rate and growth of cells, exogenous pathogenic microorganisms, as well as the basic feature detection of stem cells. For foreign source of stem cells, or after complex in vitro culture and operation of autologous sources of stem cells, as well as direct cell bank for preclinical and clinical research work (such as libraries) in the cell, besides the above tests, also should carry out a comprehensive, detailed internal and external source of pathogenic microorganism stem cell feature detection, and the analysis of the cell purity. Stem cell properties including specific cell surface markers of expression, and product differentiation potential, etc.

(2) the preparation of stem cell preparations

1. Medium

Stem cell preparations used in the preparation of culture medium should have adequate purity and is in line with the sterile, no pathogenic microorganism and endotoxin quality standard, the residue medium of recipients should be no bad influence; In stem cells under the condition of normal growth, does not affect the stem cells of biological activities, namely the "dry" and differentiation of stem cells. In the process of preparing stem cell preparations, should try to avoid the use of antibiotics.

If using a commercial source of culture medium, should choose qualified manufacturers and is provided by its composition of culture medium and related quality certificate. When necessary, to improve the quality of each batch of medium for inspection.

In addition to the special situation, should avoid as far as possible in the process of stem-cell cultures use anthropogenic or animal serum that shall be used for allogeneic serum or plasma. If must use animal serum, should ensure that their specific animal virus contamination. It is forbidden to use sponge encephalopathy popular area source of bovine serum.

If the medium containing human blood components, such as albumin, transferrin and various cytokines, etc., should be clear its origin, batch number, quality testing qualified report, and try to adopt the national has been approved by the clinical application of the product.

2. The trophoblastic cells

Used for in vitro culture and the establishment of embryonic stem cells and iPS cells of anthropogenic trophoblastic cells or animal origin, need to be used according to the exogenous cells in the body of related risk factors, introduced the process of cell source of donor, establish the risk of exogenous pathogenic microorganism and other related inspection and quality control. Proposed the trophoblast cells library, and to conduct a comprehensive inspection, according to the requirements of cell bank inspection especially for important inspection or animal source specific virus.

3. The preparation technology of stem cell preparations

Should make stem cell preparation preparation process of standard operation procedure and standard operating procedures (SOP) for each process and regularly review and revision; Stem cell preparation preparation including collection, separation, purification and amplification of stem cells and extend, the establishment of the stem cells (department), directional differentiation into functional cells, culture medium, the choice of auxiliary materials and packaging standards and use, cells cryopreserved, recovery, packing and marking, and residue removal, etc. From the preparation of the whole preparation process to enter (or) implanted into subjects and need tracking observation and record in detail. The unqualified and the stem cell preparation need to discard, to discard process specification management and record. Preparations for the rest of the stem cells must be legal and ethical requirements of processing, including formulate related SOP and strictly implemented. Stem cells in the preparation of relevant data to document and long-term preservation.

To deal with the whole process of preparation, including cell harvest, batches, operation, packing, etc., to conduct a comprehensive technology research and validation, make the appropriate process parameters and quality standards, ensure the effective control of each process.

(3) stem cells in the preparation of inspection

1. The basic requirement of stem cell preparations quality inspection

In order to ensure the safety and efficacy of stem cell therapy, each batch of stem cell preparation shall be in line with the existing stem cells under the condition of knowledge and technology comprehensive quality requirements. Preparation of test content, must in the guiding principle, on the basis of reference at home and abroad related to matrix and stem cell preparation quality control guidelines, a comprehensive quality, safety and effectiveness of test cells. At the same time, according to the characteristics, in vitro cell source and processing level and different situation, such as clinical indication for the required inspection items to do necessary adjustment. In addition, with the increasing understanding of the stem cells of knowledge and technology, cell inspection content should be updated accordingly.

In view of the different types of stem cell preparations, according to the input or differentiation before implantation in the human body needs, must and terminal differentiation of undifferentiated cells necessary test respectively. For embryonic stem cells and iPS cells preparations used during the preparation of sertoli cell, according to its cell source, also need to according to related risk factors of quality control and inspection.

In order to ensure the quality of preparation and its controllability, stem cells in the preparation of test can be divided into the quality inspection and release. Quality inspection is to ensure the safety of stem cells after in vitro specific treatment, efficacy and quality control of the overall quality inspection. Customs inspection is on the basis of the complete quality inspection, for each type of each batch stem cell preparation, before clinical application should be relatively rapid and simplified cell test.

In order to ensure the stability of the preparation technology and quality, batches on stem cell preparations quality inspection; In the preparation process, site or changes, such as size need to batches of stem cell preparations for quality inspection. Preparation of batch is to point to by the same donor, the source of the same group, at the same time, using the same technology collection and separation or create stem cells. Of embryonic stem cells or iPS cells preparations, should see a differentiation of transplanted cells available for the same batch preparation. To mix by multiple donor stem cell preparations, mix before should regard each independent source of donor or organization in the same time collection for the same batch of cells.

To source from different donors or organizations of the stem cell preparation, the need to mix, to quality inspection, all independent sources of cells as possible to avoid mixed cell preparation may be risk factors.

2. The cell test

2.1 the quality inspection

(1) to identify

Shall be by cell morphology, genetics, metabolic enzymes and the spectrum analysis, surface markers and specific gene expression product detection, such as for different donor and different types of stem cells for comprehensive identification.

(2) the activity of survival and growth

Have different cell biology active detection methods, such as living cells count, cell doubling time, cell cycle, clone formation rate, telomerase activity and cell activity and growth.

(3) the purity and uniformity

By detecting the cell surface markers, genetic polymorphism and specific biological activity, etc., in preparation for cell purity or homogeneity test. For embryonic stem cells and iPS cells implanted in front of the final product differentiation, cell purity must be conducted and/or differentiation of homogeneity test.

For the stem cell preparation need to mix, to the cell surface markers among independent cell source, cell activity, purity and biological activity test uniformity and control.

(4) the sterility test and mycoplasma detection

Should be based on the current edition of the "pharmacopoeia of the People's Republic of China" in the biological products sterile test and mycoplasma detection procedures, against bacteria, fungi, and mycoplasma contamination for testing.

(5) the source of pathogenic factors and outside cells

Should be using the method of in vivo and in vitro, according to the characteristics of each cell preparation of anthropogenic and specific pathogenic factor of the detection of animal origin. Such as using bovine serum, shall be NiuYuan specific virus detection; Such as the use of pancreatic enzymes and other pig source material, should be at least detection of porcine parvovirus. Such as embryonic stem cells and iPS cells of animal origin used in the process of preparation of sertoli cell, cell source needed related to a particular animal virus detection. Also should detect retroviruses.

(6) endotoxin detection

Should be in accordance with the current edition of the "pharmacopoeia of the People's Republic of China" the endotoxin detection procedures, to test the endotoxin.

(7) abnormal immunological reaction

Formulation of detection of foreign sources of stem cells to total lymphocyte proliferation and on the influence of different lymphocyte subsets proliferation, or influence on related cytokine secretion, to detect stem cell preparations may cause the abnormal immune response.

(8) tumorigenicity

Preparation for foreign source of stem cells or by a complex operation of autologous stem cells in vitro preparations, must by immunodeficiency animals tumorigenic test, test cell tumorigenicity.

(9) biological efficacy test

Through detection of stem cell differentiation potential of the differentiation and cell structure and physiological function, the adjustment ability of immune cells, secretion of specific cytokines, expression of specific genes and proteins, and other functions, preparation and treatment related to determine stem cell biology is effective.

Ectomesenchymal stem cells for, whatever the source, should be a variety of cell types in vitro (e.g., into fat cells, cartilage cells, osteoblasts, etc.) the differentiation ability of detection, to determine the differentiation of pluripotent cells (Multipotency). Of undifferentiated embryonic stem cells and iPS cells, by fitting in vitro embryo formation ability, and ability to form teratomas in SCID mouse, detecting the cell differentiation of pluripotent (Pluripotency). In addition, as a specific biological effect test, should be related to its treatment indications of biological effect test.

(10) medium residual amount of testing and other additives

Cope with preparation of residual in the process of preparation, influence the composition of the stem cell preparation quality and safety, such as bovine serum albumin, antibiotics, such as cytokines.

2.2 release test

Project applicants should be the quality inspection of each project according to the specific test content and standard, according to the characteristics of each type of stem cell preparations, formulate release inspection items and standards. Customs inspection items should be in a relatively short period of time, reflect the quality of the cell preparation and safety information.

3. The stem cell preparation quality review

By professional inspection institutions/lab for stem cell preparation quality review, inspection, and issue inspection report.

(4) the quality of the stem cell preparation research

In meet the requirements of the stem cell preparations quality inspection, on the basis of advice in each stage of preclinical and clinical research, using different methods of in vitro experiments on safety, efficacy and stability of the stem cell preparations to conduct a comprehensive study.

1. The quality and characteristics of the stem cell preparations

1.1 growth activity and state

Detection: growth factor dependent on the cultivation growth factor dependent stem cells, the need for continuous detection of cell growth behavior, to determine the different generation of cells to the dependence of the growth factor. If the cells in the process of wedding, especially near the high generation time, lose their dependence on growth factors, it can no longer see it as a qualified stem cells and continue to develop and use.

1.2 tumorigenicity and promote tumor

Ectomesenchymal stem cells as most preparation have relatively weak tumorigenicity, suggest tumorigenicity in animal experiment, for different types of stem cells, choose the necessary number of cells and the necessary long observation period.

Tumorigenicity experiments on animals can effectively judge the tumorigenicity, suggested that detection and tumorigenicity related biological character change, such as cells of growth factor dependency changes, the stability of the genome, are closely associated with tumorigenicity of protein (such as the key regulatory proteins in a cancerous signal path expression level or the change of the activity, sensitivity to apoptosis induced change, etc., to indirectly determine the possibility of a stem cell malignant transformation.

Ectomesenchymal stem cell at present, is generally believed that "from tumor" or a "weak" tumorigenicity, but does not rule out their existing tumor "sex" to promote tumor effect. Therefore, we suggest that according to their respective organization source of mesenchymal stem cells in the preparation and clinical indications of different, design the corresponding test method, to determine the preparation of "sex" to promote tumor.

1.3 biological effects

With the progress of the study, Suggestions for clinical treatment of indications, constantly update research biological effect testing method. As the mediated the clinical effect of the expression of key genes or proteins, and on this basis, put forward alternative biomarkers associated with the desired biological effect (Surrogate Biomarker).

2. Stem cell preparation stability studies and the determination of the period of validity

Should carry on the stem cell preparations in storage (liquid nitrogen frozen storage and temporary storage before the implanted cells and the stability studies in the process of transportation. Test items should include the cell activity, density, purity, aseptic, etc.

According to the result of stem cell preparation stability test, determine the preparations of the liquid ingredients and recipes, preservation and transportation conditions, the period of validity, and determine the corresponding to the period of validity of shipping containers and tools, facilities and qualified cells cryopreserved and conditions.

3. The fast test method of research and development

Based and experimental technology should be based on new stem cell research, aiming at the characteristic of each stem cell preparation, specific clinical indications, research and development of new rapid testing method, used for the quality control of stem cells in different stages of the preparation process and the preparation of customs inspection.

3. Stem cell preparation of preclinical studies

Should stem cell preparations of preclinical studies, provide support for the safety and efficacy of treatment and basis.

In preclinical research plan should be designed and proposed indications related disease animal model, used for predicting the outcome of treatment of stem cells in the human body may, action mechanism, adverse reactions, appropriate input or implanted way and dose of information on clinical study.

It is necessary to study on the basis of appropriate animal models, and the establishment of effective stem cell markers and animal stem cells in the body tracer technique, in order to study the content, especially the stem cells of the body alive, distribution, homing, differentiation and integration functions such as research organization. Based on comprehensive animal model study, the response to the safety of stem cell preparations and reasonable evaluation on the biological effect.

In view of the particularity of stem cell therapy, the effectiveness of preclinical safety evaluation has more difficulty and limitations, here only puts forward some basic principles and concrete research plan according to these principles. Such as to carry on the related research work does not accord with the following principles, should provide the basis and supporting materials.

(a) safety evaluation

1. The toxicity test

You can observe stem cell preparations by appropriate animal test model a variety of possible toxic effects, such as when implanted cells and implanted after the toxicity reaction of local and overall.

As difficult to adopt related animal stem cell toxicity, can consider to simulate clinical application as much as possible, using animal source corresponding stem cell preparations, with higher than that of clinical application of dose to lose animals, observe its toxic effects.

2. The abnormal immune response

For stem cell preparations, especially foreign sources, the in vitro subculture and special processing of preparation, autograft or allograft sources shall be through the abnormal immune response in vitro and animal test evaluation, including the influence of different immune cells subtypes and related factors. For embryonic stem cells and iPS cells, in vitro differentiation to express the donor HLA antigen molecules, after implanted may form of immune rejection, effective evaluation.

3. The tumorigenicity

Complex processing of high generation of time or in vitro and modified autologous sources as well as a variety of sources of stem cells in the preparation, shall conduct pre-clinical stage animal tumorigenicity. Advised to choose a suitable animal model, using the appropriate number of stem cells, the implant way of reasonable sampling period and long enough, with effective evaluation of tumorigenicity.

4. The expected differentiation

Not expected differentiation including differentiation of target cells or target area. Suggest the use of specific detection technology, in vivo animal experiment research, evaluate and monitor the possibility of unexpected differentiated.

(2) the effectiveness evaluation

1. The cell model (see the the quality of the stem cell preparation research)

Animal models of 2.

Used to observe implanted stem cells or their product change pathological process in the model; The research of stem cells homing ability and immune regulating function; After through analysis of stem cells into specific cytokines and/or specific gene expression, put forward an alternative biological effect markers.

If applied research plan, the current international stem cell biology knowledge and technical limitations, cannot put forward effective in vivo animal models in research content, should be in preclinical research report to conduct a comprehensive and detailed instructions.

Noun explanation:

Stem cell preparations (Stem cell - -based medicinal products) : refers to is used to treat disease or improving health, different types of Stem cells as the main component, with the corresponding quality and safety standards, and has the biological effects of clear cell preparation.

Embryonic stem cells, Embryonic stem cell) : from 5 to 7 days of Embryonic cells within the initial undifferentiated cells, can be in in vitro non-dividing state "unlimited self-renewal", and has the potential to all three layer cell differentiation, but not form the extraembryonic tissues, such as the placenta.

Adult stem cells (Somatic stem cell) : in all kinds of undifferentiated stem cells in the differentiation of organization, this kind of stem cells have a limited self-renewal and differentiation potential.

Mesenchymal stem cells (Mesenchymal stem/stromal cells, MSC) : a class exists in a variety of organizations, such as bone marrow, umbilical cord blood and umbilical cord, the placenta tissue and adipose tissue, etc.), with multi-directional differentiation potential, the adult stem cells, hematopoietic stem cells. Ectomesenchymal series of this kind of stem cells to a variety of cells, such as ossification, into cartilage and into fat cells, etc.) or ectomesenchymal series of cell differentiation potential, and has a unique cytokine secretion.

Progenitor cells (Progenitors) : a class can only to a particular series of cell differentiation, proliferation capacity and have only limited division of adult cells.

Precursor cells (Precursors) : a class can only to a particular terminal differentiation of cell differentiation, more limited than progenitor cell proliferation ability of adult cells.

Hematopoietic stem cells (Hematopoietic stem cell) : with high ability to self-renewal and multi-directional differentiation potential of Hematopoietic precursor cells that can differentiate into red blood cells, white blood cells, platelets, and lymphocytes.

Induced pluripotent stem cells (Induced pluripotent stem cell, iPS) : a class by gene transfection cell reprogramming technologies such as artificial induction, are similar to embryonic stem cell pluripotency stem cell differentiation potential.

Embryonic stem cell lines (Embryonic stem cell line) : under the condition of in vitro culture, continuous proliferation of Embryonic stem cells can maintain undifferentiated state.

Totipotency (Totipotent) : it is in the early days of the embryo, have all types of cells to differentiate into the body and form a full capacity for embryonic stem cells.

The totipotency (Pluripotent) : is a form the body cell types, namely the ability to source of all three layer cells, but does not have the ability to form embryos tues tissue cells.

Pluripotency (Multipotent) : refers to a form in the body's ability to more than one type of cell, but are often for a specific series of cells.

Trophoblast cells (Feeder layer) : refers to by cell - cell interactions, or secreted proteins or other substances, located at the bottom of the cultivation of the embryonic stem cells and iPS cells, these stem cells to support the growth of animal origin or anthropogenic sex cells.

Teratoma (Teratoma) : a contains three layer tissue cells and differentiation of benign tumors of the organization.

References:

1. The human body cell therapy research and preparation quality control technical guidelines "(2003).

2. The law of the People's Republic of China pharmacopoeia 2010 edition, the third department.

3. Jun-zhi wang, etc. The biotech drugs research and development and quality control (second edition), science press, 2007.

4. The European Pharmacopeia - Method - Cell 5.2.3 requires substrates for the production of vaccines for human use.

5. The WHO - Recommendations for the evaluation of animal cell cultures as substrates for the manufacture of in medicinal products and for the characterization of cell Banks (2010).

6. The FDA Guidance for Industry - Characterization and Qualification of Cell Substrates and other in Materials, informs the in the Production of Viral Vaccines for Infectious diseases Indications (2010).

7. ICH Guidelines - Viral Safety Evaluation of Biotechnology Products Derived from Cell lines of Human or Animal Origin - Q5A (R1) - 1999.

8. ICH Guidelines - Derivation and Characterization of Cell Substrates, informs the for Production of Biotechnological/in Products - Q5D - 1997.

9. Dominici m., et al. The Minimum criteria for defining multipotent stem cells - The ISCT position statement. 2006; 8 (4) : 315-317 (2006).

10. ISSCR Guidelines for clinical translation of stem cells (2008).

11. The FDA Guidance for human somatic cell therapy and gene therapy (1998).

12. FDAGuidance - the Content and review of the CMC information for human somatic cell therapy IND application (2008).

13. The FDA Guidance - Potency Tests for Cellular and Gene Therapy (2011).

14. The FDA Guidance for Industry - Current Good Tissue Practice ((CGTP) and Additional Requirements for Manufactures of Human Cells, Tissues, and Cellular and Tissue - -based Products (HCT/Ps).

15. EMA Guideline on human cell - -based medicinal products (2007).