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CELL-BASED AND GENE THERAPY MEDICINAL PRODUCTS: 
             RAW MATERIALS OF BIOLOGICAL ORIGIN FOR THEIR PRODUCTION 

GENERAL INFORMATION

        The purpose of this general information is to provide information and guidance on the development of appropriate qualification strategies for raw materials, or ancillary materials (according to the United States Pharmacopeia), used in the manufacture of cell-based/gene therapy (gene transfer) medicinal products. Due to the diverse nature of the raw materials that may require special considerations, the provisions of the chapter are extensive and do not exclude the use of different production and control methods.  The manufacturer of a raw material shall take responsibility in qualifying (proving to be suitable for the intended use) the raw material in accordance with the requirements given herein.  Nevertheless, the user of a raw material plays an ultimate role in ensuring that the material is of suitable quality for the specific use.
The quality of the raw material may be considered according to the stage of development of the cell-based/gene therapy medicinal product, thereby acknowledging the inherent evolution of the quality profile of the product during its pharmaceutical and clinical development. Typically, the extent of control increases as clinical development progresses.  The patient’s safety needs to be ensured in early phases of clinical development. When assessing the impact of the raw material on the quality, safety and efficacy of the cell-based/gene therapy medicinal product, a risk-based approach is employed. To minimize risks, the use of raw materials free from human or animal substances is preferred.
Due to its biological nature, the quality of a raw material used for manufacturing a cell-based/gene therapy medicinal product may impact the safety, potency, and purity of the final product. Thus, the quality requirements for this type of material are to be established.  Examples of the critical quality attributes specific to each type of raw material are addressed in this general information.
It should be noted that changes in raw materials during the life cycle of the cell-based/gene therapy medicinal product may affect the quality of the medicinal product and thus require additional studies to demonstrate comparability.

INTRODUCTION

In addition to chemical medicinal products, rapid development in biology, biotechnology, and medical sciences has brought to light an Advanced Therapy Medicinal Product (ATMP): a medicinal product that is gene-, tissue-, or cell-based. This new type of medicinal product shows a high potential in the treatment of various diseases, especially those rare or serious ones where there is a previously unmet medical need.
Cells used in cell-based medicinal products may be self-renewing stem cells, more committed progenitor cells, or terminally differentiated cells exerting a specific defined physiological function. They may be autologous or of allogeneic origin.
Gene therapy or gene transfer medicinal products for human use contain genes that lead to a therapeutic, prophylactic or diagnostic effect by inserting 'recombinant' genes into the body to modify or manipulate the expression of the body’s genes or to alter the biological properties of living cells. Similar to cell-based medicinal products, this type of product can treat a wide range of diseases, including genetic disorders, cancer, or long-term diseases.
Since these products are not usually amenable to extensive purification, filtration, and terminal sterilization, procedures, reagents, and materials qualification are critically important to ensuring the products’ quality.
The term “raw materials” refers to materials that come into contact with the cellular starting material, product intermediate, or final product manufacturing, but are not intended to be present in the final product.

Scope

This chapter applies to raw materials of biological origin used for the production of cell-based/gene therapy medicinal products. The raw materials used in the manufacture of active substances are not intended to form part of the active substance. The raw materials can be extracted from various biological sources or produced by recombinant DNA technology.

Classification of raw materials cell-based/gene therapy medicinal products  Raw materials cell-based/gene therapy medicinal products to be included in this Pharmacopoeia are classified as follows:
—  Sera and serum replacements;
—  Proteins produced by recombinant DNA technology such as growth factors, cytokines,

hormones, enzymes, and monoclonal antibodies;
—  Proteins extracted from biological material such as enzymes and polyclonal antibodies;
—  Vectors.
The principles of this chapter may also be applied to other classes of biological rawmaterials where appropriate.
Medical devices, plastics and chemically synthesized raw materials, such as basal media (purely composed of chemicals), synthetic peptides, or synthetic polynucleotides, are not within the scope of this chapter.

Risk Assessment

Evaluation of the impact of the raw material on the quality, safety, and efficacy of cell-based/gene therapy medicinal products must be performed by the user of the raw material. No single measure or combination of measures can guarantee the quality, functionality, and safety of a raw material for its intended use.  Therefore, a risk assessment must consider the biological origin and traceability of the raw material, the production steps applied to it, and the ability of the drug product manufacturing process to control or remove the raw material from the final medicinal product.
Any risk factor must be evaluated in relation to the clinical benefit/risk of the cell-based or gene therapy medicinal product. The evaluation of risk should be based on scientific knowledge, with the ultimate goal of patient protection. When evaluating the risk posed by the raw material to the final medicinal product, the exposure of a patient to residual amounts of raw material with potential harmful effects (e.g., adverse immune reactions) should be considered in relation to the clinical benefit/risk of the cell-based or gene therapy medicinal product.

General Requirements

Origin

The origin of the raw material and if relevant any biological substances used for the production of the raw material must be known. Special attention must be paid to risks related to the sourcing (including pooling) of the substances used for the production of the raw material. Depending on the source of the raw material and the substances used in its production, raw materials can be divided into three categories:
1)  raw materials of human or animal origin;
2)  raw materials produced using substances of human or animal origin;
3)  raw materials free from substances of human or animal origin.
Traceability of all raw materials is required, with particular attention to those materials with an inherent safety concern, i.e., those of human or animal origin.
Due to the inherent risk of transmitting adventitious agents, it is recommended to minimize, wherever possible, the use of raw materials of human or animal origin. If such raw materials are required for the production of cell-based/gene therapy medicinal products, appropriate measures are taken to minimize the risks of transmitting adventitious agents such as viruses, prions, bacteria, and protozoa.
For human blood and tissue-derived materials, only carefully evaluated donors who have been adequately tested for infectious measures enable each donation to be followed from the donation to the raw material and to the final product, and vice-versa.
When raw materials of animal origin are used, these animals fulfil specific health requirements and should be fit for human consumption and reared under controlled conditions, when applicable. If the origin of the animals is not fully traceable (e.g., animals collected from the wild), information on their geographic location at the time of sourcing should be considered.
When vectors or proteins produced by recombinant DNA technology are used as raw materials, traceability to the master cell bank/virus seed lot is required.
For all raw materials of human or animal origin, or raw materials produced using substances of human or animal origin, a viral risk assessment is performed according to the requirements of “Viral Safety”.  The extent of viral safety testing is dependent on the results of the initial risk assessment. In addition, a risk assessment with respect to transmissible spongiform encephalopathies is carried out and suitable measures are taken to minimize such risks as described in “Minimizing the Risk of Transmitting Animal Spongiform Encephalopathy Agents via Human and Veterinary Medicinal Products”.

Production

All raw materials are produced within a suitable quality management system and production facilities.
Suitable in-process controls are in place to ensure that the production process is under control and consistently produces raw materials of defined quality.
Quality attributes for raw materials include identity, purity and biological activity where applicable, and they are to be demonstrated using appropriate, qualified control methods. Relevant specifications in terms of identity, purity/impurity profile and assays are to be established.
The production process is optimized to consistently minimize and/or remove adventitious agents and harmful impurities, whilst retaining the quality of the raw material. This can be achieved using one or a combination of the following measures:
—  using validated inactivation/removal procedures such as gamma sterilization or low pH during chromatography, where possible;
—  demonstrating the ability of a production process to minimize, remove, or inactivate adventitious agents or harmful impurities;
— testing for adventitious agents or harmful impurities.
A raw material is sterile and produced under aseptic conditions and/or subject to terminal sterilization, unless otherwise justified.  If the raw material is not sterile, the level of microbial contamination must be known.
Additives, such as stabilizers, may be added to the raw material.  In cases where antibiotics and stabilizers of biological origin are used in the production of the raw material, their presence is justified and careful consideration is given to their selection, use, quality, and concentration in the raw material, as well as their impact on the actual raw material itself.

General Quality Requirements

Raw materials must meet pre-defined quality requirements for identity, purity, and biological activity.  In order to ensure the function of the raw material, it is subject to testing using appropriately qualified methods. The identity test must reflect the uniqueness of the raw material and distinguish it from other related or similar substances. Impurities include both process-related substances (e.g., in the case of recombinant proteins:  host-cell-derived proteins (HCP), host-cell-derived DNA and vector-derived DNA (residual DNA), other biological or chemical substances), and product-related substances (e.g., aggregates and degradation products).  The content of a raw material may be expressed either in absolute or relative terms.  The assay for determination of biological activity may be used to establish the content.

Identification

The identity tests are specific for the particular raw material and address the molecular structure/composition or other relevant physico-chemical, biological or immunochemical properties. Methods used in the determination of biological activity and purity may also serve to identify the raw material. Identification may be carried out by comparison with a defined reference material or a  representative batch of the raw material.

General Testing Methods

Tests that may be applicable to raw materials include the followings (see also the sections below that are for specific raw materials, i.e., Sera and serum replacements, Vectors):
     Appearance Liquid or reconstituted freeze-dried raw materials comply with the limits defined for the particular raw material with regard to “Clarity of Solution” (Appendix 4.1) and “Colour of Solution” (Appendix 4.2).
     Solubility  Freeze-dried raw materials dissolve completely in the prescribed volume of reconstituting liquid within a specified time, at a specified temperature, as defined for the particular raw material.
     Osmolality  Within the limits defined for the particular raw material (Appendix 4.35).
     pH  Within the limits defined for the particular raw material (Appendix 4.11).
     Elemental impurities  Within the limits defined for the particular raw material.
     Total protein  Within the limits defined for the particular raw material.
     Related substances  The content of product-related substances is within the limits defined for the particular raw material.
     Microbiological control  Depending on the raw material concerned, it complies with the “Sterility Test” (Appendix 10.1) or the “Limits for Microbial Contamination” (Appendix 10.5).
     Viral contaminants  Depending on the raw material concerned, relevant virus contamination is determined.
     Bacterial endotoxins  Less than the limit defined for the particular raw material (Appendix 8.5).
     Mycoplasmas  Raw materials are free from mycoplasmas.
     Stabilizer  Where applicable, it complies with the limits defined for the particular raw material.

     Water  Freeze-dried raw materials comply with the limits defined for the particular raw material (Appendix 4.12).
     ASSAY
     Content  The content (e.g., protein content)/composition of the raw material is determined by an appropriate qualified method.
     Biological activity  Where relevant, the biological activity is determined by a suitable assay. Where relevant (e.g., for enzymes), the biological activity is expressed per milligram of total protein (specific activity).

REFERENCE MATERIAL OR REFERENCE BATCH

An appropriate reference material or a representative batch of the raw material is used to perform the above-mentioned identification, tests, and assay.  Where available, the use of established reference substances or reference standards, such as European Pharmacopoeia reference standards or WHO International Standards, is recommended.

Storage

The shelf life and storage conditions are defined.

Labelling

The label states the expiration date, conditions for storage and use, and any code that may be required for traceability including the biological origin of the raw material.
Statements required to appear on the “label on the container” may appear on a label fastened to the immediate container, or in indelible writing in or on the body of the container itself.  They should also be demonstrated on the label on the package.

SERA AND SERUM REPLACEMENT

DEFINITION
     Sera from human or animal sources and serum replacements (including platelet lysates and other undefined growth additives, conditioned media, blood and other cellular components) are used as growth additives for cell culture. Sera and serum replacements used to promote cellular growth are typically complex biological mixtures, whose exact composition is not always possible to define. Due to this complex nature, special attention is given to verifying the consistency and performance of every batch.
     Bovine serum If bovine serum is used, use bovine serum.
     Human serum and platelet lysates  Human serum and platelet lysates used as raw materials for the production of cell-based/gene therapy medicinal products are human blood-derived materials, which can originate from the recipient (autologous) or from another individual (allogeneic).
     Conditioned media  Conditioned media, isolated and purified from cultured cell supernatant, may also be used to enhance cell proliferation due to various growth factors and cytokines secreted by the cells into the medium.
     Other growth additives with undefined composition  Cell and/or tissue lysates may be used as growth additives.
     Composite media  Composite media contain growth additives such as bovine serum, growth factors, etc.  The principles described in this section of the appendix apply to individual ingredients of biological origin and/or biologically active ingredients of the composite media.

PRODUCTION
Due to potential differences in quality between batches of serum, cell, or tissue lysate, suitable measures are implemented to verify the consistency of each batch before using them as raw materials for the production of cell-based/gene therapy medicinal products.
Because of the inherent risk of transmitting infectious agents from pooled plasma, pooled sera, or other derivatives from pooled allogeneic human blood or plasma, consideration is given to limit the number of donations which are pooled, unless sufficient methods for inactivation/removal of viruses are applied during production, where applicable.
For conditioned media, a cell bank system is preferred. The removal of the cells from the media must be ensured and potential impurities originating from these cells determined if possible.

IDENTIFICATION

It is recognized that the exact qualitative composition of sera and serum replacements may be difficult to determine. However, the approximate protein composition in both cases may be determined by, for example, protein electrophoresis. Where relevant, tests for total protein content or any chemical additives are performed. For human serum, the electrophoretic pattern corresponds to that of an appropriate serum reference batch. Alternatively, identity may be determined by comparison of albumin content with an appropriate serum reference batch. For serum replacements, the electrophoretic pattern or the use of markers secreted by cells/platelets may be used. Human origin is determined by a suitable “Immunochemical Methods” (Appendix 14.5), unless otherwise justified.

TESTS

See under General Testing Methods.
     Haemoglobin  Where relevant, within the limits defined for the particular raw material.
     Cell-derived impurities Where relevant, within the limits defined for the particular raw material.
     Specific tests for viral contaminants  For bovine serum, the tests for viral contaminants specified in bovine serum apply.  For human serum, the tests for viral safety specified in the Plasma for Fractionation apply.

ASSAY

The serum or serum replacement must show cell growth promoting properties that are within the limits defined for the particular raw material. More than one type of assay may be necessary to show suitability for the intended use.

PROTEINS PRODUCED BY RECOMBINANT DNA TECHNOLOGY

DEFINITION

Proteins and peptides produced by recombinant DNA technology, which are used as raw materials, include growth factors, cytokines, hormones, enzymes, and monoclonal antibodies.
Growth factors, cytokines and hormones  They are substances typically used for stimulation or inactivation, growth promotion, or differentiation of cells in cell culture systems.
Other proteins  Enzymes (e.g., collagenases), as raw materials, may be used for extraction of active substances from tissues and/or fluids. Other proteins (e.g., fibronectin) may be used as culture supports or media components.
Monoclonal antibodies  Used as raw materials, they include immunoglobulins and fragments of an immunoglobulin with defined specificity. Antibodies can either be conjugated (chemically modified) or non-conjugated. Typical chemical modifications include fluorescent labelling and conjugation to magnetic beads. Antibodies, as raw materials, may be used for selection, activation/stimulation, isolation or purification of cells in cell culture.

PRODUCTION

Production of proteins using recombinant DNA technology is based on a well-characterized host-vector system, using a master cell bank and, if applicable, a working cell bank derived from the master cell bank. The expressed protein is extracted and purified using a variety of techniques, such as extraction, precipitation, centrifugation, concentration, filtration and/or chromatography.
During protein production using recombinant DNA technology, process-related impurities including residual host-cell or vector DNA and host-cell proteins must be reduced to acceptable levels. Particular attention must also be given to product-related impurities.

IDENTIFICATION
Identity is established by appropriate, qualified methods, such as “Electrophoresis” (Appendix 3.7), “Peptide Mapping”, “Isoelectric Focusing” or “Liquid Chromatography” (Appendix 3.5). For antibodies, identification is based on immunoglobulin class, isotype and/or specificity. In addition to the above-mentioned methods, “Immunochemical methods” (Appendix 14.5) and determination of activity are also considered suitable for identification.

TESTS

See under General Testing Methods.
Host-cell-derived proteins and residual host-cell or vector DNA Where relevant for the particular raw material, the content of residual host-cell or vector DNA and/or protein is determined using a suitable method unless the production process has been qualified to demonstrate suitable clearance. The content is within the limits defined for the particular raw material.
Related proteins Related proteins (e.g., polyclonal antibodies with undefined specificities, glycoforms, degradation and oxidation products, oligomers, and aggregates) are determined using liquid chromatography, electrophoretic, or immunological methods and are within the limits defined for the particular raw material.

ASSAY

Content  The protein content is determined by an appropriate qualified method, for example by “Liquid Chromatography” (Appendix 3.5) or “Ultraviolet and Visible Spectrophotometry” (Appendix 2.2).
Biological activity  The biological activity of a recombinant protein is determined using, for example, cell proliferation, cell differentiation, or an enzyme assay. Several acceptable bioassays may exist for a particular protein.  For antibodies, cell-based immunoassays and assays based on ligand-binding and affinity may be used.
Where relevant, the biological activity is expressed per milligram of total protein (specific activity).

PROTEINS EXTRACTED FROM BIOLOGICAL MATERIAL

DEFINITION

Proteins extracted from biological material and used as raw materials include enzymes (e.g., porcine-derived trypsin and endonucleases), polyclonal antibodies, other proteins of biological origin (e.g., albumin and transferrin), and peptides of biological origin. They may be of human, animal, plant, or microbiological origin.
Proteins extracted from biological material are used in a wide range of applications such as growth promotion, differentiation or purification of cultured cells and extraction of active substances from tissues and/or fluids.

PRODUCTION

Proteins are extracted from the blood or tissue of animals or humans, or from plant or microbiological sources using mechanical and/or chemical techniques. They are then subjected to further purification processes using a variety of techniques such as centrifugation, filtration, chromatography, and concentration.
Polyclonal antibodies are produced by immunization with a specific antigen, followed by purification. Antibody purification involves selective enrichment or specific isolation of antibodies from serum based on physico-chemical fractionation, class-specific affinity, and/or antigen-specific affinity.
During production of these proteins, process-related impurities, such as blood components, tissue fragments, or contaminating proteins, must be reduced to acceptable levels. Particular attention is given to product-related impurities.

IDENTIFICATION

Identity is established by appropriate, qualified methods, such as “Electrophoresis” (Appendix 3.7), “Isoelectric Focusing”, “Peptide Mapping”, or “Liquid Chromatography” (Appendix 3.5) and “Immunochemical methods” (Appendix 14.5). 

TESTS

See under General Testing Methods.
Process-related impurities  Substances derived from the starting material (e.g., blood components, tissue fragments, or contaminating proteins) are determined using suitable methods and are within the limits defined for the particular raw material.
Related proteins  Related proteins (e.g., antibodies with undefined specificity, degradation and oxidation products, oligomers, and aggregates) are determined using suitable methods and are within the limits defined for the particular raw material.

ASSAY

Content  The protein content is determined using an appropriate qualified method, for example by Liquid Chromatography (Appendix 3.1), or UV spectrophotometry (Appendix 2.2).
Biological activity  Where relevant, the biological activity of a protein is determined using, for example, enzyme assays, immunoassays, or assays based on cell proliferation/differentiation. For trypsin, the assay may be performed as described in the Trypsin. Where relevant, the biological activity is expressed per milligram of total protein (specific activity).

VECTORS

Vectors that may be used as raw materials in the production of cell-based and gene therapy medicinal products include DNA vectors (e.g., plasmids and transposon vectors) as well as viral vectors and bacteria (e.g., modified Lactococcus species).  Vectors are usually considered as starting materials, thus not under the scope of this appendix.  In cases where vectors are not considered as starting materials, such as vectors used as helper plasmids or helper viruses, the principles of this appendix and the principles of production and quality control as outlined in chapter “Gene Transfer Medicinal Products for Human Use” are to be followed.