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I. II. A. B. C. III. A. ? B. C. D. E. IV. A. B. C. V.

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- (cellular and gene therapy, CGT) 2) 3) . FDA Application,5)

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II A. ? - , , (42 U.S.C. 262, , , (Federal Food, Drug and Cosmetic Act, FDC Act(21 U.S.C. 321 et seq.); 21CFR 601.2). / (product conformance testing, 21 CFR 601.20(a)) (21CFR 601.20(c)) (21 CFR Part 600 et seq.) (21 CFR Parts 210, 2117)) FDA cGMP ( , Current Good Manufacturing Practice) .

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, , . . - (potency) (21 CFR 600.3(s)). (strength) , (21 CFR 210.3(b)(16)(ii)). 600.3(S) (21 cfr 610.10) . - FDA . . cGMP . ( /) (21 CFR 600.3(s), 610.10 21 CFR 210.3(b)(16)(ii)) (21 CFR 610.1; 21 CFR 211.165(a)) (21 CFR 211.194; 21 CFR 600.3(kk) ; 21 CFR 211.165(d); 211. 165(e)) (acceptance) / (rejection) (reference), , / (control) (21 CFR 210.3(b)(16)(ii), 211.160) (validation) , , (21 CFR 211.165(e) 211.194(a)(2)) (active ingredients) () (21 (21 CFR 211.165(a): 21 CFR 210.3(b)(7) ) (dating 610.53(a) ) (21 CFR 610.61(g)(3) 610.61(r)) B. ? - (. 3,4,8). period) CFR 600.3(1)8)

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D. - , , () . , , / , . , . - . , (multiple assay) , E. ? 1. - , 1 2 . . , (21 CFR 312.23(a)(7) ). 2. - (: 3, 13)) . . . . - (expiry dating) (21 CFR 610.53; Ref. 7 ). 3. -

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4. - , / . , (21 CFR 211.165(e)). (21 CFR 601.12(b)(3)(vi)). - (factor) . ( Section ILA ) - () ( ) - (: , , ) . . - , (. 14 ), () . , .

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2. , (Draft, 2008, FDA) _ Potency Tests for Cellular and Gene Therapy Products (Draft Guidance)

Table of ContentsI. INTRODUCTION ................................................................................................................. 1 II. BACKGROUND .................................................................................................................. 2 A. What Are the Regulatory Requirements for Potency of Licensed Biological Products? .2 B. What are the Potency Requirements for Investigational CGT Products? ................. 3 C. What Is the Relationship Between Potency and Clinical Effectiveness for CGT Products? ... 4 III. RECOMMENDATIONS FOR POTENCY MEASUREMENTS ................................... 5 A. How to Determine What to Measure for Potency? ................................................... 5 B. What Methods May be Used to Measure Potency? ................................................... 6 1. Biological assays ........................................................................................................ 6 2. Non-biological analytical assays ............................................................................... 6 3. Multiple assays (assay matrix) ................................................................................. 7 C. What is Necessary to Correlate an Analytical Assay with Biological Activity? .... 7 D. When Should Potency Assay Development Initiate? .................................................. 8 E. What is Progressive Potency Assay Implementation? ................................................. 8 1. Early product development: ...................................................................................... 8 2. Later phase product development: ........................................................................... 9 3. Biological License ...................................................................................................... 9 IV. ASSAY DESIGN AND VALIDATION ......................................................................... 9 A. What Should be Considered During Assay Design? .................................................. 9 B. How Should Reference Materials and Controls be Utilized? .................................. 10 C. What Should be Considered for an Assay Validation Plan? .................................. 11 1. Regulations ................................................................................................................ 11 2. Statistical design and analysis ................................................................................ 11 3. Validation of qualitative assays .............................................................................. 12 4. Assay evaluation and modification ........................................................................ 12 V. REFERENCES ................................................................................................................... 14

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Guidance for Industry Potency Tests for Cellular and Gene Therapy ProductsThis draft guidance, when finalized, will represent the Food and Drug Administrations (FDAs) current thinking on this topic. It does not create or confer any rights for or on any person and does not operate to bind FDA or the public. You can use an alternative approach if the approach satisfies the requirements of the applicable statutes and regulations. If you want to discuss an alternative approach, contact the appropriate FDA staff. If you cannot identify the appropriate FDA staff, call the appropriate number listed on the title page of this guidance.

I. INTRODUCTION We, FDA, are issuing this guidance to provide you, manufacturers of cellular and gene therapy (CGT) products, with recommendations for developing tests1 to measure potency.2 These recommendations are intended to clarify the potency information that could support an Investigational New Drug Application3 (IND) or a Biologics License Application4 (BLA). Because potency measurements are designed specifically for a particular product, this guidance does not make recommendations regarding specific types of potency assays, nor does it propose criteria for product release. This guidance is intended to supplement related documents (Refs. 1 through 11, and 15) and does not replace or supersede any existing documents. This guidance applies only to CGT products5 reviewed by FDAs Office of Cellular, Tissue and Gene Therapies (OCTGT), CBER under Section 351 of the Public Health Service Act (PHS Act) (42 U.S.C. 262) (Refs. 1 and 2). This guidance does not apply to human cells, tissues, and cellular and tissue products (HCT/Ps), which are regulated solely under section 361 of the PHS Act (42 U.S.C. 264) as described under 21 CFR 1271.10 or to products regulated as medical devices under 21 CFR Part 820. This guidance also does not apply to biological products reviewed by CDER or by CBERs Office of Vaccine Research and Review (OVRR) or Office of Blood Research and Review (OBRR). FDAs guidance documents, including this guidance, do not establish legally enforceable responsibilities. Instead, guidances describe the FDAs current thinking on a topic and

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should be viewed only as recommendations, unless specific regulatory or statutory requirements are cited. The use of the word should in FDAs guidances means that something is suggested or recommended, but not required.

BACKGROUND What Are the Regulatory Requirements for Potency of Licensed Biological Products? All biological products must meet prescribed requirements of safety, purity and potency for BLA approval (42 U.S.C. 262, Federal Food, Drug and Cosmetic Act, (FDC Act) (21 U.S.C. 321 et seq.); 21 CFR 601.2). For CGTs, product conformance testing (21 CFR 601.20(a)) and control of the manufacturing process (21 CFR 601.20(c)) are required to comply with FDAs Current Good Manufacturing Practice (CGMP) For Finished Pharmaceuticals regulations (21 CFR Parts 210 and 2116) as well as the biologics regulations (21 CFR Part 600 et seq.). No lot of any licensed product may be released by the manufacturer prior to the completion of tests for conformity with standards applicable to such product, (21 CFR 610.1), which include tests for potency, sterility, purity, and identity (21 CFR Part 610, Subpart B). These requirements apply to all biological products, including autologous and single patient allogeneic products, where a lot may be defined as a single dose. Potency is defined as the specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the product in the manner intended, to effect a given result. (21 CFR 600.3(s)). Strength7 is defined as potency, that is, the therapeutic activity of the drug product as indicated by appropriate laboratory tests or by adequately developed and controlled clinical data. . . . (21 CFR 210.3(b)(16)(ii)). Regulations stipulate that [t]ests for potency shall consist of either in vitro or in vivo tests, or both, which have been specifically designed for each product so as to indicate its potency in a manner adequate to satisfy the interpretation of potency given by definition in 600.3(s) of this chapter. (21 CFR 610.10). FDA regulations allow for considerable flexibility in determining the appropriate measurement(s) of potency for each product. Potency is determined based on individual product characteristics; therefore, the adequacy of potency assays is evaluated on a

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case-by-case basis. All potency assays used for release testing of licensed biological drug products must comply with applicable biologics and CGMP regulations including: Indicate potency (biological activity/activities) specific to the product (21 CFR 600.3(s) and 610.10; and 21 CFR 210.3(b)(16)(ii)); Provide test results for release of the product (21 CFR 610.1; 21 CFR 211.165(a)) Provide quantitative data (21 CFR 211.194; see also 21 CFR 600.3(kk); 21 CFR 211.165(d); 211.165(e);); Meet pre-defined acceptance and/or rejection criteria (21 CFR 211.165(d); see also 21 CFR 600.3(kk); and 21 CFR 210.3(b)(20)); Include appropriate reference materials, standards, and/or controls (see; 21 CFR 210.3(b)(16)(ii) and 211.160); Establish and document the accuracy, sensitivity, specificity and reproducibility of the test methods employed through validation (21 CFR 211.165(e) and 211.194(a)(2)); Measure identity and strength (activity) of all active ingredients (21 CFR 211.165(a); see also 21 CFR 210.3(b)(7)); Provide data to establish dating periods (see 21 CFR 600.3(l) and 610.53(a)) Meet labeling requirements (21 CFR 610.61(g)(3) and 610.61(r)) What are the Potency Requirements for Investigational CGT Products? In early clinical phase investigations, it may not be possible to meet all of the requirements described above for licensed biological products (Refs. 3, 4, 8).

Nonetheless, you must submit data to assure the identity, quality, purity and strength (21 CFR 312.23(a)(7)(i)) as well as stability (21 CFR 312.23(a)(7)(ii)) of products used during all phases of clinical study. [T]he amount of information needed to make that assurance will vary with the phase of the investigation, the proposed duration of the investigation, the dosage form, and the amount of information otherwise available. (21 CFR 312.23(a)(7)(i)). Potency measurements are necessary for product characterization testing,8 comparability studies (Ref. 6), and stability protocols (Ref. 7), which are used to establish that a consistently manufactured product is administered during all phases of clinical

investigation. However, the complexity of CGT products can present significant challenge(s) to establishing potency assays (see Table 1). To facilitate the development

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of CGT products, we recommend an incremental approach to product characterization testing, including the development of potency assays. General recommendations for progressive potency assay implementation are outlined in Section III.E. As described in Sections III.A, III.E, and IV.C.4 of this document, your potency measurement will evolve and may change significantly as you develop your product. Table 1:

C. What Is the Relationship Between Potency and Clinical Effectiveness for CGT Products? There is no single test that can measure adequately those product attributes that predict clinical efficacy. Clinical effectiveness is demonstrated by adequate and well-controlled clinical investigations conducted with a consistently manufactured quality product. Clinical effectiveness may be correlated to product potency, but clinical study data is not a practicable quantitative measure of potency to release a lot. Rather, clinical study results may be used to establish a correlation(s)9

between the products clinical efficacy

and a potency measurement(s), which can be used for lot release, stability, and/or comparability studies (see Section III.C for more details related to correlation studies). III. RECOMMENDATIONS FOR POTENCY MEASUREMENTS

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A. How to Determine What to Measure for Potency? Because of the complexity of CGT products, you need to acquire an appropriate understanding of the biological properties of your product in order to develop relevant and meaningful potency measurements. You should collect sufficient data throughout preclinical and clinical development to inform and refine your approach to measuring potency. When initially determining the biological activity or activities that will guide your potency assay design, you should consider relevant pre-clinical investigations, proof of concept studies, early clinical studies, available historical experience, and available reference materials and controls (see Section III.C). This information may provide you with a basic understanding about product characteristics and biological activities that contribute to function. Characterization data obtained during product development may provide support for the potency assay that you choose initially, or it may lead to an improved potency measurement as you prepare to market your product (see Sections III.E and IV.C.4). As you develop your product(s), you should measure a wide range of product properties in addition to those performed for routine lot release. This may help you to assess which product attribute(s) best correlate(s) with potency. Although some of the assays you evaluate may not be practical for lot release (e.g., difficult to consistently obtain quantitative results, time-consuming), most properly designed assays (see Section IV.A) have the potential to provide valuable information about product attributes related to biological activity or clinical effectiveness, or both. CGT products may present challenges for developing assays to measure specific biological attributes that quantitatively demonstrate potency (see Table 1). CGT products often have complex and/or poorly defined mechanism(s) of action (i.e., relevant therapeutic or clinical functional activity), making it difficult to determine which product attribute is most relevant to measuring potency. Nonetheless, potency measurements should reflect the relevant biological attributes. For example, a gene therapy vector should rely on at least two biological activities for its potency: the ability to transfer a genetic sequence to a cell and the biological effect of the expressed genetic sequence. Therefore, the potency assay should incorporate both a measure of the gene transfer frequency and the biological effect of the transferred gene. In addition, the proposed mechanism(s) of action for CGT products may be dependent on more than one active ingredient10 (e.g., multiple cell types, multiple vectors, multi-epitope vaccines). For some complex products (e.g., cellular tumor vaccine) there

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could be ambiguity about which ingredients contribute to potency. For products that contain more than one known active ingredient, you should design potency

measurement(s) to determine the biological activity (strength) of all active ingredients (see 21 CFR 211.165(a)). Thus, if your product contains more than one active ingredient you might need more than one assay to measure potency of the product because one assay might be insufficient to measure the activity of each of the active ingredients (Section III.B.3). Additionally, when designing your assay(s), you should also consider the potential for interference or synergy between active ingredients. B. What Methods May be Used to Measure Potency? 1. Biological assays The traditional approach for assessing the potency of biological products is to develop a quantitative biological assay (bioassay) that measures the activity of the product related to its specific ability to effect a given result, and that also meets the criteria listed in Section II.A. Bioassays measure potency by evaluating a products active ingredients within a living biological system. Bioassays can include in vivo animal studies, in vitro organ, tissue or cell culture systems, or any combination of these. You may use in vitro or in vivo assays; however, we encourage the responsible limitation of animal use whenever possible (Ref. 12). 2. Non-biological analytical assays11 Development of a quantitative bioassay for some CGT products may be complicated by properties of the product and/or technical limitations (see Table 1). In cases where bioassay development is not feasible, it may be necessary to identify a surrogate of biological activity. For example, you may need to use an analytical assay(s) that is practical and reliable for lot release. Analytical assays can provide extensive product characterization data by evaluating immunochemical, biochemical, and/or molecular attributes of the product. These attributes may be used to demonstrate potency if the surrogate measurement(s) can be substantiated by correlation to a relevant

product-specific biological activity(s) (see Section III.C, Refs. 13 and 14). To establish meaningful correlations, you should conduct rigorous product characterization testing, as recommended throughout this document. 3. Multiple assays (assay matrix) In many cases, a single biological or analytical assay may not provide an adequate measure of potency. The following are some potential reasons:

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Product has complex mechanism of action Product has multiple active ingredients and/or multiple biological activities Limited product stability Biological assay is not quantitative, not sufficiently robust, or lacks precision If one assay is not sufficient to measure the product attribute(s) that indicates potency, then an alternative approach could be used to develop multiple complementary assays that measure different product characteristics associated with quality, consistency and stability. When used together and when results are correlated with a relevant biological activity, these complementary assays should provide an adequate measure of potency. Such a collection of assays (referred to as an assay matrix) might consist of a combination of biological assays, biological and analytical assays, or analytical assays alone (Refs. 13 and 14). The assay matrix may include assays that give a quantitative readout (e.g., units of activity) or qualitative readout (e.g., pass/fail). If qualitative assays are used as part of an assay matrix to determine potency for lot release, stability or comparability studies, they should be accompanied by one or more quantitative assays (see SectionC. What is Necessary to Correlate an Analytical Assay with Biological Activity? To demonstrate potency using an analytical assay as a surrogate measurement of biological activity, you should provide sufficient data to establish a correlation between the surrogate measurement(s) and the biological activity(ies) that is related to potency. The relationship between the surrogate measurement and biological activity may be established using various approaches, including comparison to preclinical/proof of concept data, in vivo animal or clinical data, or in vitro cellular or biochemical data. If you choose to use an analytical assay as a surrogate measurement of biological activity to meet the potency requirements for licensed biological products, you should meet criteria listed above in Section II.A. This could necessitate that you stress the product (i.e., show that the assay can detect an inactive or degraded product) and perform sufficiently controlled studies (see Section IV.). The suitability of data used to support surrogate assays for biological activity is evaluated on a case-by-case basis and depends on or is influenced by the following: Type and relevance of the correlation(s) being made;

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The amount of product information you have accumulated; How well the biological activity of the product is understood; and How well the surrogate measurement(s) reflects biological activity. If you intend to demonstrate potency by correlating a surrogate assay(s) to a relevant biological activity, you should start collecting product and assay characterization data during early investigational phases. D. When Should Potency Assay Development Initiate? As discussed throughout this document, thorough product characterization is necessary to understand the product parameter(s) that affect quality, consistency, and stability. Moreover, understanding and controlling these parameters will be necessary to

demonstrate consistency between production lots, to assess comparability of different manufacturing processes and/or various assays, and may also be necessary to allow you to determine which product attributes are related to an effective product. Thus, because the ability to measure potency is essential to product characterization, you should initiate potency assay development during preclinical and early clinical investigations to obtain as much product information as possible. In addition, measuring potency during early product development has a number of advantages, such as allowing you to: Demonstrate product activity, quality and consistency throughout product development; Generate a collection of data to support specifications for lot release; Provide a basis for assessing manufacturing changes; Evaluate product stability; Recognize technical problems or reasons a different assay might be preferable; Evaluate multiple assays; and Collect sufficient data to support correlation studies, if necessary. E. What is Progressive Potency Assay Implementation 1. Early product development: For some products in pre-clinical, Phase 1 and early Phase 2 studies, limited quantitative information on bioactivity may be sufficient. Potency assays performed on product lots used for early clinical studies are likely to have wider acceptance ranges

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than assays used in later phase investigations. Nevertheless, as clinical studies progress and product knowledge increases, you should develop and implement improved potency measurement(s) that quantitatively assesses relevant biological product attribute(s) (see 21 CFR 312.23(a)(7)). 2. Later phase product development: The primary objective of later phase investigational studies (i.e., Phase 3, pivotal12) is to gather meaningful data about product efficacy. Efficacy is determined by adequate and well-controlled clinical study(ies). Therefore, your potency assay design and acceptance criteria should be sufficient to assure that a well-characterized, consistently manufactured product was administered during your pivotal study(ies). Conformance to established limits for potency should thus provide reasonable confidence that future product lots will perform as expected at a given dose in patients. In addition, you should use a well-characterized potency assay with established limits during stability testing of conformance lots used to establish expiry dating for licensure (see 21 CFR 610.53; Ref. 7). 3. Biological License To market a biological product, a validated potency assay with defined acceptance criteria must be described and justified in the BLA (21 CFR 601.2(a) and 211.165(e), see also Section II.A). The acceptance criteria should be based on knowledge gained through manufacturing experience and data collected from assays performed during all phases of product development and clinical investigation (Ref. 5). As you evaluate product conformance lots or lots manufactured explicitly for use in your pivotal clinical studies, acceptance criteria should be refined to reflect these data. The potency assay acceptance criteria defined in your BLA, which are intended for subsequent lot release testing, should depict the potency limits established for product lots used in the pivotal clinical studies demonstrating clinical effectiveness (see FDC Act, Section 505(d), 21 U.S.C. 351). IV. ASSAY DESIGN AND VALIDATION A. What Should be Considered During Assay Design? In accordance with CGMP regulations, assay design should allow you to collect data that will permit you to evaluate your assay(s). This includes incorporating a sufficient number of replicates to allow for statistical analysis, using sample randomization to reduce biases (e.g., sources of bias associated with placement in a 96-well plate), and

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including appropriate controls. Assay design should also reflect knowledge of the factors that influence assay variability. Therefore, you should consider sources of variability in the assay method and take steps to limit them in your assay design. General principles for reducing variability include using well-defined reagents, well-calibrated equipment, and adequately trained operators. Assay variability can also be substantially reduced by following detailed standard operating procedures (SOPs) and having appropriate controls in place. Assay-specific controls will depend on the product being analyzed as well as the assay used. You should also consider the long-term availability of critical reagents, including reference materials and controls. Manufacturers may refer to several resources for a more detailed discussion of assay design strategies (e.g., Refs. 13 through 20). B. How Should Reference Materials and Controls be Utilized? As with all well designed experiments, developing a potency assay should include appropriate controls and a comparison to an appropriate reference material, when available. Running a reference material and/or control samples in parallel with the product helps ensure that the assay is performing as expected. In addition, controls help establish that the equipment and reagents are working within established limits. A well designed set of control samples can substantially increase confidence that results are meaningful and reproducible. Reference materials and standards can help with assay development and can be used to develop and qualify more relevant in house reference materials and/or controls. A number of reference materials, standards, and controls are available or are being developed for characterizing biologics. For instance, there are fluorescent bead/antibodies and particle size standards13 and guidelines14 available to help calibrate equipment and help define acceptable parameters for quantitative flow cytometry analysis (Ref. 18). Reference materials are also currently available for adenovirus type 5 (Ref. 19)15 and retrovirus16 vectors. A reference material for adeno-associated virus type 2 vectors17 is under development. Standard materials and controls for lentivirus vectors have also been described (Ref. 20). In the event that a universal standard or reference material is not available, you should develop your own in house reference material(s) (Refs. 9 through 11). These may include well characterized clinical lots or other well characterized materials prepared by you or another resource (e.g., a well characterized cell line with a profile similar to your product). There should be a clear rationale for how and why the reference material (including in house reference material/control) was developed. We encourage you to

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consult with your CBER review team when developing or obtaining reference materials. Because you will use reference materials at various stages of product development and characterization, you should subject them to stability studies in parallel with your product stability studies (Ref. 7). Moreover, you should appropriately characterize each new batch of reference material, compare it with the original, and establish appropriate procedures to qualify and eventually validate new reference materials. When possible, you should retain samples (Refs. 6 through 8) of each lot of reference material for comparison with newly manufactured reference material and prepare in advance for depletion or expiration of reference materials. C. What Should be Considered for an Assay Validation Plan? 1. Regulations To obtain a biologics license, you must submit data in your BLA demonstrating, among other things, that your product meets prescribed requirements of potency (21 CFR 601.2), which requires that you validate your potency assay with predefined acceptance criteria (see 21 CFR 211.165(e)). The validation process identifies potential sources of errors and quantifies them within the assay method. Numerous resources are available for analytical methods validation (Refs. 9 through 11). You should perform analysis and validation of all relevant assay parameters (Refs. 9 through 11), including: Accuracy Precision (Repeatability, Reproducibility) Sensitivity (Limit Of Detection/Quantitation) Specificity Linearity and Range System Suitability Robustness/Ruggedness 2. Statistical design and analysis It is critically important to apply sound and appropriate statistical methods to the design and analysis of laboratory experiments for potency measurements. Otherwise, inferences drawn from such experimental data might not be valid. Potential sources of assay variability and variations from replicates should be taken into account when reporting results. You should fully describe your methods of analysis, including your justification and rationale. These descriptions should be sufficiently clear to permit independent

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statistical analysis and evaluation of the results presented in the study reports. Data collected from potency assay validation studies, when provided in electronic format, can facilitate statistical evaluations by the CBER review committee. The results of validation studies should address the targeted validation parameters and their

conformance to acceptance criteria. We encourage you to initiate early discussions with the review team to receive feedback on the design and analysis of potency experiments. (See 21 CFR 211.194 for requirements pertaining to the laboratory records you must keep.) 3. Validation of qualitative assays As discussed in Section III.B.3, qualitative assays may be used as part of an assay matrix to assess potency, provided that you conduct suitable correlation studies. You should validate all parameters relevant to your qualitative assay and provide a rationale for those parameters that you determine are not relevant. For example, although certain assay validation parameters (e.g., linearity) may not be applicable to a qualitative assay with a pass or fail readout, appropriate control samples should be used to characterize the assay for specificity and sensitivity as well as for other features of acceptable performance (e.g., robustness, system suitability). Without quantitative data, demonstrating accuracy and precision could be challenging; however, with proper assay design (e.g., sufficient replicates), you might be able to demonstrate reproducibility. For semi-quantitative assays (assays with highly variable quantitative readout, e.g., response in an animal model), broader acceptance ranges may be considered for determining assay robustness and reproducibility. Also, limits of detection and/or quantitation may be built into the assay design suitability criteria. For example, if a reasonable amount of the control or reference material does not exhibit the desired activity with sufficient statistical justification, the assay would not generally be considered acceptable. Importantly, because of the complex nature of CGT products, specific circumstances for determining assay suitability will vary from assay to assay. Therefore, we encourage you to discuss planned experiments with your CBER review team before you initiate specific assay designs and/or detailed experimental analyses of potency measurements. 4. Assay evaluation and modification Manufacturing and testing practices evolve during product development or post-licensure, or both, making it necessary and/or beneficial to re-evaluate your potency assay. If you

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plan to modify an assay that is used in an approved application or propose a new assay, you must perform validation studies to demonstrate that the modified/new assay continues to be an appropriate measure of potency (21 CFR 211.165(e)). These changes must be submitted as a supplement to an approved application (21 CFR

601.12(b)(3)(vi)). The quantity of data needed to support changes to potency measurements(s) will depend upon a number of factors, including: Stage of product development Type of change within an existing assay Whether the assay is being used to measure a different product attribute(s) Whether the proposed assay meets assay criteria outlined above (see above and Section II.A) If you modify the potency measurement used during an investigational study, you should qualify the assay and provide justification for the proposed change(s) (e.g., more relevant, more practical, more quantitative). These recommendations further emphasize the importance of maintaining retention samples (e.g., product, reference materials, critical reagents) whenever possible. It will be difficult to compare assays or determine if new assays are performing appropriately without analyzing appropriate retention samples. As this guidance indicates, a considerable amount of data might be necessary to develop a suitable measurement of potency for your product (see also Ref. 14), and your assay(s) might change over time as you develop your product and learn new information and methods. We recommend that you have timely discussions with your review team as you design, evaluate and validate your potency measurement.

V. REFERENCES 1. Application of Current Statutory Authorities to Human Somatic Cell Therapy Products and Gene Therapy Products. October 14, 1993; 58 FR 53248. Available at

http://www.fda.gov/cber/genadmin/fr101493.pdf. 2. Guidance for Industry: Source Animal, Preclinical, and Clinical Issues Concerning the

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Use

of

Xenotransplantation

Products

in

Humans.

(April

2003).

Available

at

http://www.fda.gov/cber/gdlns/clinxeno.htm. 3. Guidance for FDA Review Staff and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs). (April 2008). Available at

http://www.fda.gov/cber/gdlns/gtindcmc.htm. 4. Guidance for Reviewers: Instructions and Template for Chemistry, Manufacturing, and Control (CMC) Reviewers of Human Somatic Cell Therapy Investigational New Drug Applications (INDs). (April 2008). Available at

http://www.fda.gov/cber/gdlns/cmcsomcell.htm. 5. International Conference on Harmonisation: Guidance on Specifications: Test

Procedures and Acceptance Criteria for Biotechnological/Biological Products (ICH Q6B). 64 FR 44928, August 18, 1999. Available at http://www.fda.gov/cber/gdlns/ichtest.pdf. 6. International Conference on Harmonisation: Guidance for Industry: Q5E Comparability of Biotechnological/Biological Products Subject to Changes in Their Manufacturing Process. (June 2005). Available at http://www.fda.gov/cder/guidance/6677fnl.pdf. 7. International Conference on Harmonisation: Final Guidelines on Stability Testing of Biotechnological/Biological Products (ICH Q5C). 61 FR 36466, July 10, 1996. Available at http://www.fda.gov/cber/gdlns/ichq5c071096.pdf. 8. Guidance for Industry: CGMP for Phase 1 Investigational Drugs. (July 2008). Available at http://www.fda.gov/cber/gdlns/indcgmp.htm. 9. Draft Guidance for Industry: Analytical Procedures and Methods Validation

Chemistry, Manufacturing, and Controls Documentation. (August 2000). Available at http://www.fda.gov/cber/gdlns/methval.htm. 10. International Conference on Harmonisation Guideline Validation of Analytical Procedures: Text and Methodology Q2(R1). (November 2005). Available at

http://www.ich.org/LOB/media/MEDIA417.pdf.

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11. Chapter Validation of Compendial Methods. US Pharmacopeia 28, United States Pharmacopeia Convention, Inc., Rockville, MD: 2005. 12. The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) Mission, Vision and Strategic Priorities. (February 2004). Available at http://iccvam.niehs.nih.gov/about/ni_Mission.htm. 13. Kawakami K, Puri, RK. Regulatory Expectations During Product Development for Tumor Vaccines. Brown F, Petricciani J editors. Development of therapeutic cancer vaccines. Dev. Biol, Basel, Karger, 2004, vol 116, pp. 53-9.

14. Potency Measurements for Cellular and Gene Therapy Products, Cellular, Tissue and Gene Therapies Advisory Committee (CTGTAC) Meeting. Gaithersburg Hilton, February 9, 2006. Available at

http://www.fda.gov/ohrms/dockets/ac/cber06.html#CellularTissueGeneTherapies. 15. Chapter Design and Analysis of Biological Assays. US Pharmacopeia 28, United States Pharmacopeia Convention, Inc., Rockville, MD: 2005. 16. Brown, F., Mire-Sluis A. (Eds.), The Design and Analysis of Potency Assays for Biotechnology Products. Brown, F. (ed.), Developments in Biologicals, Vol. 107, Basel: Karger (2002). 17. Montgomery, D. C. Design and Analysis of Experiments. John Wiley & Sons; 6th edition (2005). 18. Stelzer, GT., et al., U.S.-Canadian of Consensus Recommendations by Flow on the

Immunophenotypic

Analysis

Hematologic

Neoplasia

Cytometry:

Standardization and Validation of Laboratory Procedures. Cytometry (Comm Clin Cytometry) 30:214-230 (1997). 19. Hutchins, B., et al., Working Toward an Adenovirus Vector Testing Standard. Molecular Therapy Vol. 2, No. 6, (December 2000).

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20. Kiermer, V., et al., Report from the Lentivirus Vector Working Group: Issues for Developing Assays and Reference Materials for Detecting Replication-Competent

Lentivirus in Production Lots of Lentivirus Vectors. BioProcessing Journal, March/April 2005: 39-42.

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GUIDELINE ON POTENCY TESTING OF CELL BASED IMMUNOTHERAPY MEDICINAL PRODUCTS FOR THE TREATMENT OF CANCER(EMEA/CHMP/BWP/271475/2006)

TABLE OF CONTENTS

1. EXECUTIVE SUMMARY .................................................................................................. 3 2. INTRODUCTION (BACKGROUND) ............................................................................. 3 3. SCOPE .................................................................................................................................... 3 4. LEGAL BASIS ...................................................................................................................... 4 5. ASPECTS TO POTENCY TESTING OF CELL BASED IMMUNOTHERAPY PRODUCTS ................................................................................................................................ 4

5.1 IN VIVO (ANIMAL) POTENCY TESTING .............................................................................. 5 5.2 IN VITRO POTENCY TESTING .............................................................................................. 5 5.3 VIABLE CELL COUNT .......................................................................................................... 6 5.4 AUTOLOGOUS CELL BASED PRODUCTS .............................................................................. 6 5.5 REFERENCE PREPARATION .................................................................................................. 6 5.6 ADJUVANT CONTAINING IMMUNOTHERAPY PRODUCTS. ................................................... 6

DEFINITIONS ........................................................................................................................... 7 REFERENCES (SCIENTIFIC AND / OR LEGAL)

................................................... 8

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1. EXECUTIVE SUMMARY Licensed biological medicinal products must meet specifications for appearance, identity, purity, biological activity and/or quantity of the drug substance. Determining the biological activity of cell based immunotherapy products is not easy since the active ingredient is usually composed of whole cells and the activity of these products can generally not be attributed to one specific cell characteristic. The potency (i.e., the quantitative measure of biological activity) of cell based immunotherapy products can be measured using in vivo or in vitro tests. An appropriately validated potency assay should be based on a defined biological effect as close as possible to the mechanism(s) of action/clinical response. Surrogates for potency may be developed to demonstrate biological activity of the test sample. Development and validation of such assays for cell based immunotherapy products need special considerations. This document represents CHMP current thinking on these issues. 2. INTRODUCTION (background) Cell based immunotherapy aims at treating patients by stimulating their immune system using autologous or allogeneic cells. Immunotherapy of cancer is based on an immune response targeted against tumour-specific/tumour associated antigen(s), leading to destruction of malignant cells. The targeting of interactions between the immune system and the tumour constitute a complex approach of which the precise mechanisms of action are often not fully understood. In the scientific literature, cell based immunotherapy products for the treatment of cancer are sometimes called cell based tumour vaccines or cancer vaccines. Assessment of the biological properties constitutes an essential step in establishing a complete characterisation profile of a biological medicinal product. Due to their complexity, cell based immunotherapy products cannot be fully characterised like products derived by recombinant DNA techniques. Nevertheless, as for any biological medicinal product, the biological activity is an important characteristic and needs to be determined for cell based immunotherapy products. According to the ICH guideline1 the biological activity describes the specific ability or capacity of a product to achieve a defined biological effect. Potency is the quantitative

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measure of biological activity based on the attribute of the product, which is linked to the relevant biological properties. Current guidance on cell therapy based medicinal products is found in the Guideline for Human Cell based Medicinal Products (CHMP/410869/2006) replacing the CPMP Points to consider (PtC) on the manufacture and quality control of human somatic cell therapy medicinal products (CPMP/BWP/41450/98). According to these guidelines the final cell therapy product should be subjected to quality control and lot release testing as well as to tests to evaluate the shelf-life of the product. This should include a potency assay, which should be properly validated. However, specific guidance related to the development and validation of such assays is not available. This document intends to provide further guidance on specific requirements related to the development and validation of potency assays for cell based immunotherapy products. Other existing guidelines related to testing may be relevant and should be consulted1,2. 3. SCOPE This guidance document covers viable cell products for cancer-immunotherapy from autologous or allogeneic origin, consisting of e.g. whole tumour cells or autologous dendritic cells loaded with tumour antigens, all intended to induce tumour-specific cytotoxity although the immunological pathway may differ between products. Tumour-specific cells intended for adoptive transfer (i.e. passive immunisation strategies) are also included, for example ex-vivo primed T-cells. Some principles outlined in this document may also be applicable to tumour cell lysates. The cells may be chemically treated or genetically modified in vitro to immortalize them or to express certain gene products like growth factors or tumour antigens. If the medicinal product is to be considered as a gene therapy medicinal product3, further guidance can be found in the Note for Guidance on the Quality, Preclinical and Clinical Aspects of Gene Transfer Medicinal Products. 4. LEGAL BASIS

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This guideline has to be read in conjunction with the introduction and general principles (4) and Part I: Standardised marketing authorisation dossier requirements as well as Part IV: Advanced therapy medicinal products of the Annex I to Directive 2001/83/CE as amended. 5. ASPECTS TO POTENCY TESTING OF CELL BASED IMMUNOTHERAPY PRODUCTS Appropriately designed potency assays provide an accurate, reliable and consistent demonstration of the biological activity of the active ingredient either at the level of drug substance and/or drug product. In principle the results of a potency assay should provide assurance that the amount of the active ingredient is sufficient to induce a meaningful response and that the amount is consistent from batch to batch. As such, the potency assay should be able to detect clinically meaningful changes in the amount of active ingredient in a human dose of a product. Determining the biological activity of cell based immunotherapy products is not easy since the active ingredient is usually composed of whole cells and the activity of these products can generally not be attributed to one specific cell characteristic. Potency assays for immunotherapy products will be based on complex immune mechanisms which are often poorly or incompletely understood and which may be complicated by multi-antigen formulations and inherent variability of the starting material. Nevertheless, to assure a consistent functional activity of the medicinal product in the recipient, the potency of the product within justified limits should be demonstrated by a bioassay based on a defined biological effect as close as possible to the mechanism(s) of action/clinical response. To define the biological effect, a proper understanding of the biology of these cells is necessitated. Therefore, phenotypic and functional properties of the cells should be extensively characterised. Based on these characteristics and the mode(s) of actions established in non-clinical studies the concept of the analytical assay should be deduced. One or more antigens may be selected that are linked to the defined mechanisms of action. It is generally acknowledged that cellular immunity plays a key role in the immunological destruction of tumours. Therefore, several assays under development have been based on this principle. The mechanisms of action may be more

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complex involving both a cellular and humoral immune response. Assays based on antibody formation against selected antigens or assay based on quantitative antigen expression could thus be considered as well. However, the results of the pivotal studies should ultimately support the chosen assay. Induction of a non-relevant immune response (e.g. an antibody response that is not relevant as regards to the defined biological effect) in animals following administration of the medicinal product is generally not accepted as a measurement of potency. Ideally, one single properly developed and validated assay is sufficient to cover both characterisation issues and batch release testing. However, different kinds of assays may be needed depending on the purpose of the assay, e.g. to characterise the active substance, to validate the production process, to show batch-to-batch consistency, and to determine the stability during shelf life. A potency assay is an extremely valuable tool to provide assurance of unaltered biological characteristics of the product throughout the development of the product. This is especially important when changes to the manufacturing process are introduced after production of material for non-clinical studies or pivotal clinical studies. It may be prudent to develop in parallel different potency assay most suitable for their intended use. These may comprise for example functional bioassays or, where justified, assays based on quantitative antigen expression. Preferably, a suitable potency assay should be in place already when material for the first clinical trial is produced and it should be validated prior to phase III clinical trials unless otherwise justified. Lot release and shelf life specifications for potency should be determined and amended during product development, as appropriate. It is strongly recommended that the development of a suitable potency assay be started as soon as possible. Potency of cell based immunotherapy products can be measured in a number of different assays including in vivo and in vitro test systems.

5.1 In vivo (animal) potency testing

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An in vivo potency assay is a useful tool to verify the biological activity of the active ingredient. However, the development of a relevant biological in vivo potency assays for cell based immunotherapy products may be hampered by the lack of a relevant animal model due to the inherent immunological differences between man and animals. In addition to the lack of suitable animal models, it is acknowledged that such assays very often suffer from wide inherent biological viability. In vivo potency testing may also be particularly lengthy to perform and as such may not be practical for lot release. However, the use of relevant animal models should be fully explored for their applicability for routinely performed assays. Moreover, they might be useful as a product characterization tool, e.g., after the introduction of a process change or any other change that may impact the quality of the medicinal product. For example, animals which are transgenic for human major histocompatibility antigens can be used to present human antigens to the immune system of these animals. Also, immuno-compromised animals (e.g., athymic mice) might be used to determine the functional response of adoptively transferred human T-cells as the measurement of potency. As for any animal based potency assay, suitable conditions for conducting in vivo animal testing should be set after appropriate validation. Some principles outlined in current available guidance for biological assays of prophylactic vaccines and their statistical analysis may be useful (e.g. Ph.Eur. 2.7 & 5.3.6).

5.2 In vitro potency testingWith in vitro assays, a biochemical or physiological response can be measured at the cellular level. Such assays may be suitable as a direct measure of the biological activity on a routine basis, i.e. for monitoring product consistency in batch release testing. Measurable parameters are, for example, in vitro lysis of target cells by tumour-specific (CD8) T-cells, in-vitro cytokine production by specific cells, e.g. lymphocytes in response to the product, and co-stimulatory capacity of dendritic cells (DCs). Where a direct measure of potency is not possible, surrogates for potency may be developed to verify biological activity of the test sample provided that a correlation between the surrogate and the defined biological activity has been demonstrated. Surrogate analysis may comprise different kind of tests including determination of cell

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surface markers, activation markers, secretion of factors, expression of a single gene product or protein expression pattern. Surrogate for potency may be developed for both in vitro and in vivo potency tests. If the mechanism of action of the medicinal product can be clearly related to specific antigens (i.e., tumour-specific antigens, tumour-associated antigens), the potency assay could be based on quantification of these antigens by suitable methods (e.g. flow cytometry analysis). However, special consideration should be given to the validation of non-standard methods if used for batch release testing. The possibilities of using combinations of certain parameters (e.g. viability, cell marker expression, antigen expression) could be envisaged.

5.3 Viable cell countOne of the requirements included in Directive 2003/63/EC (Annex I, part IV) is that human somatic cell therapy medicinal products are made of a defined number (pool) of viable cells. Cell viability is an important parameter of product integrity and may be used as an in-process control after manipulation of certain cell characteristics e.g. up-regulation of cell surface expression of specific antigens after cytokine treatment. Cell viability may also be an important element of the potency of cell based products. However, it should be linked with other measures of potency that demonstrate the potential for biological activity of the product, such as quantitative antigen expression or biological activity as measured in the bioassay.

5.4 Autologous cell based productsFor cell based immunotherapy products comprised of autologous cells, sample and time constraints may hamper complete batch control testing at release. In addition, there may be an inherent variability within the sourced autologous cell population, which cannot be fully rectified by the manufacturing process. In this case the use of variable cell populations may be clinically justified. This variability in cell characteristics could pose difficulties in validation of the potency assay and in assigning acceptance limits for potency.

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Nevertheless, whenever a manipulation generates a more homogeneous subpopulation, the development of an appropriate potency assay should be fully explored, which could effectively be applied either as a characterisation tool or batch release test, or both. In this situation, the absence of a suitable potency assay is not accepted without proper justification, as this will pose difficulties in demonstrating production consistency of autologous cell preparations after changes in manufacture or product composition have been implemented.

5.5 Reference preparationIn general, potency assays on biological medicinal products rely heavily on the use of reference preparations with an established potency. Most likely, no international reference preparation will be available for highly specific cell based immunotherapy products and it may be difficult to generate such preparations for autologous products. n-housereference materials should be characterised in terms of their composition, purity and biological activity as thoroughly as possible by physicalchemical- biological methods. The in-house reference material should preferably be clinically qualified or shown to be comparable to materials shown to be efficacious in clinical trials.

5.6 Adjuvant containing immunotherapy productsThere may be cases, where immunotherapy products will require an adjuvant to raise their low immunogenicity. However, it should be kept in mind that these adjuvants may exert activities that may interfere with the intended potency assay. For example, Mycobacterium bovis (bacillus Calmette- Guerin - BCG)5 has been used as an adjuvant but one of the BCG activities is associated with activation of monocytes/macrophages6. Where the adjuvant is combined with the active cellular moiety prior to performing the potency assay and the adjuvant may interfere with the specific biological activity, special considerations should be given to this issue during assay development. Compounds that are given separately and/or at a different time point in order to pre-condition the immune system and that may be needed for biological activity, are not considered to be adjuvants. As such, those compounds are outside the scope of this specific section.

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DEFINITIONS Biological activity: The specific ability or capacity of the product to achieve a defined biological effect. Potency: The measure of the biological activity using a suitably quantitative biological assay (also called potency assay or bioassay), based on the attribute of the product, which is linked to the relevant biological properties.REFERENCES (scientific and / or legal)1. ICH Topic Q6B, Step 4 Note for Guidance on Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. CPMP/ICH/365/96 - Adopted March 99. 2. ICH Topic Q5C, Step 4 Note for Guidance on Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological Products. CHMP/ICH/138/95

Adopted Dec. 95.

3 EU Commission Directive 2003/63/EC, Annex I, Part IV: Advanced Therapy Medicinal Products 4 EMEA/CHMP Note for Guidance on the Quality, Preclinical and Clinical Aspects of Gene Transfer Medicinal Products. CPMP/BWP/3088/99 5 Mesa C., Fernandez L. Challenges facing adjuvants for cancer immunotherapy. Immunology and Cell Biology 82 (2004): 644-650 6 Suttmann H., Jacobsen M., Reiss K., Jocham D., Bohle A,. Brandau S. Mechanisms of bacillus Calmette-Guerin mediated natural killer cell activation. J Urol. Oct 174 (2004): 1490-1495 7 CHMP Explanatory note on immunomodulators for the guideline on adjuvants in vaccines for human use. EMEA/CHMP/VWP/244894/2006 July 2006,

Adopted 27

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GUIDELINE ON HUMAN CELL-BASED MEDICINAL PRODUCTS TABLE OF CONTENTS EXECUTIVE SUMMARY ....................................................................................................... 3 1. INTRODUCTION (BACKGROUND) ................................................................................ 3 2. SCOPE .................................................................................................................................. 3 3. LEGAL BASIS .................................................................................................................... 4 4. MAIN GUIDELINE TEXT ................................................................................................ 4 4.1 RISK ANALYSIS ................................................................................................................... 4 4.2 QUALITY AND MANUFACTURING ASPECTS ........................................................................ 5 4.2.1 Starting and raw materials ............................................................................................ 5 4.2.2 Manufacturing process ................................................................................................... 8 4.2.3 Characterisation ............................................................................................................ 10 4.2.4 Quality control .............................................................................................................. 13 4.2.5 Validation of the manufacturing process ................................................................... 14 4.2.6 Development Pharmaceutics ......................................................................................... 15 4.2.7 Traceability ................................................................................................................... 17 4.2.8 Comparability ................................................................................................................ 17 4.3 NON-CLINICAL DEVELOPMENT ......................................................................................... 18 4.3.1. Pharmacolog y............................................................................................................... 18 4.3.2. Toxicology ..................................................................................................................... 19 4.4 CLINICAL DEVELOPMENT .................................................................................................. 21 4.4.1 General aspects ............................................................................................................. 21 4.4.2 Pharmacodynamics ....................................................................................................... 21 4.4.3 Pharmacokinetics .......................................................................................................... 21 4.4.4 Dose finding studies ..................................................................................................... 21 4.4.5 Clinical Efficacy ............................................................................................................ 22 4.4.6 Clinical Safety ............................................................................................................... 22 4.4.7 Pharmacovigilance and Risk Management Plan ....................................................... 23 REFERENCES (SCIENTIFIC AND / OR LEGAL) .......................................................... 23

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EXECUTIVE SUMMARY This guideline replaces the Points to Consider on the Manufacture and Quality Control of Human Somatic Cell Therapy Medicinal Products (CPMP/BWP/41450/98). It takes into account the current legislation and the heterogeneity of human cell-based products, including combination products. A risk analysis approach can be used by the applicants to justify the development and evaluation plans and can be a basis for the preparation of a risk management plan. In the quality and manufacturing section, guidance is provided on the criteria and testing of all starting materials, on the design and validation of the manufacturing process, on characterisation of the human cell-based medicinal products, on quality control aspects, on the development programme, traceability and vigilance* and on comparability issues. Guidance specific to the matrix/device/scaffold component in combination products is The guideline acknowledges that conventional non-clinical pharmacology and toxicology studies may not be appropriate for cell-based medicinal products. Therefore the guideline addresses which non-clinical studies are necessary to demonstrate proof-of-principle and to define the pharmacological and toxicological effects predictive of the human response. Special problems might be associated with the clinical development of human cell-based medicinal products. Guidance is therefore provided on the conduct of

pharmacodynamic/pharmacokinetic studies, dose finding and clinical efficacy and safety studies. The guideline describes the special consideration that should be given to pharmacovigilance aspects and the risk management plan for these products. 1. INTRODUCTION (background) Rapid development in the fields of biology, biotechnology and medicine has led to the development of new treatments and highly innovative medicinal products, including medicinal products containing viable cells. These new cell-based medicinal products have a high potential in the treatment of various diseases where there is a previously unmet medical need. Human cell-based medicinal products are heterogeneous with regard to the origin and type of the cells and to the complexity of the product. Cells may be self-renewing stem cells, more committed progenitor cells or terminally differentiated cells exerting a specific defined physiological function. Cells may be of autologous or allogeneic origin.

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In addition, the cells may also be genetically modified. The cells may be used alone, associated with biomolecules or other chemical substances or combined with structural materials that alone might be classified as medical devices (combined advanced therapy medicinal products). 2. SCOPE This multidisciplinary guideline will address development, manufacturing and quality control as well as non-clinical and clinical development of cell-based medicinal products (CBMP) including somatic cell therapy medicinal products as defined in Directive 2001/83/EC, Part IV, Annex I1 and tissue engineered products as defined in Regulation 1394/2007/EC2. This guideline is intended for products entering the Marketing

Authorisation (MA) procedure. However, the principles laid down in the guideline should be considered by applicants entering into clinical trials. Cell-based medicinal products discussed in this document have the following

characteristics: - They contain viable human cells of allogeneic or autologous origin undergoing a manufacturing process; - They may be combined with non-cellular components; - The cells may be genetically modified. The present document applies only to the cellular component of the cell based medicinal products containing genetically modified cells. Although this document does not cover non-viable cells and cellular fragments originating from human cells, the underlying scientific principles may be applicable. This guideline does not cover xenogeneic cell-based medicinal products. 3. LEGAL BASIS This guideline should be read in conjunction with the introduction and general principles (4) and part 4 of the Annex I to Directive 2001/83/EC1 as amended and the Regulation on Advanced Therapy Medicinal Products 1394/2007/EC2. Also, donation, procurement and testing of cells from human origin must comply with

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overarching Directive 2004/23/EC4 and technical directives drawn from it, Directives 2006/17/EC5 and 2006/86/EC6. 4. MAIN GUIDELINE TEXT 4.1 Risk analysis The risk posed by the administration of a cell-based medicinal product is highly dependent on the origin of the cells, the manufacturing process, the non-cellular components and on the specific therapeutic use. The variety of cell-based medicinal products can lead to very different levels of risks for the patients, the medical personnel or the general population. Therefore the development plans and evaluation requirements need to be adjusted on a case by case basis according to a multifactorial risk based approach (see Annex I to Directive 2001/83/EC2). At the beginning of the product development, an initial risk analysis may be performed based on existing knowledge of the type of product and its intended use. This should be updated by the applicant throughout the product life cycle as data are collected to further characterise the risk. The comprehensive risk analysis should be used to justify the product development. It should also serve as a basis for the preparation of a risk management plan in accordance with the guideline on risk management systems for medicinal products for human use (EMEA/CHMP/96268/2005)7. In particular, the results of the comprehensive risk analysis should be used: to identify risk factors associated with the quality and safety of the product; to determine the extent and focus of the data required during non-clinical and clinical development; when establishing the need for risk minimisation activities;

when determining the post market risk management activities specified in the pharmacovigilance plan.

The following general risk criteria (non-exhaustive) can be used in the estimation of the

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overall risk of the product: origin (autologous-allogeneic); ability to proliferate and/or differentiate; ability to initiate an immune response (as target or effector); level of cell manipulation (in vitro/ex vivo expansion/activation/ differentiation /genetic manipulation/ cryo-conservation); mode of administration (e.g. ex vivo perfusion, local or systemic surgery); duration of exposure or culture (short to permanent) or life span of cell; combination product (cells and bioactive molecules or structural materials); availability of clinical data on or experience with similar products. 4.2 Quality and manufacturing aspects This part of the guideline describes activities by manufacturers following procurement of the cells and tissues. The manufacture of cell-based medicinal products should be in compliance with the principles of good manufacturing practices, as set out in Directive 2003/94/EC8 and its Annex 29. The active substance of a cell-based medicinal product (CBMP) is composed of the engineered (manipulated) cells and/or tissues. Additional substances (e.g. scaffolds, matrices, devices, biomaterials, biomolecules and/or other components) when combined as an integral part with the manipulated cells are considered part of the active substance and are therefore considered as starting materials, even if not of biological origin. CBMP often contain, or consist of cell samples of limited size and many are intended to be used in a patient-specific manner. This can raise specific issues pertaining to quality control testing designs for each product under examination. Since this document covers a variety of CBMPs, processes involved can vary from very simple to highly complex. For certain CBMPs, the starting material, the active substance and the finished product can be closely related or nearly identical. For such products, some requirements listed below could be inadequate and in those cases only relevant sections and items should be addressed. 4.2.1 Starting and raw materials

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The manufacturing process of CBMP usually does not include terminal sterilisation, purification steps, viral removal and/or inactivation steps. Therefore, stringent sourcing requirements and acceptance criteria for all materials derived from human or animal origin should be adequately defined according to their intended use. 4.2.1.1. Cells Donated cellular material from single or pooled donors, once processed (see 4.2.2.1) may be: A single primary cell isolate used directly for the CBMP; Primary cells cultured for a few passages before being used for the CBMP; Cells based on a well-defined cell bank system consisting of a master cell bank and a working cell bank. An adequately controlled cell storage system should be established to allow proper maintenance and retrieval of cells without any alteration of their intended final characteristics. Storage conditions should be optimised to ensure cell viability, density, purity, sterility and function. Identity should be verified by relevant genotypic and/or phenotypic markers and the proportion of cells bearing these identity markers evaluated as an indicator of the intended cell population. A. Cells of primary origin The specific requirements for donation, procurement and testing laid down in Directive 2006/17/EC5 shall be met. Procedures and standards employed for the selection of appropriate donors and the exclusion of high-risk or otherwise unsuitable candidate donors should be clearly delineated and justified. If it is necessary to pool cells from different donors, the risk analysis should address the possibility that pooling of allogeneic cell populations may increase the risk of undesired immunological responses in the recipient and compromise its therapeutic activity. In addition, pooling of cells may increase the risk of disease transmission. Depending on the nature of the source of the cells and tissues, other risk

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factors, e.g. previous radiation exposure, should be also considered and addressed. On receipt of the cells for use in a medicinal product, a specific microbiological screening programme should be in place, adapted to the type of cells, with validated assays capable of detecting human infectious agents with appropriate sensitivity and taking into consideration the medium components that might interfere with the assays (e.g. antibiotics). When cells originate from non-healthy tissues, the product specific acceptance criteria should be defined according to the intended use. Quality parameters aimed at the definition of acceptance criteria for a given organ or tissues should be specified, taking into consideration general aspects such as shipment and storage conditions. In the case of autologous donation, the testing regimen of the starting material should be justified, taking into account the autologous use. Where allogeneic primary cells are collected and expanded for use in multiple patients, the cell lot should be appropriately characterised. The same characterisation programme shall be applied to each new cell lot. B. Banking system for established cell lines Where cell lines are used, an appropriately characterised Master Cell Bank (MCB) and Working Cell Bank (WCB) should be established, whenever possible. Cell banking and characterisation and testing of the established cell banks should comply with the ICH guideline Q5D10. 4.2.1.2. Other materials, reagents and excipients Various materials are needed for collection, selection, culture or even genetic or phenotypic modification of cells, such as other cells, enzymes, antibodies, cytokines, sera and antibiotics. Exposure to such materials can also impact on the quality, safety and efficacy of the final therapeutic product. As a consequence, each substance used in the procedure should be clearly specified and evaluated as to its suitability for the intended

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use. The microbial purity and low endotoxin level of these materials should be ensured. Materials, including cells that function as support for growth and adhesion e.g. feeder cells should be evaluated and/or validated as to their suitability for the intended use. The quality of biologically active additives in culture media such as growth factors, cytokines and antibodies, should be documented with respect to identity, purity, sterility and biological activity and absence of adventitious agents. It is recommended to keep the use of such materials to a minimal and to avoid the use of reagents with sensitisation potential e.g. -lactam antibiotics. For viral safety aspects, the guidelines on viral safety11, and Eudralex vol. 2B13 should

12

be taken into consideration. The principles laid down in the general text of the European Pharmacopoeia on viral safety14 should be followed for every substance of animal and human origin that is used during the production.

Measures should be taken to reduce the risk of transmissible spongiform encephalopathy according to the relevant European legislation and guidelines15. Where appropriate, the Note for Guidance on the Production and quality control of medicinal products derived by recombinant DNA technology16 and the Note for Guidance on the Production and quality control of Monoclonal Antibodies17 should be taken into account. When the raw materials, reagents and/or excipients have a marketing authorisation or mentioned in a Pharmacopoeia, appropriate references may be given. The following information must be added for materials of human or animal origin: A. Human derived materials Reagents of human origin (e.g. albumin, immunoglobulins) should be evaluated for their suitability in a manner identical to that employed for plasma-derived products as recommended in the CPMP Note for guidance on plasma-derived medicinal products18. The use of synthetic alternatives should be investigated. If serum is required in the

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culture media, the use of serum isolated from the same individual who donated the cells is preferred, where possible, to alternate allogeneic serum. B. Animal derived material Where cells or tissues of animal origin are used e.g. as supportive cells, the guidance given in Points to consider on Xenogeneic Cell Therapy Medicinal Products19 should be followed. Animal derived reagents may harbour infectious agents and may increase undesirable immunological responses in the recipient. When applicable, the use of animal reagents should be avoided and replaced by non animal derived reagents of defined composition. When bovine serum is used, the recommendations of the Note for Guidance on the Use of Bovine Serum in the Manufacture of Human Biological Medicinal Product20 should be followed. The use of irradiated sera and/or alternative synthetic media is encouraged and should be considered. For viral safety testing of materials of other animal species, the table of extraneous agents to be tested for in relation to the general and species-specific guidelines on production and control of mammalian veterinary vaccines21 and Note for Guidance on Production and Quality Control of Animal Immunoglobulins and Immunosera for Human use22 should be consulted. C. Special considerations Special recommendations for the starting materials of cell-based Gene Therapy Medicinal Products When the cells in the active substance are genetically modified, the Note for Guidance on the quality, preclinical and clinical aspects of gene transfer medicinal products23 should be followed, which gives details on the quality control, characterisation and preclinical testing of gene transfer vectors. Cell populations which are transformed should be assayed for appropriate and reproducible expression of the newly acquired

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characteristics. Special attention should be paid to the level and length of expression and quality of the gene product(s) produced by the cells. As far as applicable and practicable, the new characteristics of the cells should be quantified and controlled. Special recommendations for matrix/device/scaffold components of combined products Cell-based medicinal products may incorporate structural components which

independently are medical devices or active implantable medical devices. Those devices should meet the essential requirements laid down in Directive 93/42/EEC24 concerning medical devices and Directive 90/385/EEC25 on the approximation of the laws of the Member States relating to active implantable medical devices, respectively, and this information shall be provided in the marketing authorization application. In the case where a Notified Body has evaluated the device part, the result of this assessment shall be included in the dossier. Cell-based medicinal products may also incorporate structural components which are not identical to, or used in the same way as in a medical device. All structural components should be appropriately characterised and evaluated for their suitability for the intended use (See sections on Characterisation and Development Pharmaceutics). Any matrices, fibers, beads, or other materials that are used in addition to or in combination with the cells should be described and their function underpinned by means of chemical, biological, physical (e.g. structure and degradation) and mechanical properties. Inclusion of additional bioactive molecules should also be described and their impact should be evaluated.

4.2.2 Manufacturing process The manufacturing process of cell-based medicinal products should be carefully designed and validated to ensure product consistency. The requirements should be defined and justified. A detailed description of the manufacture of the active substance and of the finished product should be provided. The type of manipulation(s) required for cell processing and

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the physiological function of the cells shall be described. A flow diagram of the entire process starting from biological fluid/tissue/organ or from cell banks should be prepared indicating critical steps and intermediate products (e.g. intermediate cell batches), as well as operating parameters, in-process controls and acceptance criteria. Manufacture of combined medicinal products consisting of cells and matrices/devices/scaffolds, require additional consideration regarding the cell-matrix/scaffold interactions and quality issues raised there from. Attention should be paid to biodegradable materials, which may possess the potential for environmental changes (e.g. raising pH) for the cells during the manufacture or after administration. Information on procedures used to transport material during the manufacturing process of the product, including transportation and storage conditions and holding times, should be provided. The manufacturing area should be physically separated from the procurement area If different tissues and cellular products are processed and stored in the same

manufacturing area there is an increased risk of cross contamination during each step of the procedure, e.g. via processing equipment or in storage containers such a liquid nitrogen tanks, and therefore, adequate control measures to prevent cross-contamination should be put into place. Equipment and premises used for manufacturing of CBMP should be suitable and qualified for aseptic production. It is recommended that dedicated, product-specific or single-use equipment are used in the production, whenever possible. 1. Cell preparation procedures All cell preparation procedures should be justified in terms of their intended purpose. Inappropriate handling and improper processing of cells/tissues must be avoided as they can impair or destroy the integrity and/or function of the cells and thus result in therapeutic failure. Microbiological control is a pivotal aspect of process control and quality evaluation of all cell preparations. Monitoring of in vitro cell culturing at selected stages of the production should be performed where feasible. The culture should be examined for any microbial contamination in accordance with the culturing procedure

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and growth characteristics of the cells. After appropriate controls have been performed / implemented, the biological fluid / tissue /organ can undergo one or more of the following steps: Organ/tissue dissociation The procedure to obtain the cells from the organ/tissue has to be described (with respect to the type of enzyme, media, etc.) and validated. Consideration should be given to the degree of disruption applied to the tissue in order to preserve the intended functional integrity of the cellular preparation and to minimize cell-derived impurities in the product (cell debris, cross contamination with other cell types). Isolation of the cell population of interest Any procedure used to isolate and / or purify the cell population of interest should be described. Its effectiveness should be addressed in relation to the intended use and the method(s) should be validated. Cell culture During in vitro cell culture, consideration should be given to ensure acceptable growth and manipulation of the isolated cells. The processing steps should be properly designed to preserve the integrity and control the function of the cells. The procedures for any manipulation should be documented in detail and closely monitored according to specific process controls. The duration of cell culture and maximum number of cell passages should be clearly specified and validated. The relevant genotypic and phenotypic characteristics of the primary cell cultures, of the established cell lines10

and the

derived cell clones should be defined and their stability with respect to culture longevity determined. Consistency/repeatability of the cell culture process should be demonstrated and the culture conditions including the media and the duration should be optimised with respect to the intended clinical function of the cells. Special consideration should be given to the growth potential of cells in response to

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growth factors since cell subpopulations may gain a growth advantage under defined in vitro culturing conditions. Cell modification Various treatments (physical, chemical or genetic) can be applied to cells. The method used to modify the cells should be fully described. In the case of genetic modification of cells, requirements set up in the Note for guidance on Quality, preclinical and clinical aspects of gene transfer medicinal products23 should be followed. Cells cultured in or on a matrix/device/scaffold If the cells are grown directly inside or on a matrix/device/scaffold, the quality of the combined advanced therapy medicinal product relies predominantly on the properly controlled manufacturing process. For such products, the cell culture process has to be thoroughly validated and the effect of the device on the cell growth, function and integrity has to be taken into account. The effect that the cells may exert on the device (e.g. on rate of degradation) should also be considered (see also 4.2.6. Development Pharmaceutics). 2. In-process controls The manufacturing process needs to be controlled by several in-process controls at the level of critical steps or intermediate products. Intermediate cell products are products that can be isolated during the process; specifications of these products should be established in order to assure the reproducibility of the process and the consistency of the final produc