This Guideline has been developed by the appropriate ICH Expert Working Group and has been subject to consultation by the regulatory parties, in accordance with the ICH Process. At Step 4 of the Process the final draft is recommended for adoption to the regulatory bodies of European Union, Japan and USA.
This guidance document provides general principles on the setting and justification, to the extent possible, of a uniform set of international specifications for biotechnological and biological products to support new marketing applications.
A specification is defined as a list of tests, references to analytical procedures, and appropriate acceptance criteria which are numerical limits, ranges, or other criteria for the tests described. It establishes the set of criteria to which a drug substance, drug product or materials at other stages of its manufacture should conform to be considered acceptable for its intended use. “Conformance to specification” means that the drug substance and drug product, when tested according to the listed analytical procedures, will meet the acceptance criteria. Specifications are critical quality standards that are proposed and justified by the manufacturer and approved by regulatory authorities as conditions of approval.
Specifications are one part of a total control strategy designed to ensure product quality and consistency. Other parts of this strategy include thorough product characterization during development, upon which many of the specifications are based, adherence to Good Manufacturing Practices, a validated manufacturing process, raw materials testing, in-process testing, stability testing, etc.
Specifications are chosen to confirm the quality of the drug substance and drug product rather than to establish full characterization and should focus on those molecular and biological characteristics found to be useful in ensuring the safety and efficacy of the product.
The principles adopted and explained in this document apply to proteins and polypeptides, their derivatives, and products of which they are components (e.g.,conjugates). These proteins and polypeptides are produced from recombinant or non-recombinant cell-culture expression systems and can be highly purified and characterized using an appropriate set of analytical procedures.
The principles outlined in this document may also apply to other product types such as proteins and polypeptides isolated from tissues and body fluids. To determine applicability, manufacturers should consult with the appropriate regulatory authorities.
This document does not cover antibiotics, synthetic peptides and polypeptides, heparins, vitamins, cell metabolites, DNA products, allergenic extracts, conventional vaccines, cells, whole blood, and cellular blood components. A separate ICH Guideline, ”Specifications: Test Procedures and Acceptance Criteria for New Drugs Substances and New Drug Products: Chemical Substances” addresses specifications, and other criteria for chemical substances.
This document does not recommend specific test procedures or specific acceptance criteria nor does it apply to the regulation of preclinical and/or clinical research material.
Characterization of a biotechnological or biological product (which includes the determination of physicochemical properties, biological activity, immunochemical properties, purity and impurities) by appropriate techniques is necessary to allow relevant specifications to be established. Acceptance criteria should be established and justified based on data obtained from lots used in preclinical and/or clinical studies, data from lots used for demonstration of manufacturing consistency and data from stability studies, and relevant development data.
Extensive characterization is performed in the development phase and, where necessary, following significant process changes. At the time of submission, the product should have been compared with an appropriate reference standard, if available. When feasible and relevant, it should be compared with its natural counterpart. Also, at the time of submission, the manufacturer should have established appropriately characterized in-house reference materials which will serve for biological and physicochemical testing of production lots. New analytical technology and modifications to existing technology are continually being developed and should be utilized when appropriate.
2.1.1 Physicochemical properties
A physicochemical characterization program will generally include a determination of the composition, physical properties, and primary structure of the desired product. In some cases, information regarding higher-order structure of the desired product (the fidelity of which is generally inferred by its biological activity) may be obtained by appropriate physicochemical methodologies.
An inherent degree of structural heterogeneity occurs in proteins due to the biosynthetic processes used by living organisms to produce them; therefore, the desired product can be a mixture of anticipated post-translationally modified forms (e.g., glycoforms). These forms may be active and their presence may have no deleterious effect on the safety and efficacy of the product (section 2.1.4). The manufacturer should define the pattern of heterogeneity of the desired product and demonstrate consistency with that of the lots used in preclinical and clinical studies. If a consistent pattern of product heterogeneity is demonstrated, an evaluation of the activity, efficacy and safety (including immunogenicity) of individual forms may not be necessary.
Heterogeneity can also be produced during manufacture and/or storage of the drug substance or drug product. Since the heterogeneity of these products defines their quality, the degree and profile of this heterogeneity should be characterized, to assure lot-to-lot consistency. When these variants of the desired product have properties comparable to those of the desired product with respect to activity, efficacy and safety, they are considered product-related substances. When process changes and degradation products result in heterogeneity patterns which differ from those observed in the material used during preclinical and clinical development, the significance of these alterations should be evaluated.
Analytical methods to elucidate physicochemical properties are listed in Appendix 6.1. New analytical technology and modifications to existing technology are continually being developed and should be utilized when appropriate.
For the purpose of lot release (section 4), an appropriate subset of these methods should be selected and justified.
2.1.2 Biological activity
Assessment of the biological properties constitutes an equally essential step in establishing a complete characterization profile. An important property is the biological activity that describes the specific ability or capacity of a product to achieve a defined biological effect.
A valid biological assay to measure the biological activity should be provided by the manufacturer. Examples of procedures used to measure biological activity include:
• Animal-based biological assays, which measure an organism's biological response to the product;
• Cell culture-based biological assays, which measure biochemical or physiological response at the cellular level;
•Biochemical assays, which measure biological activities such as enzymatic reaction rates or biological responses induced by immunological interactions.
Other procedures such as ligand and receptor binding assays, may be acceptable.
Potency (expressed in units) is the quantitative measure of biological activity based on the attribute of the product which is linked to the relevant biological properties, whereas, quantity (expressed in mass) is a physicochemical measure of protein content. Mimicking the biological activity in the clinical situation is not always necessary. A correlation between the expected clinical response and the activity in the biological assay should be established in pharmacodynamic or clinical studies.
The results of biological assays should be expressed in units of activity calibrated against an international or national reference standard, when available and appropriate for the assay utilized. Where no such reference standard exists, a characterized in-house reference material should be established and assay results of production lots reported as in-house units.
Often, for complex molecules, the physicochemical information may be extensive but unable to confirm the higher-order structure which, however, can be inferred from the biological activity. In such cases, a biological assay, with wider confidence limits, may be acceptable when combined with a specific quantitative measure. Importantly, a biological assay to measure the biological activity of the product may be replaced by physicochemical tests only in those instances where:
• sufficient physicochemical information about the drug, including higher-order structure, can be thoroughly established by such physicochemical methods, and relevant correlation to biologic activity demonstrated; and
• there exists a well-established manufacturing history.
Where physicochemical tests alone are used to quantitate the biological activity (based on appropriate correlation), results should be expressed in mass.
For the purpose of lot release (section 4), the choice of relevant quantitative assay (biological and/or physicochemical) should be justified by the manufacturer.
2.1.3 Immunochemical properties
When an antibody is the desired product, its immunological properties should be fully characterized. Binding assays of the antibody to purified antigens and defined regions of antigens should be performed, as feasible, to determine affinity, avidity and immunoreactivity (including cross-reactivity). In addition, the target molecule bearing the relevant epitope should be biochemically defined and the epitope itself defined, when feasible.
For some drug substances or drug products, the protein molecule may need to be examined using immunochemical procedures (e.g., ELISA, Western-blot) utilizing antibodies which recognize different epitopes of the protein molecule. Immunochemical properties of a protein may serve to establish its identity, homogeneity or purity, or serve to quantify it.
If immunochemical properties constitute lot release criteria, all relevant information pertaining to the antibody should be made available.
2.1.4 Purity, impurities and contaminants
• Purity
The determination of absolute, as well as relative purity, presents considerable analytical challenges, and the results are highly method-dependent. Historically, the relative purity of a biological product has been expressed in terms of specific activity (units of biological activity per mg of product) which is also highly method-dependent. Consequently, the purity of the drug substance and drug product is assessed by a combination of analytical procedures.
Due to the unique biosynthetic production process and molecular characteristics of biotechnological and biological products, the drug substance can include several molecular entities or variants. When these molecular entities are derived from anticipated post-translational modification, they are part of the desired product. When variants of the desired product are formed during the manufacturing process and/or storage and have properties comparable to the desired product, they are considered product-related substances and not impurities (section 2.1.1).
Individual and/or collective acceptance criteria for product-related substances should be set, as appropriate.
For the purpose of lot release, (section 4), an appropriate subset of methods should be selected and justified for determination of purity.
• Impurities
In addition to evaluating the purity of the drug substance and drug product, which may be composed of the desired product and multiple product-related substances, the manufacturer should also assess impurities which may be present. Impurities may be either process or product-related. They can be of known structure, partially characterized, or unidentified. When adequate quantities of impurities can be generated, these materials should be characterized to the extent possible and, where possible, their biological activities should be evaluated.
Process-related impurities encompass those that are derived from the manufacturing process, i.e., cell substrates (e.g., host cell proteins, host cell DNA), cell culture (e.g., inducers, antibiotics, or media components), or downstream processing (see “Appendix”, section 6.2.1). Product-related impurities (e.g., precursors, certain degradation products) are molecular variants arising during manufacture and/or storage, which do not have properties comparable to those of the desired product with respect to activity, efficacy, and safety.
Further, the acceptance criteria for impurities should be based on data obtained from lots used in preclinical and clinical studies and manufacturing consistency lots. Individual and/or collective acceptance criteria for impurities (product-related and process-related) should be set, as appropriate. Under certain circumstances, acceptance criteria for selected impurities may not be necessary (section 2.3).
Examples of analytical procedures which may be employed to test for the presence of impurities are listed in Appendix 6.2. New analytical technology and modifications to existing technology are continually being developed and should be utilized when appropriate.
For the purpose of lot release (section 4), an appropriate subset of these methods should be selected and justified.
• Contaminants
Contaminants in a product include all adventitiously introduced materials not intended to be part of the manufacturing process, such as chemical and biochemical materials (e.g., microbial proteases), and/or microbial species. Contaminants should be strictly avoided and/or suitably controlled with appropriate in-process acceptance criteria or action limits for drug substance or drug product specifications (section 2.3). For the special case of adventitious viral or mycoplasma contamination, the concept of action limits is not applicable, and the strategies proposed in ICH Harmonised Tripartite Guidelines “Quality of Biotechnological/Biological Products: Viral Safety Evaluation of Biotechnology Derived Products Derived from Cell Lines of Human or Animal Origin” and “Quality of Biotechnological/Biological Products: Derivation and Characterization of Cell Substrates Used for Production of Biotechnological/Biological Products“ should be considered.
2.1.5 Quantity
Quantity, usually measured as protein content, is critical for a biotechnological and biological product and should be determined using an appropriate assay, usually physicochemical in nature. In some cases, it may be demonstrated that the quantity values obtained may be directly related to those found using the biological assay. When this correlation exists, it may be appropriate to use measurement of quantity rather than the measurement of biological activity in manufacturing processes, such as filling.
2.2.1 Reference standards and reference materials
For drug applications for new molecular entities, it is unlikely that an international or national standard will be available. At the time of submission, the manufacturer should have established an appropriately characterized in-house primary reference material, prepared from lot(s) representative of production and clinical materials. In-house working reference material(s) used in the testing of production lots should be calibrated against this primary reference material. Where an international or national standard is available and appropriate, reference materials should be calibrated against it. While it is desirable to use the same reference material for both biological assays and physicochemical testing, in some cases, a separate reference material may be necessary. Also, distinct reference materials for product-related substances, product-related impurities and process-related impurities, may need to be established. When appropriate, a description of the manufacture and/or purification of reference materials should be included in the application. Documentation of the characterization, storage conditions and formulation supportive of reference material(s) stability should also be provided.
2.2.2 Validation of analytical procedures
At the time the application is submitted to the regulatory authorities, applicants should have validated the analytical procedures used in the specifications in accordance with the ICH Harmonised Tripartite Guidelines “Validation of Analytical Procedures: Definitions and Terminology” and “Validation of Analytical Procedures: Methodology", except where there are specific issues for unique tests used for analyzing biotechnological and biological products.
2.3.1 Process-related considerations
Adequate design of a process and knowledge of its capability are part of the strategy used to develop a manufacturing process which is controlled and reproducible, yielding a drug substance or drug product that meets specifications. In this respect, limits are justified based on critical information gained from the entire process spanning the period from early development through commercial scale production.
For certain impurities, testing of either the drug substance or the drug product may not be necessary and may not need to be included in the specifications if efficient control or removal to acceptable levels is demonstrated by suitable studies. This testing can include verification at commercial scale in accordance with regional regulations. It is recognized that only limited data may be available at the time of submission of an application. This concept may, therefore, sometimes be implemented after marketing authorization, in accordance with regional regulations.
2.3.2 In-process acceptance criteria and action limits
In-process tests are performed at critical decision making steps and at other steps where data serve to confirm consistency of the process during the production of either the drug substance or the drug product. The results of in-process testing may be recorded as action limits or reported as acceptance criteria. Performing such testing may eliminate the need for testing of the drug substance or drug product (section 2.3.1). In-process testing for adventitious agents at the end of cell culture is an example of testing for which acceptance criteria should be established.
The use of internal action limits by the manufacturer to assess the consistency of the process at less critical steps is also important. Data obtained during development and validation runs should provide the basis for provisional action limits to be set for the manufacturing process. These limits, which are the responsibility of the manufacturer, may be used to initiate investigation or further action. They should be further refined as additional manufacturing experience and data are obtained after product approval.
2.3.3 Raw materials and excipient specification
The quality of the raw materials used in the production of the drug substance (or drug product) should meet standards, appropriate for their intended use. Biological raw materials or reagents may require careful evaluation to establish the presence or absence of deleterious endogenous or adventitious agents. Procedures which make use of affinity chromatography (for example, employing monoclonal antibodies), should be accompanied by appropriate measures to ensure that such process related impurities or potential contaminants arising from their production and use do not compromise the quality and safety of the drug substance or drug product.
Appropriate information pertaining to the antibody should be made available. The quality of the excipients used in the drug product formulation (and in some cases, in the drug substance), as well as the container/closure systems, should meet pharmacopoeial standards, where available and appropriate. Otherwise, suitable acceptance criteria should be established for the non-pharmacopoeial excipients.
Pharmacopoeias contain important requirements pertaining to certain analytical procedures and acceptance criteria which, where relevant, are part of the evaluation of either the drug substance or drug product. Such monographs, applicable to biotechnological and biological products, generally include, but are not limited to tests for sterility, endotoxins, microbial limits, volume in container, uniformity of dosage units and particulate matter. With respect to the use of pharmacopoeial methods and acceptance criteria, the value of this guidance is linked to the extent of harmonisation of the analytical procedures of the pharmacopoeias. The pharmacopoeias are committed to developing identical or methodologically equivalent test procedures and acceptance criteria.
The concept of release limits vs. shelf-life limits may be applied where justified. This concept pertains to the establishment of limits which are tighter for the release than for the shelf-life of the drug substance or drug product. Examples where this may be applicable include potency and degradation products. In some regions, the concept of release limits may only be applicable to in-house limits and not to the regulatory shelf-life limits.
Appropriate statistical analysis should be applied, when necessary, to quantitative data reported. The methods of analysis, including justification and rationale, should be described fully. These descriptions should be sufficiently clear to permit independent calculation of the results presented.
The setting of specifications for drug substance and drug product is part of an overall control strategy which includes control of raw materials and excipients, in-process testing, process evaluation or validation, adherence to Good Manufacturing Practices, stability testing, and testing for consistency of lots. When combined in total, these elements provide assurance that the appropriate quality of the product will be maintained. Since specifications are chosen to confirm the quality rather than to characterize the product, the manufacturer should provide the rationale and justification for including and/or excluding testing for specific quality attributes. The following points should be taken into consideration when establishing scientifically justifiable specifications.
• Specifications are linked to a manufacturing process.
Specifications should be based on data obtained from lots used to demonstrate manufacturing consistency. Linking specifications to a manufacturing process is important, especially for product-related substances, product-related impurities and process-related impurities. Process changes and degradation products produced during storage may result in heterogeneity patterns which differ from those observed in the material used during preclinical and clinical development. The significance of these alterations should be evaluated.
• Specifications should account for the stability of drug substance and drug product.
Degradation of drug substance and drug product, which may occur during storage, should be considered when establishing specifications. Due to the inherent complexity of these products, there is no single stability-indicating assay or parameter that profiles the stability characteristics. Consequently, the manufacturer should propose a stability-indicating profile. The result of this stability-indicating profile will then provide assurance that changes in the quality of the product will be detected. The determination of which tests should be included will be product-specific. The manufacturer is referred to the ICH Harmonised Tripartite Guideline: “Stability Testing of Biotechnological/Biological Products”.
• Specifications are linked to preclinical and clinical studies.
Specifications should be based on data obtained for lots used in pre-clinical and clinical studies. The quality of the material made at commercial scale should be representative of the lots used in preclinical and clinical studies.
• Specifications are linked to analytical procedures.
Critical quality attributes may include items such as potency, the nature and quantity of product-related substances, product-related impurities, and process-related impurities. Such attributes can be assessed by multiple analytical procedures, each yielding different results. In the course of product development, it is not unusual for the analytical technology to evolve in parallel with the product. Therefore, it is important to confirm that data generated during development correlate with those generated at the time the marketing application is filed.
Selection of tests to be included in the specifications is product specific. The rationale used to establish the acceptable range of acceptance criteria should be described. Acceptance criteria should be established and justified based on data obtained from lots used in preclinical and/or clinical studies, data from lots used for demonstration of manufacturing consistency, and data from stability studies, and relevant development data.
In some cases, testing at production stages rather than at the drug substance or drug product stages may be appropriate and acceptable. In such circumstances, test results should be considered as in-process acceptance criteria and included in the specification of drug substance or drug product in accordance with the requirements of the regional regulatory authorities.
Generally, the following tests and acceptance criteria are considered applicable to all drug substances (for analytical procedures see section 2.2.2). Pharmacopoeial tests (e.g., endotoxin detection) should be performed on the drug substance, where appropriate. Additional drug substance specific acceptance criteria may also be necessary.
4.1.1 Appearance and description
A qualitative statement describing the physical state (e.g., solid, liquid) and color of a drug substance should be provided.
4.1.2 Identity
The identity test(s) should be highly specific for the drug substance and should be based on unique aspects of its molecular structure and/or other specific properties. More than one test (physicochemical, biological and/or immunochemical) may be necessary to establish identity. The identity test(s) can be qualitative in nature. Some of the methods typically used for characterization of the product as described in section 2.1 and in Appendix 6.1 may be employed and/or modified as appropriate for the purpose of establishing identity.
4.1.3 Purity and impurities
The absolute purity of biotechnological and biological products is difficult to
determine and the results are method-dependent (section 2.1.4.). Consequently, the purity of the drug substance is usually estimated by a combination of methods. The choice and optimization of analytical procedures should focus on the separation of the desired product from product-related substances and from impurities.
The impurities observed in these products are classified as process-related and product-related:
• Process-related impurities (section 2.1.4) in the drug substance may include cell culture media, host cell proteins, DNA, monoclonal antibodies or chromatographic media used in purification, solvents and buffer components. These impurities should be minimized by the use of appropriate well-controlled manufacturing processes.
• Product-related impurities (section 2.1.4) in the drug substance are molecular variants with properties different from those of the desired product formed during manufacture and/or storage.
For the impurities, the choice and optimization of analytical procedures should focus on the separation of the desired product and product-related substances from impurities. Individual and/or collective acceptance criteria for impurities should be set, as appropriate. Under certain circumstances, acceptance criteria for selected impurities may not be required (section 2.3).
4.1.4 Potency
A relevant, validated potency assay (section 2.1.2) should be part of the specifications for a biotechnological or biological drug substance and/or drug product. When an appropriate potency assay is used for the drug product (section 4.2.4), an alternative method (physicochemical and/or biological) may suffice for quantitative assessment at the drug substance stage. In some cases, the measurement of specific activity may provide additional useful information.
4.1.5 Quantity
The quantity of the drug substance, usually based on protein content (mass), should be determined using an appropriate assay. The quantity determination may be independent of a reference standard or material. In cases where product manufacture is based upon potency, there may be no need for an alternate determination of quantity.
Generally, the following tests and acceptance criteria are considered applicable to all drug products. Each section (4.2.1 - 4.2.5) is cross-referenced to respective sections (4.1.1 - 4.1.5) under Drug Substance. Pharmacopoeial requirements apply to the relevant dosage forms. Typical tests found in the pharmacopoeia include, but are not limited to sterility, endotoxin, microbial limits, volume in container, particulate matter, uniformity of dosage units, and moisture content for lyophilized drug products. If appropriate, testing for uniformity of dosage units may be performed as in-process controls and corresponding acceptance criteria set.
4.2.1 Appearance and description
A qualitative statement describing the physical state (e.g., solid, liquid), color, and clarity of the drug product should be provided.
4.2.2 Identity
The identity test(s) should be highly specific for the drug product and should be based on unique aspects of its molecular structure and for other specific properties. The identity test(s) can be qualitative in nature. While it is recognized that in most cases, a single test is adequate, more than one test (physicochemical, biological and/or immunochemical) may be necessary to establish identity for some products. Some of the methods typically used for characterization of the product as described in section 2.1 and in Appendix 6.1 may be employed and/or modified as appropriate for the purpose of establishing identity.
4.2.3 Purity and impurities
Impurities may be generated or increased during manufacture and/or storage of the drug product. These may be either the same as those occurring in the drug substance itself, process-related, or degradation products which form specifically in the drug product during formulation or during storage. If impurities are qualitatively and quantitatively (i.e., relative amounts and/or concentrations) the same as in the drug substance, testing is not necessary. If impurities are known to be introduced or formed during the production and/or storage of the drug product, the levels of these impurities should be determined and acceptance criteria established.
Acceptance criteria and analytical procedures should be developed and justified, based upon previous experience with the drug product, to measure changes in the drug substance during the manufacture and/or storage of the drug product.
The choice and optimization of analytical procedures should focus on the separation of the desired product and product-related substances from impurities including degradation products, and from excipients.
4.2.4 Potency
A relevant, validated potency assay (section 2.1.2) should be part of the specifications for a biotechnological and biological drug substance and/or drug product. When an appropriate potency assay is used for the drug substance, an alternative method (physicochemical and/or biological) may suffice for quantitative assessment of the drug product. However, the rationale for such a choice should be provided.
4.2.5 Quantity
The quantity of the drug substance in the drug product, usually based on protein content (mass), should be determined using an appropriate assay. In cases where product manufacture is based upon potency, there may be no need for an alternate determination of quantity.
4.2.6 Genenral tests
Physical description and the measurement of other quality attributes is often important for the evaluation of the drug product functions. Examples of such tests include pH and osmolarity.
4.2.7 Additional testing for unique dosage forms
It should be recognized that certain unique dosage forms may need additional tests other than those mentioned above.
Acceptance Criteria
Numerical limits, ranges, or other suitable measures for acceptance of the results of analytical procedures which the drug substance or drug product or materials at other stages of their manufacture should meet.
Action Limit
An internal (in-house) value used to assess the consistency of the process at less critical steps.
Biological Activity
The specific ability or capacity of the product to achieve a defined biological effect. Potency is the quantitative measure of the biological activity.
Contaminants
Any adventitiously introduced materials (e.g., chemical, biochemical, or microbial species) not intended to be part of the manufacturing process of the drug substance or drug product.
Degradation Products
Molecular variants resulting from changes in the desired product or product-related substances brought about over time and/or by the action of, e.g., light, temperature, pH, water, or by reaction with an excipient and/or the immediate container/closure system. Such changes may occur as a result of manufacture and/or storage (e.g., deamidation, oxidation, aggregation, proteolysis). Degradation products may be either product-related substances, or product-related impurities.
Desired Product
(1) The protein which has the expected structure, or (2) the protein which is expected from the DNA sequence and anticipated post-translational modification (including glycoforms), and from the intended downstream modification to produce an active biological molecule.
Drug Product (Dosage Form; Finished Product)
A pharmaceutical product type that contains a drug substance, generally, in association with excipients.
Drug Substance (Bulk Material)
The material which is subsequently formulated with excipients to produce the drug product. It can be composed of the desired product, product-related substances, and product- and process-related impurities. It may also contain excipients including other components such as buffers.
Excipient
An ingredient added intentionally to the drug substance which should not have pharmacological properties in the quantity used.
Impurity
Any component present in the drug substance or drug product which is not the desired product, a product-related substance, or excipient including buffer components. It may be either process- or product-related.
In-house Primary Reference Material
An appropriately characterized material prepared by the manufacturer from a representative lot(s) for the purpose of biological assay and physicochemical testing of subsequent lots, and against which in-house working reference material is calibrated.
In-house Working Reference Material
A material prepared similarly to the primary reference material that is established solely to assess and control subsequent lots for the individual attribute in question. It is always calibrated against the in-house primary reference material.
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.
Process-Related Impurities
Impurities that are derived from the manufacturing process. They may be derived from cell substrates (e.g., host cell proteins, host cell DNA), cell culture (e.g., inducers, antibiotics, or media components), or downstream processing (e.g., processing reagents or column leachables).
Product-Related Impurities
Molecular variants of the desired product (e.g., precursors, certain degradation products arising during manufacture and/or storage) which do not have properties comparable to those of the desired product with respect to activity, efficacy, and safety.
Product-Related Substances
Molecular variants of the desired product formed during manufacture and/or storage which are active and have no deleterious effect on the safety and efficacy of the drug product. These variants possess properties comparable to the desired product and are not considered impurities.
Reference Standards
Refer to international or national standards.
Specification
A specification is defined as a list of tests, references to analytical procedures, and appropriate acceptance criteria which are numerical limits, ranges, or other criteria for the tests described. It establishes the set of criteria to which a drug substance, drug product or materials at other stages of its manufacture should conform to be considered acceptable for its intended use. “Conformance to specification” means that the drug substance and drug product, when tested according to the listed analytical procedures, will meet the acceptance criteria. Specifications are critical quality standards that are proposed and justified by the manufacturer and approved by regulatory authorities as conditions of approval.
This appendix provides examples of technical approaches which might be considered for structural characterization and confirmation, and evaluation of physicochemical properties of the desired product, drug substance and/or drug product. The specific technical approach employed will vary from product to product and alternative approaches, other than those included in this appendix, will be appropriate in many cases. New analytical technology and modifications to existing technology are continuously being developed and should be utilized when appropriate.
6.1.1 Structural characterization and confirmation
a) Amino acid sequence
The amino acid sequence of the desired product should be determined to the extent possible using approaches such as those described in items b) through e) and then compared with the sequence of the amino acids deduced from the gene sequence of the desired product.
b) Amino acid composition
The overall amino acid composition is determined using various hydrolytic and analytical procedures, and compared with the amino acid composition deduced from the gene sequence for the desired product, or the natural counterpart, if considered necessary. In many cases amino acid composition analysis provides some useful structural information for peptides and small proteins, but such data are generally less definitive for large proteins. Quantitative amino acid analysis data can also be used to determine protein content in many cases.
c) Terminal amino acid sequence
Terminal amino acid analysis is performed to identify the nature and homogeneity of the amino- and carboxy-terminal amino acids. If the desired product is found to be heterogeneous with respect to the terminal amino acids, the relative amounts of the variant forms should be determined using an appropriate analytical procedure. The sequence of these terminal amino acids should be compared with the terminal amino acid sequence deduced from the gene sequence of the desired product.
d) Peptide map
Selective fragmentation of the product into discrete peptides is performed using suitable enzymes or chemicals and the resulting peptide fragments are analyzed by HPLC or other appropriate analytical procedure. The peptide fragments should be identified to the extent possible using techniques such as amino acid compositional analysis, N-terminal sequencing, or mass spectrometry. Peptide mapping of the drug substance or drug product using an appropriately validated procedure is a method that is frequently used to confirm desired product structure for lot release purposes.
e) Sulfhydryl group(s) and disulfide bridges
If, based on the gene sequence for the desired product, cysteine residues are expected, the number and positions of any free sulfhydryl groups and/or disulfide bridges should be determined, to the extent possible. Peptide mapping (under reducing and non-reducing conditions), mass spectrometry, or other appropriate techniques may be useful for this evaluation.
f) Carbohydrate sturcture
For glycoproteins, the carbohydrate content (neutral sugars, amino sugars, and sialic acids) is determined. In addition, the structure of the carbohydrate chains, the oligosaccharide pattern (antennary profile) and the glycosylation site(s) of the polypeptide chain is analyzed, to the extent possible.
6.1.2 Physiochemical properties
a) Molecular weight or size
Molecular weight (or size) is determined using size exclusion chromatography, SDS-polyacrylamide gel electrophoresis (under reducing and/or non-reducing conditions), mass spectrometry, and other appropriate techniques.
b) Isoform pattern
This is determined by isoelectric focusing or other appropriate techniques.
c) Extinction coefficient (or molar absorptivity)
In many cases it will be desirable to determine the extinction coefficient (or molar absorptivity) for the desired product at a particular UV/visible wavelength (e.g., 280 nm). The extinction coefficient is determined using UV/visible spectrophotometry on a solution of the product having a known protein content as determined by techniques such as amino acid compositional analysis, or nitrogen determination, etc. If UV absorption is used to measure protein content, the extinction coefficient for the particular product should be used.
d) Electrophoretic patterns
Electrophoretic patterns and data on identity, homogeneity and purity can beobtained by polyacrylamide gel electrophoresis, isoelectric focusing, SDS-polyacrylamide gel electrophoresis, Western-blot, capillary electrophoresis, orother suitable procedures.
e) Liquid chromatographic patterns
Chromatographic patterns and data on the identity, homogeneity, and purity can be obtained by size exclusion chromatography, reverse-phase liquid chromatography, ion-exchange liquid chromatography, affinity chromatography or other suitable procedures.
f) Spectroscopic profiles
The ultraviolet and visible absorption spectra are determined as appropriate. The higher-order structure of the product is examined using procedures such as circular dichroism, nuclear magnetic resonance (NMR), or other suitable techniques, as appropriate.
This appendix lists potential impurities, their sources and examples of relevant analytical approaches for detection. Specific impurities and technical approaches employed, as in the case of physicochemical characterization, will vary from product to product and alternative approaches, other than those listed in this appendix will be appropriate in many cases. New analytical technology and modifications to existing technology are continuously being developed, and should be applied when appropriate.
6.2.1 Process-related impurities and contaminants
These are derived from the manufacturing process (section 2.1.4) and are classified into three major categories: cell substrate-derived, cell culture-derived and downstream-derived.
a) Cell substrate-derived impurities include, but are not limited to, proteins derived from the host organism, nucleic acid (host cell genomic, vector, or total DNA). For host cell proteins, a sensitive assay e.g., immunoassay, capable of detecting a wide range of protein impurities is generally utilized. In the case of an immunoassay, a polyclonal antibody used in the test is generated by immunization with a preparation of a production cell minus the product- coding gene, fusion partners, or other appropriate cell lines. The level of DNAf rom the host cells can be detected by direct analysis on the product (such as hybridization techniques). Clearance studies, which could include spiking experiments at the laboratory scale, to demonstrate the removal of cell substrate-derived impurities such as nucleic acids and host cell proteins may
sometimes be used to eliminate the need for establishing acceptance criteria for these impurities.
b) Cell culture-derived impurities include, but are not limited to, inducers antibiotics, serum, and other media components.
c) Downstream-derived impurities include, but are not limited to, enzymes, chemical and biochemical processing reagents (e.g., cyanogen bromide, guanidine, oxidizing and reducing agents), inorganic salts (e.g., heavy metals, arsenic, non metallic ion), solvents, carriers, ligands (e.g., monoclonal antibodies), and other leachables.
For intentionally introduced, endogenous and adventitious viruses, the ability of the manufacturing process to remove and/or inactivate viruses should be demonstrated as described in ICH Harmonised Tripartite Guideline “Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin”
6.2.2 Product-related impurities including degradation products
The following represents the most frequently encountered molecular variants of the desired product and lists relevant technology for their assessment. Such variants may need considerable effort in isolation and characterization in order to identify the type of modification(s). Degradation products arising during manufacture and/or storage in significant amounts should be tested for and monitored against appropriately established acceptance criteria.
a) Truncated forms: Hydrolytic enzymes or chemicals may catalyze the cleavage of peptide bonds. These may be detected by HPLC or SDS-PAGE. Peptide mapping may be useful, depending on the property of the variant.
b) Other modified forms: Deamidated, isomerized, mismatched S-S linked, oxidized or altered conjugated forms (e.g., glycosylation, phosphorylation) may be detected and characterized by chromatographic, electrophoretic and/or other relevant analytical methods (e.g., HPLC, capillary electrophoresis, mass spectroscopy, circular dichroism).
c) Aggregates: The category of aggregates includes dimers and higher multiples of the desired product. These are generally resolved from the desired product and product-related substances, and quantitated by appropriate analytical procedures (e.g., size exclusion chromatography, capillary electrophoresis).
