Questions and answers for biological medicinal products
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This question and answer page is developed and maintained by the CHMP Biologics Working Party (BWP) and provides agreed positions by the Biologics Working Party position on issues that can be subject to different interpretation or require clarification, typically arising from discussions or correspondence during assessment procedures of biological human medicinal products.
In order to obtain information on a topic, please click on the topics/questions. Please note that this page has been produced to provide transparency and additional information, and should be read in conjunction with the European Pharmacopoeia, CHMP guidelines on quality and other guidance documents.
Storage Sites for Master cell bank/ Working cell bank/ Active substance/ Finished product/ Intermediates (3.2.S.2.1, 3.2.P.3.1)
Storage sites for master cell banks (MCB) and working cell banks (WCB) should be stated in CTD Section 3.2.S.2.1 However, there is no need to register storage sites for active substance/finished product or intermediates in the dossier. For these, only the storage conditions need to be stated in the CTD.
Reprocessing (3.2.S.2.2, 3.2.P.3.3)
If reprocessing is not mentioned in the dossier, it is assumed it is not applied for. However, Applicants are encouraged to explicitly state in section 3.2.S.2.2 or 3.2.P.3.3 either
- at which step(s) reprocessing may be performed. This should be supported by appropriate process validation data,
- that there is no reprocessing at any step in the manufacturing process. Reprocessing may be added post-approval through a variation procedure.
Raw materials and media components (3.2.S.2.3)
Where media are sourced from an external supplier and quantitative details of the media components are not available, a qualitative composition of media components is sufficient. In such cases, a confirmation that an agreement is in place with the supplier to notify the MAH in case of changes to the medium should be included in section 3.2.S.2.3.
Cell culture reagents should be of appropriate microbial purity. However assurance of microbial quality is considered to fall under GMP and handled by the company’s pharmaceutical quality system. Therefore, it is not a formal requirement to register a test for sterility of cell culture reagents in the CTD.
Cleavable purification tag (His-tag) (3.2.S.2, 3.2.S.4)
Some proteins are expressed with a cleavable purification tag (e.g. ‘His-tag’) which is removed after initial purification steps. Such purification tags may represent a possible immunogenicity risk. Therefore, the immunogenic/toxicological risk of these impurities should be carefully evaluated and clinically justified. Sufficient data should be provided demonstrating that the manufacturing process and associated control strategy are capable of consistently removing the tag to a sufficiently low and appropriately justified toxicological level. If residual purification tag impurities are present at measurable levels, then an acceptance criterion in the active substance specification or a limit for in-process control (IPC) should be proposed.
Method identification numbers (3.2.S.4.1, 3.2.P.5.1)
Unequivocal identifiers for in-house analytical methods should be included in the specification table. The method descriptions and the method validation summaries should include these in-house method identifiers for the non-compendial methods. This information is required in order to provide a clear link between the release and stability specifications and the descriptions and validations of analytical procedures used for testing. Applicants are free to choose any type of identifier (e.g. name, code, number etc.) provided it is clear and unambiguous. These identifiers are considered different from SOP numbers, which may be site specific and/or subject to changes after minor revisions which do not otherwise require the submission of a variation.
Process validation, removal of process related impurities, process related impurities testing (3.2.S.2.5, 3.2.P.3.5)
It is acceptable not to routinely test for process related impurities if consistent elimination has been demonstrated by validation studies and sufficient batch data is available. Preferably, the reduction of impurities is studied in process evaluation studies by spiking experiments or demonstration of robust reduction capacity in the relevant manufacturing steps. It should be justified that the relevant manufacturing step is capable of removing impurities to a sufficient level when run under worst-case conditions.
This is not applicable to impurities with a high safety risk and proteinaceous impurities (e.g. HCPs), which normally require routine control.
Process validation; column/resin re-use qualification (3.S.2.5)
Full-scale data for chromatography column/resin re-use qualification are normally not required at time of MAA, instead a validation protocol for the post-approval full-scale validation should be provided. However, this protocol and the intended number of re-uses should be supported with appropriate small-scale / process characterisation data provided at MAA.
Active substance specification Host cell proteins testing, (3.2.S.3.2, 3.2.S.4.1, 3.2.S.4.5)
Host cell protein (HCP) testing is typically part of the active substance specification, as appropriate. Routine control of HCP is deemed important as this may be a relevant safety parameter, i.e. it is not considered appropriate omit HCP testing based on process validation studies only. Until adequate manufacturing experience together with extensive batch data demonstrating consistent low levels of HCP is available, a release specification should be included in S.4.1 for HCP with a clinically qualified upper limit justified in S.4.5. HCP may be tested on an intermediate and reported as a critical in-process control, if the following is fulfilled:
- the Applicant has extensive knowledge and control of the manufacturing process (critical process parameters affecting HCP levels should be identified),
- scale-down process qualification studies followed by at-scale process validation and batch data results demonstrate HCP clearance,
- it is confirmed that exceeding the acceptance criterion for the IPC will lead to batch rejection,
- the proposed IPC limit is clinically qualified.
- HCP testing will be part of comparability assessments of future process changes where relevant.
Regardless whether HCP is routine tested as an IPC or as a release test, the HCP assay should be well described and validated and acceptance limits should be justified; all documentation should be included in the CTD (either in section S.4 or section S.2.4). The suitability of the detecting polyclonal antibody (coverage of HCP representative of the process, as mentioned in Ph.Eur.2.6.34) should be assessed. In addition, other recommendations from Ph. Eu 2.6.34, should also be addressed by the Applicant.
Active substance specifications, sterility (3.2.S.4)
For biological active substances, information on microbial control is normally expected in the dossier (e.g. in-process or release tests for bioburden, endotoxin, mycoplasma etc., as appropriate). However, unless the biological active substance is explicitly claimed to be sterile by the Applicant, there is no requirement for a sterility specification for a biological active substance, irrespective of its storage conditions (frozen, refrigerated, other).
Biosimilar medicinal products: characterization, side-by-side comparison, specification, biosimilarity, analytical comparability (3.2.S, 3.2.S.3.1, 3.2.S.4.1, 3.2.P.5.1, 3.2.R)
Since for most biosimilar applications only a limited number of clinical batches are used, it is not possible to set and clinically justify specification limits solely based on clinical batches of the proposed biosimilar. Therefore, characterisation results of the reference product (as obtained by the applicant using their own qualified/validated test methods intended for the proposed biosimilar) may be used for clinical justification of the specification limits of the proposed biosimilar. Result ranges obtained for marketed reference product batches can be assumed to represent a clinically qualified range for the respective quality parameters.
As regards process-related impurities (e.g. host cell DNA and HCP), since processes are as such not expected to be similar, in most cases the applicant cannot refer to characterisation results from the reference product to justify the specification limits and the control strategy for process-related impurities should be established based on the manufacturing experience for the biosimilar.
In order to demonstrate biosimilarity/analytical comparability, it is not generally required to perform a side by side comparison (i.e. testing data from a single analytical experiment/run). A side by side comparison is important only for those tests with high intrinsic between-run variability, in order to minimize such variability. In other cases, data from different experiments are acceptable.
Where an Applicant can demonstrate that a monoclonal antibody (mAb) binds to a soluble target which has no transmembrane variant or is not known to be cell/membrane-associated, then characterisation testing for Fc functionality assays (ADCC/CDC/ADCP) can be omitted. In such cases, Fc functionality testing is not required to demonstrate comparability after manufacturing changes or for biosimilarity testing.
The evaluation of ADCC activity should not be based solely on reporter gene assays. A “classical” ADCC assay approach using a two cell format, i.e. target cells plus effector cells (such as NK cells or PBMCs), is required for demonstrating biosimilarity of mAbs with ADCC function.
Where binding to Fc?RIIa is a relevant quality parameter, the biosimilarity exercise should include binding to both polymorphic receptor variants 131H and 131R.
Where binding to Fc?RIIIa is a relevant quality parameter, the biosimilarity exercise should include binding to both polymorphic receptor variants 158V and 158F.
Concerning Fc effector functions for mAbs of the IgG2 subclass, it is acknowledged that they are expected to show low binding activity to Fcγ receptors. However, in case of a biosimilar application, to confirm analytical similarity between a reference product and a biosimilar, binding to C1q and relevant FcγRs should be investigated. In case similar binding to Fc receptors and C1q is demonstrated, a comparative assessment of functional assays for Fc-dependent activities may not be required. However, if relevant differences in FcγRs or Cq1 binding are observed, then relevant activities (antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC)) should be evaluated.
Monoclonal antibodies specification, ADCC activity (3.2.S.4.1, 3.2.P.5.1)
For mAbs with Antibody Dependent Cell-mediated Cytotoxicity (ADCC) activity, it is sufficient to only have one parameter in the specifications that verifies this function. Possible tests include a cellular ADCC assay, Fc?RIIIa binding, or relevant testing of the glycoprofile; the two latter parameters being more reliable, one of them is to be preferred to the ADCC assay depending on their correlation to ADCC assay. Indeed, where control of effector function is based on testing of the Fc?RIIIa binding or the glycoprofile, a correlation should be demonstrated between ADCC and the proposed Fc?RIIIa binding assay or the glycosylation parameters. Depending on the specific molecule, control may be based on afucosylation (G0+G1+G2), high mannose forms (e.g. M5+M6+M7+M8), total afucosylation (G0+G1+G2+high mannose forms) or a combination of several of these parameters, whatever is most relevant as indicator for ADCC.
Transport validation (3.2.P.3.5)
For biological products, a product-specific transport validation study is generally not required. Instead, stability during transport can be justified based on (a combination of) prior knowledge and simulated/laboratory transport studies. The Applicant should also confirm that the temperature is routinely monitored throughout shipping. However, for products that may be more susceptible to the effects of transport, such as cell therapies, product-specific data is typically expected. The use of surrogate markers should be justified.
Polysorbate testing in finished product specification (3.2.P.5)
Where polysorbate is included in the finished product formulation and it has been demonstrated in formulation development studies that polysorbate is a relevant component to prevent particle formation, or that particle formation is linked to polysorbate degradation, a test for polysorbate should be included not only in the routine release but also in the stability specifications. Alternatively, sufficient pharmaceutical/formulation development data to demonstrate that polysorbate levels remain stable over the proposed shelf life of the finished product, could be provided to substantiate omission of such a test during stability studies.
Low Endotoxin Recovery, Endotoxin masking effect (3.2.P.5.3)
LER (low endotoxin recovery) refers to the reduced ability to detect spiked endotoxin in products tested using the compendial Limulus Amebocyte Lysate (LAL) assay. This phenomenon may be due to endotoxin masking, and its causes are not completely understood. Current knowledge suggests that LER is inherent to certain product formulations. Thus, for product formulations that contain a combination of a surfactant (e.g. polysorbate) and a chelator (e.g. EDTA, citrate, phosphate, histidine), LER studies should be submitted in the MAA dossier.
LER studies, requested in a MAA, should be performed by spiking undiluted samples with known amounts of endotoxin and by monitoring the recovery over specific time intervals. LER studies should reflect conditions relevant for the manufacturing process, including relevant temperatures, and should reflect potential hold times during manufacture.
Based on current scientific state of the art, a minimum of four time-points should be evaluated to ensure valid and accurate results. Two consecutive data points falling below 50% recovery (lower compendial limit), should be considered as LER.
If LER is detected, the Applicant should propose an adequate mitigation strategy (e.g. the compendial method should be optimized or an alternative method developed). For LER exhibiting products, it is strongly recommended to set the finished product specification limit for bacterial endotoxin as low as reasonably achievable, according to actual manufacturer`s data. This approach would help to ensure that a larger safety margin is built into the finished product endotoxin specification limit. The recommended location of the LER studies is CTD section 3.2.P.5.3 (validation of analytical procedures) or CTD 3.2.P.2.3 (manufacturing process development) with a cross-reference in 3.2.P.5.3, as appropriate.
Of note, quality control (QC) sample hold-times and storage conditions are distinct from hold time applied during LER studies. Indeed, QC samples may be diluted and/or frozen prior to testing, while this is not the case for LER studies. The appropriateness of the QC samples respective hold time i.e. maximum allowable time between sampling and testing, is a general GMP requirement1.
LER investigations should focus on the finished product (FP) as it is administered to the patient. LER studies may not be necessary for the active substance (AS) if the matrix is similar for both AS and FP. However, if there are differences in the matrix, a risk assessment should be performed to evaluate whether LER testing on the FP is sufficiently representative for the AS.
PDA Technical Report No. 82 on LER is recognised as a relevant standard for designing LER studies and could therefore be considered by Applicants when designing LER studies. Although the technical report primarily focuses on protein-based products, in the absence of specific guidance, it can also be consulted for other products such as vaccines and gene therapy.
1 EudraLex Volume 4: EU Guidelines for Good Manufacturing Practice for Medicinal Products for Human and Veterinary Use. Part 1, Chapter 6: Quality Control.
Excipient, Composition (3.2.P.1)
Where nitrogen gas is used for backfilling (head space gas), it should be included in the finished product composition. However, in such cases it should not be included in the main composition table but placed outside of this table (using an asterisk/footnote). In such cases, nitrogen should not be listed as an excipient in the SmPC.
Container closure system (3.2.P.7)
The suppliers of container closure systems for the sterile finished product(e.g. rubber stoppers and glass cartridges) should be stated in CTD 3.2.P.7.
Prior knowledge
Justifications based on prior knowledge should be included in the sections of the dossier where the product-specific data it is complementing or replacing would have been included.
Non-novel excipients manufactured using recombinant technology (3.2.P.4, 3.2.A.3)
For excipients manufactured using recombinant technology, that are not considered novel, the submission of certain manufacturing and control information is required to ensure consistent quality. In addition to the CTD module 3.2.P.4 “Control of excipients” (specifications, analytical methods, method validation and justification of specifications), applicants should also provide detailed information on the following aspects: manufacturers, description of the manufacturing process, control of materials, process validation, characterization, impurity testing, stability and viral safety assessment (including proof of process capability to remove/clear relevant model viruses). Information should be presented in section 3.2.A.3 Excipients.
Post approval change management protocol (PACMP), Comparability, (3.2.R)
A comparability exercise should be presented in a PACMP in such a manner that it would be suitable for the implementing variation submission, i.e. that all tests (routine and extended characterisation) are completely described and that similarity criteria are well-defined and unequivocal.
Adjustment to chromatographic separations test procedures as per Ph. Eur. general chapter 2.2.46
Introductory note:
This Q&A provides information on how Ph. Eur. general chapter 2.2.46Chromatographic separation techniques (in particular with respect to the adjustment of chromatographic conditions) can be used for non-compendial (in house) analytical procedures and when the conditions described in the pharmacopoeia text apply.
Background information:
When a pharmacopoeial test procedure is used as intended, the test procedure is not required to be described in the dossier. Instead a cross-reference to the current respective Ph. Eur. monograph is considered sufficient.
A pharmacopeial test procedure adjusted within the limits prescribed in Ph. Eur. general chapter 2.2.46 Chromatographic separation techniques is considered to be the pharmacopeial test procedure.
In all other cases, the test procedure is considered to be an in-house test procedure. This means that the full description of the test procedure and validation data should be provided in the dossier.
a) If the applicant is using the pharmacopoeial test procedure as described in the respective monograph
No, a variation application is not necessary.
b)If the applicant is using an in-house test procedure for the finished product, that is based on the one described in the respective monograph of the active substance
Yes, a variation must be submitted according to the relevant variation guidance and the relevant documentation should be provided.
C) If the applicant is using an in-house test procedure for the active substance/finished product/excipient (regardless of whether reference to the corresponding Ph. Eur. general chapter(s) is made)
Yes, a variation must be submitted according to the relevant variation guidance and the relevant documentation should be provided.
Where an adjustment of chromatographic test parameter(s) is made to a non-compendial (in-house) test procedure, within the limits and conditions defined in Ph. Eur. general chapter 2.2.46, it is considered that the method of analysis remains the same. The analytical procedure description in the dossier should be updated accordingly. The change can be notified to the competent authority as a minor Type IA variation (for veterinary medicines: variation not requiring assessment (VNRA)) under the relevant classification category.
Where an adjustment of chromatographic test parameter(s) is made to a non-compendial (in-house) test procedure outside the limits and conditions defined in Ph. Eur. general chapter 2.2.46, it is considered that the method of analysis has changed. The analytical procedure description in the dossier should be updated accordingly. The change can be submitted to the competent authority as a minor Type IB variation (for veterinary medicines: variation requiring assessment) under the relevant classification category.
General chapter 2.2.46 describes the extent to which various parameters of a chromatographic test procedure, as described in an individual monograph, may be adjusted without fundamentally modifying the pharmacopoeial analytical procedure.
The same principles as those described in Ph. Eur. 2.2.46 may also be applied in case of adjustments to chromatographic parameters of non-compendial (in-house) analytical procedures.
Adjustments other than those indicated in Ph. Eur. 2.2.46 require revalidation of the test procedure.
If the adjustment has been performed within the limits and conditions described in Ph. Eur. general chapter 2.2.46, compliance with system suitability test (SST) criteria and other conditions described in the chapter (e.g. selectivity and elution order of any specified impurities should be equivalent) is considered sufficient to show that the updated test procedure is at least equivalent to the former test procedure. In this case, further validation of the test procedure is not deemed necessary.
An SST covering the relevant critical analytical procedure attributes should be described in the dossier. After adjustment of an analytical procedure, it should be considered whether the SST requirements are still adequate and sufficient for the adjusted analytical procedure.