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)

The applicant/MAH needs to be aware of critical aspects affecting the quality and safety of the biological product, and information on the composition of the media used during manufacturing is required to be included in the dossier for a proper risk assessment regarding possible residual impurities in the finished product. Where media are sourced from an external supplier and quantitative details of the media components are not available, a qualitative composition of media components needs to be provided. 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.

Applicants need to make the proper timely arrangements with their supplier, in order to have this information available at the time of submission of the dossier as the information is a requirement to conclude on the quality and safety of the medicinal product for the CHMP Opinion.

As reminder, the submission of confidential information from third parties directly to EMA (apart from the ASMF for chemical active substances) is not possible and the Applicant needs to have access to this information and include it in the dossier.

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.

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.

For safety related critical in-process controls for unprocessed bulk, such as tests for mycoplasma and adventitious agents, Module 3 should clearly state that a deviation from acceptance criteria would result in batch rejection. It should be noted that the provisions outlined GMP Annex 16, section 3, which allow a QP to certify a batch despite unexpected deviations in the manufacturing process, do not apply to these safety-related critical in-process controls.

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.

These requirements primarily apply to highly purified recombinant proteins. 

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.

Effector functions (Fc functions) of monoclonal antibodies (mAbs), such as antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and complement dependent cytotoxicity (CDC) are demonstrated by testing:

  • binding of mAb to relevant cell receptors or proteins, including FcγRIIIa binding for ADCC, FcγRIIa binding for ADCP, and C1q binding for CDC 
  • the biological response after binding, using Fc functional activity testing such as classical cell-based assays and reporter gene assays. 

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.

For mAbs where Fc-mediated effector functions are a plausible mechanism of action (MoA), an assay measuring Fc functional activity should be included in the characterisation / biosimilarity exercise / comparability after manufacturing changes (where relevant). Functional assays should be performed in addition to relevant Fc receptor binding assays. Where binding to FcγRIIa is a relevant quality parameter, the biosimilarity exercise should include binding to both polymorphic receptor variants 131H and 131R. 

Where binding to FcγRIIIa is a relevant quality parameter, the biosimilarity exercise should include binding to both polymorphic receptor variants 158V and 158F.

For mAbs where Fc functions are unlikely or not known to contribute to the MoA (e.g. mAbs that bind to soluble targets, IgG2 mAbs etc.), Fc functional activity testing is not required in the characterisation / biosimilarity exercise / comparability after manufacturing changes (where relevant), provided that Fc receptor binding and C1q binding is demonstrated to be sufficiently low, excluded, or demonstrated to be highly similar to the reference product in the case of biosimilars. However, Fc functionality testing may still be warranted if Fc function cannot be excluded as a potential MoA and relevant differences are observed in binding to Fc receptors. 

For the purpose of demonstrating comparability after manufacturing changes, functionality assays can be omitted from characterisation testing if the Applicant demonstrates that the mAb binds to a soluble target which has no transmembrane variant or is not known to be cell/membrane-associated. 

Monoclonal antibodies specification, ADCC activity (3.2.S.4.1, 3.2.P.5.1)

When Antibody Dependent Cell-mediated Cytotoxicity (ADCC) is a primary mechanism of action, the specification is generally expected to include a functional potency assay that directly addresses Fc-mediated activity and reflects the mechanism of action. If an ADCC assay is included as a release test, it is recommended to also include an antigen binding assay in the specification. This is because ADCC assays often show analytical and batch-to-batch variability due to glycosylation, while antigen binding assays typically offer higher accuracy and precision, and are less affected by Fc-related variability. Using both assays together provides more robust control of biological activity. Nonetheless, in some cases, ADCC alone may be sufficient for potency determination, provided that validation studies show that the ADCC assay is sufficiently precise and that meaningful release limits are in place to detect sub-potent batches.

Alternatively, ADCC activity may also be controlled through release testing of glycan structures (see below discussion on glycoprofile) and/or FcγRIIIa binding, if a strong correlation to ADCC has been demonstrated. In such cases, an antigen binding assay should be included in the specification alongside glycan/FcγRIIIa testing.

When ADCC is a secondary mechanism of action for monoclonal antibodies, it is generally sufficient to include one parameter in the specification to verify this function. Suitable parameters may include a cellular ADCC assay, FcγRIIIa binding or glycoprofile analysis. FcγRIIIa binding and glycoprofile testing are typically more reliable than the ADCC assay, and where these assays are used, a demonstrated correlation with ADCC activity is required. Where glycoprofile is used for routine control of ADCC activity, depending on the specific mAb, this 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 an indicator for ADCC.

Reference standard qualification protocols (3.2.S.5, 3.2.P.6)

Protocols for establishing future reference standards may sometimes contain the option to assign a potency of 100% to a new reference standard if the measured results for that new standard fall within a pre-specified interval (e.g. 95% confidence interval of the mean potency measured is within 90-110%). If this option is proposed, then the dossier should explain how potential drift in potency is avoided when new reference standards are qualified.

Date of manufacture (3.2.P.3.3 )

For vaccines, since the date of manufacture and the start of the shelf life can be different, both the specific manufacturing step that defines the date of manufacture and also the step that defines the start of the shelf life of the finished product should be given in section 3.2.P.3.3 of the dossier. In principle, if the finished product is stored at a lower temperature for a period of time before release, the start of the shelf life should be considered as the date when the finished product is moved to its normal recommended storage conditions/temperature prior to release. Any exceptions to this approach should be approved by the regulatory authority.

Bioburden testing (3.2.P.3, 3.2.P.5 )

According to the EMA guideline on sterilization (EMA/CHMP/CVMP/QWP/850374/2015), bioburden testing prior to sterile filtration should meet a limit of NMT 10 CFU/100 mL. A sample volume of 100 mL is expected for products where the size of the batch allows collection of such 100 mL volume sample. Where smaller sample volumes are proposed, such as in cases of low batch size, it should be justified that any difference in sensitivity would not adversely impact the probability of detecting microbial contamination in the sample and would be sufficient to guarantee a sterile product. This justification should also consider important parameters such as filter area, batch size and microbial retention capacity. If it is confirmed that the method maintains sufficient sensitivity, then a lower sampling volume could be accepted.

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.

Visible particles (3.2.P.5 )

If a biological medicinal product has a specification for appearance of “practically free from visible particles” and Ph. Eur. 2.9.20 and 5.17.2 are followed, then no additional information on the nature of the particles needs to be included in the CTD. These requirements are considered sufficiently covered by Ph. Eur. 5.17.2, GMP standards, and the Pharmaceutical Quality System (PQS). In certain cases when intrinsic particle formation is unavoidable, e.g. formulations with a high protein concentration, a release criterion of “a few translucent to white particles per vial” may be acceptable. In these specific cases, the origin and nature of the particles should be determined, a safety risk assessment should be provided based on the intended route of administration, and the criteria for acceptable and unacceptable finished product batches should clearly defined in the dossier. 

Protein content determination (3.2.P.5.1)

Annex 1 of Directive 2001/83 (see Directive 2003/63 EUR-Lex - 32003L0063 - EN) states that ’Unless there is appropriate justification, the maximum acceptable deviation in the active substance content of the finished product shall not exceed ± 5 % at the time of manufacture.’ In view of the precision of protein determination methods and final formulation procedures, this requirement is also considered applicable to medicinal products containing recombinant proteins including mAbs. In other words, these products must comply with a specification of nominal value + 5%, unless specifically justified that this requirement is not feasible (based on e.g. method validation data for protein content determination). Lack of clinical effect is not considered sufficient justification. 

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 or recombinant Factor C based assay 1. 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 performed.

LER studies should be performed for all products where the risk is identified, 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. For new marketing authorisation applications (MAAs), data from these studies should be included in Module 3. 

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 requirement 2.

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 Schwarz, H., Gornicec, J., Neuper, T. et al. Biological Activity of Masked Endotoxin. Sci Rep 7, 44750 (2017). https://doi.org/10.1038/srep44750

2 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 with an intended biological effect and manufactured using recombinant technology (3.2.P.4, 3.2.A.3 )

For novel excipients please refer to the Guideline on excipients in the dossier for application for marketing authorisation of a medicinal product (EMEA/CHMP/QWP/396951/2006).

For non-novel excipients that have an intended biological effect (e.g. adjuvants), the excipient manufacturer(s) should be registered in the Marketing Authorisation. For such excipients, compliance with a Ph. Eur. monograph alone might not be sufficient to ensure adequate control. Therefore, the specification of the excipient, including the analytical methods and acceptance criteria, should be justified based on batch data and/or be clinically justified.

For non-novel excipients manufactured using recombinant technology (e.g. hyaluronidase used to enhance delivery and absorption), 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” (acceptance criteria, analytical methods, method validation and justification of specification), 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 2.2.46 Chromatographic 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.

Novel or complex manufacturing process in Q.II.b.1 and Q.II.b.4

In categories Q.II.b.1 and Q.II.b.4, a "novel or complex manufacturing process" refers to situations where the manufacturing process involves novel or complex technology that may present challenges during site transfer and/or scale-up activities that could impact the quality of the finished product and therefore require submission of type II variations.

It should be noted that not all non-standard processes (as defined in Annex II of the Guideline on process validation for finished products - information and data to be provided in regulatory submissions) are considered complex in this context.

For example, ‘aseptic processing’ is not a complex manufacturing process for the purpose of classifications of changes in categories Q.II.b.1 and Q.II.b.4, but as it is a non-standard manufacturing process, production scale process validation data is required.

Therefore, to help the interpretation of the term "complex manufacturing process" in the categories Q.II.b.1 and Q.II.b.4, some examples of products manufactured by processes that could be considered complex are provided below (but are not limited to):

  • Advanced Therapy Medicinal Products (ATMPs)
  • Liposomal preparations
  • Nanoparticulate preparations (e.g. Lipid nanoparticles)
  • Parenteral modified release preparations
  • Metered-dose preparations for inhalation and inhalation powders
  • Multilayer tablets

In addition, examples of "novel or innovative manufacturing processes" include (but are not limited to):

  • Continuous manufacturing
  • Decentralized manufacturing
  • Additive manufacturing
  • Use of process models in the control strategy
  • Manufacture of personalised medicines
  • Other advanced manufacturing approaches

If a change is submitted as a Type IB variation, applicants must provide a justification if they consider that the manufacturing process is not "novel or complex”, as per the above interpretation. If the justification is not accepted, the competent authority may ask the applicant to withdraw and resubmit as Type II variation. Appropriate variation categorisation can also be anticipated using Post Approval Change Management Protocols. If there is any uncertainty, applicants may consult the relevant authority before submitting the variation.

Endotoxin Limulus Amoebocyte lysate (LAL) test (3.2.S.4, 3.2.P.5[E)

The traditional LAL assay relies on horseshoe crab blood, and these species are increasingly under threat. In line with the 3Rs principles (Replacement, Reduction and Refinement), Applicants are encouraged to transition to endotoxin tests that do not rely on animal derived reagents, such as the recombinant factor C (rFC) assay and the recombinant cascade reagent (rCR) assay.

For new MAAs, Applicants who still propose to use the LAL assay should describe their plans and ongoing efforts to develop and implement an animal free endotoxin assay in the foreseeable future. These plans may be preliminary at the time of submission, but they should demonstrate a commitment to transitioning away from animal lysate–based methods.

Under the current variations guideline (C/2025/5045), replacing LAL with either rFC or rCR typically falls under a Type II variation for the active substance, excipient, or finished product, since these are considered biological, immunological, or immunochemical analytical methods (variation categories Q.I.b.2.(c), Q.II.c.2.(c) or Q.II.d.2.(c)). However, to promote wider uptake of non-animal approaches, when replacing LAL with a pharmacopoeial rFC test, Applicants may instead submit a Type IB variation (variation categories (Q.I.b.2.(z), Q.II.c.2.(z), Q.II.d.2.(z)).

The situation differs for the rCR test because it is not currently described in a Ph. Eur. monograph. This means Applicants currently must provide full analytical method validation data and therefore replacement of LAL with rCR must be submitted as a Type II variation. 

Additional details on the rFC and rCR assays can be found on the EDQM website.

Biosimilar acceptance criteria (3.2.S.4.1, 3.2.P.5.1)

The specification acceptance criteria must be justified in the dossier, as is required for any medicinal product. The acceptance criteria should typically fall within the Quality Target Product Profile (QTPP) or the Quality Range established for the reference medicinal product (RMP) during the biosimilarity exercise. When a European Pharmacopoeia (Ph. Eur.) monograph is available, compliance with such a written standard is required. Note that demonstration of biosimilarity to the reference medicinal product cannot be inferred based on compliance with the written standard. Process- and safety related limits defined by other relevant Ph. Eur. chapters (e.g. endotoxin limits) are directly applicable to biosimilars.

Vaccine potency testing (3.2.P.5.1)

For vaccines, potency testing of the finished product should generally be performed on the final container product, rather than the final bulk. Exceptions to this requirement may be considered only in cases where it can be justified that testing at the bulk stage would reduce the need for in vivo testing, and/or testing on the final container is demonstrably not feasible.

Extractable volume testing (3.P.5)

Yes, a finished product release test for extractable volume may be replaced by an in line 100% fill weight check performed as an in-process control (IPC) during the filling operation.

To justify omission of the compendial extractable volume test, the dossier should include sufficient characterisation data demonstrating that batches controlled using the 100% weight check IPC would meet the requirements of Ph. Eur. 2.9.17 if extractable volume were tested. A comparative analysis should be provided showing results from both the weight-based IPC and the extractable volume test.

Once equivalence between the two tests has been established, the 100% in process fill weight check, with its defined acceptance criteria, should be listed in the finished product specification, accompanied by a footnote stating that it is performed as an IPC during the aseptic filling step. In addition, the extractable volume test (in accordance with Ph. Eur. 2.9.17) should still be included in the finished product specification with the criterion “complies if tested”. 

Viral filtration (P.3.3, A.2)

Yes. Since different filter models may vary in material and pore size, which can affect virus‑removal performance, the brand name and model of the viral nanofilter should be specified in the dossier. Therefore, it is not acceptable to state in the dossier that “equivalent filters” may be used, since a change in filter brand or model would normally require a new virus‑clearance study. If pre‑filtration is applied before nanofiltration (e.g., to remove filter foulants), the brand name and model of the prefilter should also be included in the dossier.

Pre-use filter integrity testing (3.2.P.3.3)

The EMA Guideline on the sterilisation of the medicinal product, active substance, excipient and primary container (EMA/CHMP/CVMP/QWP/850374/2015) states for products which undergo sterile filtration, the integrity of a sterilised filter should be verified by testing before use unless specifically justified and validated. Similarly, Ph. Eur. 5.1.1 Methods of preparation of sterile products states that filtration processes should be monitored using parameters that include pre‑filtration integrity test results. Therefore, details of pre-use filter integrity testing should be included in Module 3. 

Typically, pre-use integrity testing is performed in one of two ways; (1) before assembly and sterilisation of the filter (pre‑use integrity test), or (2) after assembly and sterilisation of the filter (pre‑use post‑sterilisation integrity test, or PUPSIT).

PUPSIT differs from pre-use integrity testing because it is conducted after the filter has been installed and sterilised. PUPSIT can therefore detect defects that may have arisen during the handling, installation, or the sterilisation of the filter. 

Filter integrity testing is a standard requirement in Module 3, but applicants may choose to register either pre‑use integrity testing (which in some cases, such as for ready-to-use or single-use sterile filters, may be performed by the filter manufacturer or supplier) or PUPSIT. There is no requirement to register both pre‑use integrity testing and PUPSIT. Applicants should, however, be aware that PUPSIT is a requirement of EU GMP Annex 1, and if an applicant proposes to omit PUPSIT from the manufacturing process, this will be reviewed during GMP inspection.

Adeno associated virus (AAV) infectious titre test (3.2.S.4, 3.2.P.5)

For AAV gene therapy products, the Ph. Eur. monograph on Gene therapy medicinal products for human use (3186) specifies that an infectious particle titre must be determined for batch release, using a cell-based method. The monograph cites assays such as the median cell culture infective dose (CCID₅₀) and the infectious centre assay (ICA) as acceptable methods, and it also requires determination of the viral genome to infectious particle ratio.

The TCID50 assay is currently the most widely used infectious titre/infectivity method for AAVs. It is a cell‑based assay that determines the dilution of an AAV preparation required to infect 50% of cultured wells by exposing cells to serial viral dilutions and scoring each well as positive or negative for successful transduction. Since AAV is not capable of productive replication in the absence of a helper virus, and recombinant AAVs usually lack rep and cap genes, the assay is typically performed in a permissive cell line that provides the necessary Rep and Cap functions, with the addition of a helper virus. This provides a surrogate measure of the in vivo AAV infectious capability. 

It is recognised that infectivity assays such as the TCID50 assay can be associated with high variability and may pose challenges in a routine QC environment. Therefore, it may be acceptable to replace the TCID50 assay with alternative methods that provide equivalent information through different analytical principles, provided that suitability is sufficiently demonstrated. If, for instance, an applicant can demonstrate that their potency assay measures both transduction and transgene expression and is sensitive to changes in infectivity, it may be acceptable to omit routine release testing for infectious particle titre. In such cases, the potency assay should typically cover the efficiency of gene transfer as well as the level of expression or biological activity of the therapeutic gene product. To support such an approach, the applicant should provide a robust justification in the Marketing Authorisation Application (MAA) demonstrating that the proposed potency matrix adequately controls all the relevant aspects of biological activity. In this case, the infectivity assay should remain as characterisation test.

Such a potency testing strategy may not directly capture information on the ratio of vector particle titre to infectious vector titre. Therefore, the proportion of non-functional vector particles (i.e. those which are not capable of successfully delivering the transgene) should be determined using orthogonal approaches, including a determination of empty and partially filled capsids. 

Changes to master cell banks (MCB) (3.2.S.2.3)

Annex I of Regulation (EC) No 1234/2008 specifies which types of changes to a Marketing Authorisation must be submitted as line extensions rather than as variations. In Annex 1 it states that the following type of change to the active substance is considered a line extension:

  • Modification of the vector used to produce the antigen or the source material, including a new master cell bank from a different source, where the efficacy/safety characteristics are not significantly different.

For mammalian cell lines, a new transfection results in a new clone which may behave differently, and such a change requires an extensive data package for approval. Therefore, for mammalian cell lines if a new MCB is established through re-cloning with a new transfection, the change should be submitted as a line extension, even if the cell line remains the same. This applies regardless of whether the new transfection uses the original vector or a modified vector. 

However, if the MCB is replaced or adapted without a new transfection (for example, re-establishing an MCB from an earlier research/precursor cell bank), then the change may be submitted as a variation rather than a line extension.

Biosimilars containing recombinant hyaluronidase (3.2.R)

For excipients such as recombinant hyaluronidase, information on the manufacturing and control is required to ensure the consistent quality of the enzyme (see also the Q&A on non‑novel excipients with an intended biological effect and manufactured using recombinant technology). In addition to this quality information, biosimilarity between the recombinant hyaluronidase components of the reference product and the biosimilar should be demonstrated by comparing enzyme activity in an appropriate in vitro assay.

Reference standard qualification protocols (3.2.S.5, 3.2.P.6)

When the use of a correction factor for assigning potency to future reference standards is justified, this should be clearly defined in the reference standard qualification protocol, including an explanation of how the correction factor is calculated.

If any change to the use or calculation of a correction factor becomes necessary, this change should be submitted as a variation under category Q.I.b.3.

Page update history

An update log is available to show the date and summary of changes to this webpage. It does not include updates to linked documents or minor edits like typos or broken link fixes.

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20 July 2026

Section 'Active substance specification Host cell proteins testing' updated.

Question 'What information on characterisation, biosimilarity, and comparability after manufacturing changes should be provided for effector functions of monoclonal antibodies' updated.

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