Submitted: 22 August 2022; Revised: 29 September 2022; Accepted: 10 October 2022; Published online first: 24 October 2022

Introduction

In response to stakeholder demand, in 2014 the European Pharmacopoeia (Ph. Eur.) Commission embarked upon a project to set new public standards for therapeutic monoclonal antibodies (mAbs). The aim of the project was twofold: develop general methodologies applicable to a wide range of mAbs and, at the same time, elaborate individual monographs for mAbs using a multisource approach that takes into account the specifications of more than one marketed product (‘multisource mAb’). A ‘bottom-up’ approach was designed to help build and continually develop the strategy, progressing from specific, i.e. product-specific monographs to general, i.e. general methodologies, product-class/sub-class monographs quality attribute requirements, see Figure 1. In pursuit of these goals, a case study was carried out using infliximab as a concrete example to assess the robustness of the approach and to investigate the feasibility of establishing a product-specific monograph for a complex, multisource mAb. The knowledge gained might then be used to expand the project to develop other Ph. Eur. texts applicable to mAbs.

Infliximab monograph development: the steps

Infliximab, the first approved tumour necrosis factor-alpha (TNF-α) inhibitor, was chosen as a case study because it has been a mainstay biotherapeutic and lucrative target for biosimilar development (with two biosimilars on the market at the ­outset of the study), and has evolved into a mature subject, thus providing a sound basis from which to address standardisation of an mAb.

Collaborative study for experimental verification of analytical procedures applied to infliximab
Following a positive response to the call for participation from an infliximab manufacturer with submission of data and provision of material for testing, the Ph. Eur. conducted a collaborative study to verify the robustness, transferability and suitability of analytical procedures applied to infliximab for inclusion in a monograph. The study included experimental verification of a number of physicochemical procedures, i.e. peptide mapping, N-glycan analysis, isoelectric focusing (IEF), cation-exchange chromatography (CEX), size-exclusion chromatography (SEC) and capillary electrophoresis, as well as of a TNF-α neutralization cell-based assay. The verification study was carried out using a sample panel consisting of different infliximab preparations kindly donated to the European Directorate for the Quality of Medicines & HealthCare (EDQM) by the infliximab manufacturer, and an additional commercial batch of infliximab product purchased. Several Official Medicines Control Laboratories (OMCLs) and the EDQM laboratory took part in the study, generating a significant amount of data in support of the elaboration of a monograph for infliximab. The outcome of the study demonstrated that the candidate physicochemical test procedures and cell-based assay could be carried out without major problems and all procedures were shown to be transferable, robust and suitable for a monograph. In addition, the proposed tests and acceptance criteria were found to be widely applicable to infliximab from different manufacturing sources. Overall, this work proved that complex analytical procedures for mAbs could be standardised.

Preparation of the monograph draft
The monograph for Infliximab concentrated solution (2928) was drafted with specific consideration given to: the complexity of the manufacturing process and how best to reflect the link between this and product quality; process-dependent heterogeneity, i.e. glycosylation, charged variant profile, and consistency of production; complexity of the mAb mechanism of action; complexity and choice of analytical procedures; defining monograph acceptance criteria. The approach to setting monograph specifications was, therefore, oriented towards promoting flexibility. This flexible approach, as a means to address complexity, is discussed in detail in the ‘Lessons learned’section.

Pharmeuropa enquiry and stakeholder feedback
The stakeholder feedback gathered during the public consultation in Pharmeuropa comprised a significant number of comments provided by manufacturers, industry associations and regulatory authorities. Most comments raised technical points that led to minor amendments of the draft monograph. In general, stakeholders supported monographs for mAbs (and other biological medicines) – they considered such quality standards to be useful and valuable, on the understanding that compliance with such monographs alone does not infer biosimilarity. In order to avoid potential misunderstanding of the ‘flexibility’ built into the monograph, it was recommended to provide further guidance on this matter for greater clarity, for example in the General Notices [1] (see section Setting of monograph specifications – additional flexibility for more details).

Interested parties actively contributed to the public consultation by commenting on the text in addition to performing their own testing of the draft monograph and providing further confirmatory data (for details regarding the monograph elaboration process see [2]). This helped shape the concept and reaffirmed that the proposed monograph specifications suitably reflected EU-approved infliximab products, on the basis of the data available to the Ph. Eur. at the time (status 09/2017). Through focused communication and coordinated efforts, the Ph. Eur. not only ensured engagement of all key players, including industry, licensing authorities and OMCLs, in the monograph elaboration process, but also set a science-based and flexible approach to creating a monograph on a complex mAb, addressing concerns from industry that each manufacturer has a unique product with its own control strategy. This topic is further explained in Section Stakeholder.

Monograph adoption, publication in the Ph. Eur. and implementation
An important milestone in the mAb public standard-setting strategy was achieved with the adoption of the monograph for Infliximab concentrated solution (2928) and its publication in Ph. Eur. Supplement 9.6 (effective January 2019). Monograph implementation is an inherent element of this strategy, and close collaboration with relevant parties must be maintained so that appropriate actions are taken to ensure the monograph continues to fulfil its role and suitably cover all preparations currently on the market.

In accordance with the principles of Ph. Eur. monograph elaboration, the monograph for Infliximab concentrated solution (2928) reflects the specifications of products – innovator and biosimilar – currently authorized in the Ph. Eur. Member States. After the monograph came into force in January 2019, biosimilar manufacturers – whose products (expressed in different types of eukaryotic cells than the originally tested substances) were granted marketing authorization during monograph development – requested an update to take the new sources available on the market into account and thus feed the new regulatory approvals into the monograph revision process. In light of the new data available, and in close collaboration with all interested parties, the monograph specification limit for related proteins by capillary electrophoresis-sodium-dodecyl sulfate (CE-SDS) under reducing conditions was revised, which successfully completed the process [3].

Infliximab case study: challenges and lessons learned

The complexity of therapeutic mAbs requires assays beyond the traditional physicochemical techniques and adequate analytical methodology to characterise both their physicochemical properties and biological activity. This puts severe demands on the analytical techniques required for their comprehensive structural characterisation, as well as for identity, quality, potency and purity testing. MAbs display a large diversity of quality attributes, which in turn can be analysed using a large variety of analytical procedures. This makes it difficult to choose the tests to be included in a monograph and raises the fundamental question of how the information required for a public standard should be defined. The following sections describe the steps and approaches taken to address these challenges, see also ­Figure 2.

Setting of monograph specifications – additional flexibility
A public standard provides a set of key quality attributes – not necessarily restricted to critical quality attributes (CQAs), as per the definition of the ICH Q8(R2) Guideline on Pharmaceutical Development – and is one part of a total control strategy designed to ensure product quality and consistency. However, its scope does not generally extend to characterisation, which includes determination of physicochemical and immunochemical properties, biological activity, purity and impurities, through an extensive programme conducted using a combination of orthogonal procedures.
When setting monograph specifications, the key challenge is striking an appropriate balance between flexible expectations that will apply to a large variety of products, and comprehensive and sufficiently prescriptive requirements that will facilitate successful independent testing. The latter should include tools to evaluate system suitability, as well as acceptance criteria for key quality attributes, to facilitate standardisation and remain compatible with the development of follow-on versions.

The concept of flexibility, as enshrined in the Ph. Eur. General Notices, allows the use of alternatives to the official procedures described in the Ph. Eur., as long as they lead to the same pass/fail results and are approved by the competent authority. Also envisaged is the possibility of omitting certain tests if assurance that an article is of pharmacopoeia quality is obtained on the basis of its design, the control strategy used and the available relevant data, e.g. derived from validation studies of the manufacturing process. In addition, as defined in the General Notices, flexibility is provided by the use of process analytical technology and/or real-time release testing (including parametric release) as an enhanced approach to end-product testing alone, in circumstances deemed appropriate by the competent authority [1].

During drafting of the infliximab monograph, however, it became evident that additional flexibility (other than that defined in General Notices) was needed when setting monograph specifications in order to address complex testing methodologies and the structural complexity and heterogeneity of the monoclonal antibody, as well as the potential diversity resulting from different manufacturing processes.

Production section – tests related to process-dependent heterogeneity
In order to increase its flexibility, the Production section of the monograph was expanded to include general requirements for consistency of production and specific requirements related to process-dependent heterogeneity. N-glycosylation microheterogeneity is associated with the single site (Asn300) of N-glycosylation in the Fc-region of the heavy chains, which yields several glycoforms. As such, it plays a critical role in protein structure/conformation and its Fc-effector function associated with the secondary mechanism of action of infliximab. The primary use of glycan analysis is to monitor the consistency of oligosaccharide distribution with expected glycan structures, thus reflecting manufacturing consistency and product quality. Following the approach used in Ph. Eur. monographs for glycoproteins, e.g. Etanercept (2895), Human coagulation factor IX (rDNA) concentrated solution (2522), Human coagulation ­factor VIIa (rDNA) concentrated solution (2534), glycan analysis was included in the Production section of the monograph [4, 5]. Glycan analysis is a multi-step procedure available in a variety of formats. Generic methods of analysis are already described in the Ph. Eur. general chapter Glycan analysis of glycoproteins (2.2.59). With this in mind, the infliximab monograph prescribes the use of a ‘suitable procedure’ developed according to general chapter 2.2.59. The requirements for carrying out the analysis are flexible in order to allow the use of another methodology based on the principles stated in the general chapter and adaptation to the equipment and approach of users. In addition, a specific analytical procedure is given as an example in the monograph, with a detailed description and system suitability criteria. Lastly, as acceptance criteria in the form of numerical limits for specific glycan species may not always be suitable for all registered products, they should therefore be considered on a case-by-case basis.

Charged isoforms are multiple molecular variants attributed to differences in C-terminal lysine, site-specific deamidation and charged glycan species, and are yet another source of heterogeneity. Charged variants are determined using a combination of two orthogonal techniques, namely IEF (test procedure A) and ion exchange chromatography (IEX) (test procedure B). The monograph allows for high flexibility as regards A, by using the same approach applied to glycan analysis. The user needs to employ a suitable IEF procedure according to the Ph. Eur. general chapter on Isoelectric focusing (2.2.54), whereas a detailed, specific procedure is given as an example. In addition, the monograph allows for the use of a suitable capillary IEF procedure developed according to the general chapter on Capillary electrophoresis (2.2.47), as an alternative to IEF. This must be a validated procedure whose suitability has been demonstrated to the satisfaction of the competent authority. As regards test procedure B, the monograph prescribes the use of a specific CEX procedure. As with glycan analysis, an independent analyst is able to carry out procedures A and B and verify system suitability (by using the dedicated chemical reference substance [CRS]), but cannot assess compliance with monograph specifications, for which the use of an in-house reference preparation is required. Moreover, the limits for groups of isoforms determined by IEX are set in a flexible way, ‘as authorised by the competent authority’.

‘Suitable’ versus ‘example’ procedure
The approach outlined above is intended to provide flexibility when complex, multi-step procedures or different assay formats are to be described &ndashl it is not to be seen as a universal approach. Likewise, for example, biological assays require a certain degree of flexibility regarding the assay format, readout (see section below). For all these procedures, an outline of a suitable test procedure, e.g. essential steps in the procedure or generic methods for analysis, for example, based on Ph. Eur. general chapters, is given as a requirement, whereby the term ‘suitable’ is a conventional term defined in the General Notices. On the other hand, detailed procedures with specific instructions – including sample preparation, quantities/concentrations/composition of reagents and buffers, chromatographic conditions, plate design for cell assays, readout, use of dedicated Ph. Eur. reference standards for calibration and method performance verification – may be described as ‘examples’. As explained in the newly revised General Notices [1], the sentence ‘The following procedure is given as an example’ means that either the analytical procedure described has been validated and may be implemented as is or it may be replaced by a suitable, validated procedure (without having to demonstrate its equivalence to the ‘example’ procedure), that shall be approved by the competent authority. This new addition to the General Notices– previously defined in the Technical guide for the elaboration of monographs on synthetic peptides and recombinant DNA proteins [5] – underpins the flexibility offered by Ph. Eur. monographs to adapt, as needed, to complex biotherapeutics.

Reference preparations in tests related to process-dependent heterogeneity
The infliximab monograph prescribes the use of reference standards, which are an integral part of the quality standard. A Ph. Eur. Infliximab Chemical Reference Substance (CRS) [6], established to support glycan analysis and charged variant determination, facilitates evaluation of method performance based on specific system suitability criteria. Flexibility is added by the use of an in-house reference preparation, shown to be representative of batches tested clinically and batches used to demonstrate consistency of production, in the respective tests for comparative purposes and compliance testing, e.g. matching LC profiles.

Acceptance criteria for quality attributes
A balance has been struck in the monograph between acceptance criteria in the form of numerical limits/ranges, and acceptance criteria ‘as authorised by the competent authority’. For several quality attributes, monograph acceptance criteria are not expressed as numerical limits, as no ‘one-size-fits-all’ specification is possible for process-dependent quality attributes such as glycosylation and charged variant profiles. The same applies for process-related impurities derived from both the upstream process, namely host-cell-derived proteins and host-cell- and vector-derived DNA, and the downstream process (residual protein A). Acceptance criteria for protein content (stated in the Definition section) are also expressed as ‘authorised by the competent authority’ to avoid excluding products with a different concentration.

The monograph specifications for composition of glycosylation are linked to specific glycan families, i.e. afucosylated, fucosylated and sialylated glycans, defined based on approved specifications. Afucosylation is a relevant parameter to ensure product quality, safety and efficacy, whereas sialylation is not necessarily considered a CQA for most antibodies. With the understanding that the monograph provides a set of key quality requirements, reference to fucosylated and sialylated glycans is considered important, as these species are part of the overall glycan profile and indicators for consistency of product quality. Similar considerations apply to monograph specifications for charged variants – charge heterogeneity is generally linked to production consistency. The grouping of charged variants, which are generally referred to as acidic or basic species, depends on the manufacturing process. A typical infliximab profile consists of six charged isoforms; the approach of summation of specific isoforms is based on group criteria, namely: acidic, ­bearing charged glycans (species with differences in deamidation status); main + basic, non-sialylated (species with differences in C-terminal lysine content); basic, bearing charged glycans (species with two C-terminal lysine). The ‘grouping’ approach applied in the monograph was demonstrated to be equally suitable for all infliximab preparations tested in the collaborative study, with the proviso that there may be various ways of presenting the limits.

On the other hand, acceptance criteria for product-related impurities (namely high-/low-molecular-weight species determined by SEC) and related proteins (species other than heavy chain and light chain by CE-SDS under reducing conditions and species other than the intact IgG by CE-SDS under non-reducing conditions) are expressed in the form of numerical limits. These are quality attributes for which it was considered appropriate to define limits in the monograph. The same applies to potency expressed as specific activity, for which a numerical range is defined in the Definition section of a monograph.

Finally, the acceptance criteria for peptide mapping are defined, according to which chromatograms matching the CRS peptide map are to be obtained – there is no heterogeneity in the protein backbone and the amino acid sequence needs to be the same for all infliximab products (none of the selected marker peaks of the peptide map is associated with the C-terminal lysine or glycan variability). Importantly, the role of peptide mapping in the monograph is not to achieve complete elucidation of the primary structure – normally done by a combination/range of orthogonal techniques – but to confirm identity and provide direct evidence of the sequence based on a specific fingerprint (selected marker peaks).

UV determination at 280 nm described in the Ph. Eur. general chapter on Total protein (2.5.33) is the procedure of choice for the estimation of protein content, in accordance with the data provided by the manufacturer. The theoretical (predicted) molecular absorptivity is used in the calculation of protein content: this is a new approach, different to the common monograph practice that consists in determination of the protein content using a product-specific CRS with assigned content.

The biological activity of infliximab is categorised into two parts [7]. The primary mechanism of action involves TNF-α neutralisation, mediated through the target-specific binding by the antigen-binding fragment (Fab) domain; infliximab binds to TNF-α and inhibits it from binding to its cognate receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). The second mechanism of action involves immune-mediated effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), through the binding of the Fab region to transmembrane TNF-α and via interaction of the Fc domain with Fc receptors expressed on various cell types. TNF-α a neutralisation assay is the current lot release potency assay for infliximab products. The monograph defines a numerical range for specific activity (Definition section), expressed as International Units of potency (TNF-α neutralising activity) per milligram of protein. The specific activity is determined according to the Assay section of the monograph, from protein content determined by UV spectroscopy at 280 nm using the specific absorbance and potency estimated by a bioassay referring to infliximab biological reference preparation (BRP) [8] calibrated in International Units. In addition, both compliance with the requirements stated under Assay/Potency and the protein fingerprint generated by peptide mapping serve as identification criteria in the monograph.

Monograph specifications for potency have built-in flexibility. The requirement is to carry out a suitable cell-based assay based on the inhibitory action of infliximab on the biological activity of TNF-α (with a suitable readout for assessing this inhibitory effect), calibrated with infliximab BRP; this accommodates different formats of TNF-α neutralisation assays commonly used as potency assays for infliximab. A specific WEHI-164-based cytotoxicity assay using a tetrazolium-based colorimetric readout is given as an example – it includes tools for verification of method performance, with defined system suitability criteria based on Infliximab BRP and specific controls, and thus provides a robust methodology. Together with these, specific acceptance criteria (numeric range) are given for the relative potency estimates.

Monograph versus release specifications
The basis for monograph elaboration is approved specifications, a set of product-specific test procedures and acceptance criteria; the latter are usually carefully chosen by a specific manufacturer based on a specific control strategy in place. Therefore, the set of specifications may not cover all key quality attributes of the monoclonal antibody, and may therefore not be sufficient for a public standard.

An example is peptide mapping: generally not part of release specifications but employed during physicochemical characterisation and stability studies, peptide mapping is considered a key analytical procedure for confirming primary structure. An adapted procedure is, therefore, included in the set of identification tests given in the monograph. The use of a tryptic peptide map as a qualitative tool in the monograph does not require a complete characterisation of the individual peptide peaks and complete elucidation of the primary structure with the purpose of 100% protein sequence coverage. Instead, it provides requirements for the identification of a fingerprint by comparing the generated ‘signature peptides’ with those obtained with a dedicated CRS, whereby corresponding profiles of chromatograms are to be obtained. The system suitability requirements for the marker peaks (‘signature peptides’) usually refer to a qualitative similarity (i.e. overall elution pattern, number of peaks, relative retentions, no additional peaks) of the chromatogram obtained with the reference standard to that supplied with the CRS.

From a monograph perspective, the understanding of the product characteristics that affect quality, efficacy and safety and of the analytical procedures required to monitor them is highly dependent on a sufficient level of product knowledge, including knowledge of product characterisation. This may be reflected in the monograph by general provisions in the Production section, referring to key quality attributes that need to be monitored during the course of product development and the manufacturing process. For example, analysis of infliximab glycosylation typically employs a range of state-of-the-art methodologies, including site-specific glycosylation analysis to determine the range of glycan structures displayed at Asn300, combined with monosaccharide analysis and evaluation of the type/amount of sialic acids capping the glycan structures, all these being part of in-depth characterisation studies [7]. In addition to specific requirements for N-glycan analysis/oligosaccharide profiling, it was considered essential to include in the monograph a general requirement for consistency of expected N-glycan occupancy determined using suitably qualified assays.

As regards Fc-effector functions, which together with TNF-α binding and TNF-neutralisation are part of infliximab’s biological activity, inclusion of a specific assay (typically employed during biological characterisation studies) was not technically feasible. Therefore, a general requirement is included in the Production section, which stipulates that: ‘During the course of product development, it must be demonstrated that the manufacturing process consistently produces a product with the expected N-glycan occupancy and Fc-effector functions (antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC)) using suitably qualified assay(s).’

Experimental verification
The analytical procedures proposed for the monograph are validated in accordance with accepted scientific practice and recommendations on analytical validation (see Ph. Eur. General Notices), with validation data provided in the manufacturer data package submitted to the EDQM to support the monograph elaboration. Yet, their robustness, transferability and suitability for inclusion in a pharmacopoeial standard had to be confirmed. Importantly, the information provided by the manufacturer on robustness verification allowed a set of key parameters to be defined and potential technical issues to be anticipated. The experimental verification is also a step where the Ph. Eur. reference standard (CRS and BRP) strategy is consolidated.

Although the analytical procedures were validated by the manufacturer and robustness checks performed, experience has shown that transferring the method from the manufacturer’s QC laboratory to an external laboratory is challenging work. To this end, close collaboration and regular exchanges with the manufacturer are essential to ensure that the information needed for monograph elaboration is complete, that technical questions are responded to promptly and that practical solutions addressing technical issues are identified. The analytical procedures to be included in the infliximab monograph were subject to rigorous and extensive experimental verification. This provided a sound scientific basis for data evaluation, based on inter-laboratory comparison, as well as important knowledge for a better understanding of the information to be included in a public standard.

Cell-based biological assays (or bioassays) are considered to be among the most complex analytical procedures, with a challenging method transfer [9]. The experimental verification of the infliximab bioassay was a complex, resource-intensive and rigorous undertaking, and – as such – a project in its own right. It involved careful evaluation of critical parameters and assessment of sources of variations, e.g. dilution series and specific reagents – TNF-α, cells from specific batches, colorimetric assay kits from various commercial sources. All of these helped define a suitable set of criteria for the monograph and ensured that all elements contributing to the bioassay performance are sound and controlled.

Reference standards
The infliximab monograph specifies the use of Infliximab CRS [6] and Infliximab BRP [8]. The Infliximab CRS was established to serve qualitative purposes, namely system suitability testing and peak identification. The CRS is required for the following tests described in the monograph: glycan analysis (Production section); charged variants by IEF (Production section); charged variants by liquid chromatography (Production section); peptide mapping (Identification B); related proteins by capillary electrophoresis under both reducing and non-reducing conditions (Tests section); impurities with molecular masses differing from that of infliximab by SEC (Tests section).

Development of the World Health Organization (WHO) 1st International Standard for Infliximab was initiated while the Ph. Eur. was working on the elaboration of the infliximab monograph. This laid the foundations for EDQM-WHO joint efforts to assure compatibility of strategies for the development of infliximab standards. As a result, a collaborative study was organised jointly by WHO and the EDQM for the simultaneous establishment of the 1st WHO International Standard for Infliximab and of the 1st Ph. Eur. Infliximab BRP [8, 10]. Importantly, participation of manufacturers in the collaborative study provided an opportunity to assess the suitability of the infliximab International Standard (and Infliximab BRP) as a reference standard for infliximab products produced using different manufacturing processes and contributed to the lessons learned from the study, showing that complex molecules and complicated assays can be standardised.

Infliximab BRP is intended for use in the potency assay described in the monograph, as an assay performance indicator (system suitability), and for the estimation of relative potency. Infliximab BRP batch 1 consists of freeze-dried infliximab and has an assigned potency of 500 IU of TNF-α neutralising activity per ampoule, the same as the corresponding 1st International Standard.

As a general note, the establishment of the CRS/BRP relies solely on the provision of the necessary starting material(s) by manufacturer(s). Therefore, securing the supply by ensuring the commitment of companies at an early stage (preferably at the monograph initiation phase), is a major prerequisite for driving monograph development. In addition, an initial reflection on a CRS/BRP strategy must be undertaken early enough and adapted at a later stage, i.e. based on the outcome of method verification. Consideration should be given to the quantity of material to be donated by the manufacturer that may represent a significant amount of a manufacturing batch, as well as to the sustainability of the CRS/BRP supply throughout the lifetime of the monograph.

Stakeholder participation

The achievements against the envisaged roadmap for setting public standards for mAbs are the result of a dynamic and transparent collaborative process, based on the complex interaction of all parties involved. Firstly, the Ph. Eur. Group of Experts responsible for developing these standards is itself a multi-stakeholder platform and forum for discussion. It includes equal representations from innovator and biosimilar companies, from OMCLs and National Control Laboratories (NCLs), and from regulatory authorities; this reflects the positive stakeholder engagement and helps ensure effective communication of both regulatory and industry expectations, based on partnership and common understanding. This group has experienced dynamic growth through an increased and diversified membership, echoing the interest and willingness of stakeholders to participate actively in the work of the Ph. Eur. in this important field.

The collaboration between the EDQM and infliximab producers during the elaboration and implementation of the infliximab monograph was key to the positive outcome of the infliximab case study. Engagement of all stakeholders in the work of the Ph. Eur. as early as possible is crucial – key stakeholders got involved in monograph elaboration, responded positively to the call for participation in monograph development, provided technical support and carried out testing, in order to help set the public standard, see Figure 3. With the recognition of infliximab as a rapidly evolving target, the need to update the monograph to reflect newly approved products is to be addressed. Infliximab manufacturers offered knowledge and experience that contributed further to ensuring alignment between the Ph. Eur. monograph and the EU-approved specifications for the two biosimilars granted marketing authorisation during monograph elaboration.

Perspectives emerging from the infliximab case study; horizontal standard development

With the finalisation of the infliximab monograph, the Ph. Eur. embarked on the elaboration of an individual monograph for another multisource mAb from the same class and thus drove forward its work on TNF-α inhibitors. To this end, the elaboration of a monograph for adalimumab (a fully human antibody that targets the same TNF-α antigen as infliximab), recently added to the Ph. Eur. work programme, is a natural continuation of the work of the Ph. Eur. Commission on TNF-α antagonists, to pursue the reflection initiated with the infliximab case study and confirm the approach by addressing new product specificities. Furthermore, working on new candidate molecules will further shape the concept and enrich the understanding of analytical procedures that could serve as platform methodologies for mAb analysis.

The infliximab case study provided an important proof of concept and facilitated a better understanding of how to tackle subclasses of mAbs in a transversal manner. The Ph. Eur. has taken steps towards exploring flexible concepts of standardisation by developing new ‘horizontal standards’ or ‘performance-based standards’. These efforts are aimed at establishing general requirements/methodologies to reflect a suitable set of expectations that can be applied across various quality attributes common to (classes/subclasses of) mAbs, with one of the main focuses being the development of anti-TNF-α product-based standards. A new general chapter on Cell-based assay for potency determination of TNF-α antagonists (2.7.26) – the first of three planned new ‘horizontal standards’ for mAbs – has been just published in the Ph. Eur. [11]. This chapter will contribute to standardisation of the anti-TNF-α class of antibodies through rationalisation of methodologies and common functionalities, and thus drive forward the horizontal approach. The work in this field has also been leveraging the knowledge and experience gained from single source biotherapeutic monographs elaborated or under elaboration through a dedicated mechanism established at the Ph. Eur. [4, 12]: the Ph. Eur. published its first individual monograph for a TNF-α inhibitor, Etanercept (2895) [13], in January 2017, and is progressing with the elaboration of a new single source monograph for another TNF-α inhibitor, Golimumab concentrated solution (3103) [14].

Conclusion and future steps

The recent developments in the field of public standards for biotherapeutics have demonstrated that elaboration of Ph. Eur. monographs on complex biotherapeutics is not only possible but also very useful [15, 16]. These monographs recognise the process-specific character of such products and focus on flexible concepts of standardisation that enable innovation by manufacturers. The successful outcome of the infliximab case study demonstrated that it is feasible to establish a monograph for a highly complex, multisource mAb. The monograph is designed to be suitable for all products authorised in Ph. Eur. Member States.

The Ph. Eur. is advancing in the field of setting public standards for mAbs and considers expanding its portfolio of quality standards for this category of products by targeting product classes as well as specific drug substances, as new opportunities occur as a result of collaboration with key stakeholders. The Ph. Eur. also aims at extending the focus of horizontal standard development beyond specific product classes and functional assays, and to enlarge the analytical package with physicochemical procedures with broad application and impact.

These developments are another positive example of the advantages of close collaboration across the scientific community, bringing together Ph. Eur. experts, regulators and manufacturers, and benefiting from invaluable support contributed to the experimental work by OMCLs and NCLs.

Acknowledgements

EDQM is grateful to all the manufacturers who contributed to the elaboration of the infliximab monograph by providing data and material, and to the participants in the experimental verification study and in the collaborative studies for CRS/BRP establishment. We would like to thank the MAB Working Party of the European Pharmacopoeia for their outstanding work on monograph elaboration and for their continued engagement and enhanced support throughout the stages of the infliximab project. Finally, our thanks go to Emmanuelle Charton, Marie-Emmanuelle Behr-Gross, Sylvie Jorajuria, Louise Birrell and Sara Chauvin for their review of the manuscript and helpful discussions.

Competing interests: None.

Provenance and peer review:  Not commissioned; externally peer reviewed.

References
1. Council of Europe. European Directorate for the Quality of Medicines & HealthCare. General notices. General chapter 1. Ph. Eur. 11^th Edition [home­page on the Internet]. [cited 2022 Sep 29]. Available from: https://www.edqm.eu/en/-/new-general-chapter-on-implementation-of-pharmacopoeial-procedures-published-in-the-11th-edition-of-the-european-pharmacopoeia
2. Council of Europe. European Directorate for the Quality of Medicines & HealthCare. Guide for the work of the European Pharmacopoeia [homepage on the Internet]. [cited 2022 Sep 29]. Available from: https://www.edqm.eu/documents/52006/69195/03+Guide+for+the+work+of+the+European+Pharmacopoeia.pdf/bfd74013-47c2-eb7e-adb8-4d12c72e4ef6?t=1650374246462
3. Infliximab concentrated solution, monograph 2928. Ph. Eur. Supplement 10.3 Strasbourg, France: Council of Europe 2021. See also Knowledge Database available from: https://extranet.edqm.eu/publications/recherches_sw.shtml
4. Buda M, Wicks S, Charton E. Elaborating European Pharmacopoeia monographs for biotherapeutic proteins using substances from a single source. Pharmeur Bio Sci Notes. 2016;2016:129-34.
5. Council of Europe. European Directorate for the Quality of Medicines & HealthCare. Technical guide for the elaboration of monographs on synthetic peptides and recombinant DNA proteins (2018) [homepage on the Internet]. [cited 2022 Sep 29]. Available from: https://www.edqm.eu/documents/52006/66555/04-peptides-en.pdf/ccfbbb84-8d20-b5ac-ce43-b0b5becd8c64?t=1623150654812
6. Council of Europe. European Directorate for the Quality of Medicines & HealthCare. Ph. Eur. Infliximab chemical reference substance [homepage on the Internet]. [cited 2022 Sep 29]. Available from: https://crs.edqm.eu/db/4DCGI/View=Y0002047
7. European Medicines Agency. EMA/CHMP/589317/2013. Remsima. Assessment report. 27 June 2013 [homepage on the Internet]. [cited 2022 Sep 29]. Available from: https://www.ema.europa.eu/en/documents/assessment-report/remsima-epar-public-assessment-report_en-0.pdf
8. Wadhwa M, Rigsby P, Behr-Gross M-E. Collaborative study for the establishment of infliximab biological reference preparation batch 1. Pharmeur Bio Sci Notes. 2020;2020:49-52.
9. White JR, Abodeely M, Ahmed S, Debauve G, Johnson E, Meyer D M, et al. Best practices in bioassay development to support registration of biopharmaceuticals. Biotechniques. 2019;67(3);126-37.
10. Metcalfe C, Dougall T, Bird C, Rigsby P, Behr-Gross M-E, Wadhwa M, et al. The first World Health Organization International Standard for infliximab products: a step towards maintaining harmonized biological activity. MAbs. 2019;11(1):13-25.
11. Council of Europe. European Directorate for the Quality of Medicines & HealthCare. Cell-based assays for potency determination of TNF-alpha antagonists, general chapter 2.7.26. Ph. Eur. Supplement 11.1.
12. Council of Europe. European Directorate for the Quality of Medicines & HealthCare. Procedure 4 (P4). Everything you always wanted to know [homepage on the Internet]. [cited 2022 Sep 29]. Available from: https://www.edqm.eu/documents/52006/0/P4procedureAllyouNeedtoKnow2.pdf/85d459b5-fadb-8c98-acb3-b3a22a8c7349?t=1640092701402
13. Council of Europe. European Directorate for the Quality of Medicines & HealthCare. Etanercept, monograph 2895. Ph. Eur. Supplement 11.1.
14. Golimumab concentrated solution, monograph 3103. Pharmeuropa. 2021;33(3).
15. Charton E. The role of European Pharmacopoeia monographs in setting quality standards for biotherapeutic products. Generics and Biosimilars Initiative Journal (GaBI Journal). 2016;5(4):174-9. doi:10.5639/gabij.2016.0504.045.
16. Buda M, Kolaj-Robin O, Charton E. Biotherapeutic products in the European Pharmacopoeia: have all challenges been tackled? Generics and Biosimilars Initiative Journal (GaBI Journal). 2022;11(1):7-12. doi:10.5639/gabij.2022.1101.003

Disclosure of Conflict of Interest Statement is available upon request.

Copyright © 2022 Pro Pharma Communications International

Permission granted to reproduce for personal and non-commercial use only. All other reproduction, copy or reprinting of all or part of any ‘Content’ found on this website is strictly prohibited without the prior consent of the publisher. Contact the publisher to obtain permission before redistributing.