International Standards to Support the Consistency of Therapeutic Monoclonal Antibodies Bioactivity Over Time: Evidence from an International Collaborative Study for Rituximab

Introduction

Monoclonal antibodies (mAbs) are a highly successful class of drugs which have revolutionized the treatment of many important diseases in oncology and inflammation. Unlike chemically synthesised small molecule medicines, mAbs are complex multi-chain glycoproteins produced in living cells using sophisticated manufacturing processes.¹ These highly complex processes are tightly controlled to ensure their consistency, since small changes have the potential to alter the product characteristics with associated impact on efficacy and safety (these are known as critical process parameters (CPP) and product critical quality attributes (CQA)). A key element in ensuring product consistency is the assessment of bioactivity using bioassays, which are inherently variable and often difficult to calibrate.

The loss of market exclusivity of some of the earliest ‘blockbuster’ therapeutic mAbs like infliximab and rituximab has driven the development of ‘biosimilars’. This promises to increase price competition, lower healthcare costs and consequently improve patient access to these medicines. The specific regulatory pathway for biosimilars involves of a reduced clinical package but an emphasis on quality characterization. This consists of extended pre-clinical physicochemical and functional assessment designed to demonstrate biosimilarity.^2-5 This exhaustive comparability study with the innovator product, referred to as the reference medicinal product (RMP), uses orthogonal methods intended to define a quality product target profile, understand CQAs, and prove equivalence with the RMP. Bioassays are essential to assess bioactivity which is expressed by comparison with a reference standard.⁶ Manufacturers’ bioassay reference standards (‘in-house’ reference standards) undergo extensive characterization and play a significant role throughout the life-cycle of a mAb product, including post-approval.

Post-approval process and product changes during manufacturing are common and authorized by the regulators. Reported examples include changes affecting afucosylation levels and ADCC (Antibody dependent cellular cytotoxicity) activity of batches of innovator products Mabthera® and Herceptin® with different expiration dates.^7,8 The potential for drifting and evolution of mAb products has been acknowledged⁹ however, the situation is becoming significantly more complex as the number of authorized biosimilar products increases.¹⁰ At present robust regulatory processes rely on the use of manufacturers’ ‘in-house’ reference standards and the RMP to deliver consistently safe and efficacious mAb products. Recently, the first international standards (IS) for the bioactivity of mAbs, rituximab (NIBSC 14/210) and infliximab (NIBSC 16/170) have been established by the World Health Organization (WHO).^11,12 International collaborative studies have demonstrated that the use of a common IS preparation greatly improves global potency data harmonization. This new class of public standards can support mAb product quality and consistency overtime in an increasingly complex market place.¹⁰ In this study we explain the role of these preparations and show data from the development of the first rituximab IS to showcase the role and benefits of their use.¹⁰

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International Standards (IS) for the Biological Activity of mAbs

The World Health Organization (WHO) has recognized the need for standardization of mAbs to ensure global compliance with safety, quality and efficacy.¹³ IS for mAbs are endorsed by the WHO (Expert Committee on Biological Standards, ECBS) and established after demonstration of their fi tness for purpose using international collaborative studies. Bioassay IS for mAbs are highly stable lyophilized preparations, typically stored in sealed glass ampoules over nitrogen, that define international units (IU) of bioactivity. These preparations are used to calibrate ‘in house’ reference standards and bioassay performance during the product lifecycle facilitating potency data harmonization.¹⁴ This role is distinct from that of the RMP (Figure 1) as crucially, bioassay IS for mAbs have no regulatory function in defining biosimilarity, therapeutic dose or specificactivity (units of bioactivity per mass unit). This role lies exclusively with the RMP10 which is distinct in defining the product fingerprint or quality target product profile against which the biosimilar candidate is assessed through a comparability exercise.

The First WHO International Standard for the Biological Activities of Rituximab (NIBSC 14/210)

The benefits and role of bioassay IS for mAbs have recently been demonstrated through an international collaborative study for a rituximab preparation.¹¹ Rituximab, a chimeric IgG1 mAb, was first authorized in the United States and later in Europe as Rituxan® (1997) and Mabthera® (1998) (Genentech/Roche), respectively. It is used to treat a number of B-cell lymphoproliferative disorders, transplant rejection and autoimmunity both as mono- or combination therapies.^15,16 It binds to CD20+ cells and induces target cell death either directly or through its Fc immune effector functions which include complement dependent cytotoxicity (CDC) and antibody dependent cell-mediated cytotoxicity (ADCC).^17,18 At present six biosimilar rituximab products have been approved in Europe, the first in February 2017, Truxima® (Celltrion) followed by Rixathon® (Sandoz) in June 2017 and Riximyo®, Bitzima®, Ritemvia® and Rituzena® (http://www.ema.europa.eu) which are other available rituximab brands from Celltrion and Sandoz. Current reports indicate that more than 40 rituximab biosimilar or copy products (which are those following other regulatory pathways than those laid down by the European Medicines Agency (EMA), US Food and Drug Administration (FDA) or WHO) are at various stages of development worldwide.¹⁹

A recombinant rituximab preparation expressed in CHO cells was kindly donated to the WHO by Sandoz GmB (Austria) to develop a rituximab IS. The sample was re-formulated in human serum albumin (HSA) and lyophilized under controlled conditions to facilitate the preparation of biologically stable ampoules for long-term use.²⁰ The suitability of the preparation to serve as an IS was evaluated in an international collaborative study involving 16 laboratories from nine different countries comprising manufacturers, regulators, a Pharmacopoeia and a contract research organization. The study involved the blind testing of a coded duplicate of the candidate rituximab IS preparation and a reformulated lyophilized batch of the innovator product Mabthera® used as comparator. Each laboratory used their own qualified bioassays and ‘in-house’ reference standards where possible to report rituximab potency data. Statistical analysis of the data was performed by the National Institute for Biological Standards and Control (NIBSC). The bioassays and platforms used in the study are relevant to the mechanism of action of rituximab and reflect those used during development and manufacturing of rituximab products (Figure 2).

Statistical comparison of potency values expressed relative to the participant laboratories ‘in-house’ reference preparations used as standard, showed that the potency estimates for both the IS and the comparator were highly dispersed. There was clearly poor agreement amongst the laboratories in defining potency for the CDC, ADCC and binding activities and significant differences between their ‘in-house’ reference preparations (Figure 3, left side panels). The use of the IS preparation as reference standard (common to all laboratories) to calculate the relative potency estimates led to good agreement in the potency data (very low dispersion of GM values) amongst the participating laboratories illustrating the benefits of using a common rituximab preparation (Figure 3, right side panels).

The established IS for rituximab (NIBSC 14/210) harmonizes the reporting of rituximab bioactivities by different laboratories. This preparation has been assigned arbitrary international units of bioactivity of 1000 IU of CDC activity per ampoule, 1000 IU of ADCC activity per ampoule, 1000 IU of cell binding activity per ampoule and 1000 IU of apoptotic activity per ampoule.

To fulfill the role of an IS it is important to demonstrate stability over an extended period of time. To do this we conducted real-time stability monitoring and accelerated degradation studies which demonstrated that to date, there is no loss of CDC or ADCC activity upon storage at the recommended temperature of -20 °C. Further, accelerated thermal degradation studies predict no loss of CDC or ADCC activity when stored at -20 °C suggesting excellent long-term stability for the NIBSC 14/210 rituximab preparation. Studies over a further extended period are ongoing.

The Use of mAb IS for Bioassays to Support Consistency of mAb Products Over Time

As new biosimilars for mAbs are authorized and new markets emerge the need for public bioassay standards has become increasingly important. Furthermore, the reported drifting bioactivity of marketed mAb products and the increasing complexity of the biosimilar market place has brought this issue very much into focus. The ISs serve as an independent reference point in time to allow traceability of the bioactivity of mAbs and their biosimilars over extended product life cycles. As they have an assigned international unitage, they allow calibration and validation of ‘in-house’ reference standards and bioassays performance, a function that is independent and different from that of assessing biosimilarity (which requires the RMP as per regulatory guidelines; for the different roles of the RMP and the IS, see Figure 1). Therefore, the use of ISs for mAb provide much needed support to manufacturers during product and bioassay development, manufacturing and post-marketing surveillance (Figure 4). This will in turn support manufacturers and regulators in the identification of any changes in the relative bioactivity of these products and allow the correlation of any clinical consequences with product drifting or evolution over time.

Available ISs at the moment are rituximab (NIBSC 14/210) and infliximab (NIBSC 16/170) with trastuzumab, cetuximab, bevacizumab and adalimumab²¹ all endorsed by the WHO and currently under development at NIBSC.

Conclusion

Biotherapeutic mAbs and their corresponding biosimilars are complex and heterogenous products. This complexity is intrinsically linked to their production in living cells using sophisticated manufacturing and purification processes. Process and product changes are common and acknowledged as unavoidable due to the very nature of recombinant DNA-derived biotechnology products. This inherent structural complexity and the increasing complexity of the mAb biosimilar market place highlights the need for independent reference standards. WHO IS for the bioactivity of mAbs are uniquely placed to provide manufacturers and regulators with the additional tools required to ensure product consistency, safety and efficacy over time. This in turn will increase public confidence and global reach of this highly successful class of biologics.

Acknowledgements

We are grateful to Sandoz GmB (Austria) for donating the material for the development of rituximab IS and to the laboratories participating in the collaborative study. We thank the Standards Processing Division, Peter Rigsby and Thomas Dougall for the statistical analysis of the rituximab study data. We thank Chris Burns, Adrian Bristow, Meenu Wadhwa and Christian Schneider for their helpful discussions. This work was partially funded by the UK Department of Health’s Policy Research programme (Grant Number 044/0069).

Author Biographies

Sandra Prior, PhD joined NIBSC (UK) in 2015 working on the development of bioassay standards for therapeutic Monoclonal Antibodies (mAbs) and the investigation of structure-function in relation to the safety and efficacy of mAbs products. Member of EDQM mAb expert working party. She has over 15 years’ experience in applied immunology initially working in the field of vaccines (NIBSC, UK) and later in bioassay development and immunogenicity assessment of biotherapeutics in Lonza Biologics (Cambridge, UK).

Simon E. Hufton, PhD joined NIBSC (Potters Bar, UK) in 2009 and his interests include antibody immunotherapy and protein structure-function particularly related to the efficacy, quality and safety of biological medicines. He has over 20 years’ experience in the therapeutic monoclonal antibody field including as Director of Research at Dyax Corp (Liege, Belgium) and Wyeth (Dublin Ireland). He is a biochemist by training and has authored over 30 publications on various aspects of monoclonal antibodies.

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Author Biographies

Sandra Prior, PhD joined NIBSC (UK) in 2015 working on the development of bioassay standards for therapeutic Monoclonal Antibodies (mAbs) and the investigation of structure-function in relation to the safety and efficacy of mAbs products. Member of EDQM mAb expert working party. She has over 15 years’ experience in applied immunology initially working in the field of vaccines (NIBSC, UK) and later in bioassay development and immunogenicity assessment of biotherapeutics in Lonza Biologics (Cambridge, UK).

Simon E. Hufton, PhD joined NIBSC (Potters Bar, UK) in 2009 and his interests include antibody immunotherapy and protein structure-function particularly related to the efficacy, quality and safety of biological medicines. He has over 20 years’ experience in the therapeutic monoclonal antibody field including as Director of Research at Dyax Corp (Liege, Belgium) and Wyeth (Dublin Ireland). He is a biochemist by training and has authored over 30 publications on various aspects of monoclonal antibodies.