Biological products are stratified as diverse and complex molecules, unlike conventional small molecule drugs. This intrinsic heterogeneity poses a challenge to characterize biological medicines, underlining the need for a reference standard. A reference standard, simply translates as a formally approved biologic molecule whose biological and therapeutic effects are well established.
Manufacturers use reference standards to provide solutions for different purposes such as discovery, regulatory compliance, and process efficiency.
Different and unique purposes of reference standards vary from (a) assigning a potency value to a biologic drug, (b) confirming identity of a drug substance, (c) determining the performance of quality control (QC) assays or suitability tests, (d) testing the presence of specific product and process related impurities, (e) determining molecular weight distribution and weight- average molecular weight, and (f) assessing qualification and validation of analytical and production methods. The comparator approved types of reference standards can be sourced commercially, if available, or generated in-house.
Among biologics, biotherapeutic monoclonal antibodies (mAbs) have had a major impact on healthcare, especially in the fields of oncology and inflammation. Most of the approved therapeutic mAbs are of the IgG-class antibody, which is the common type of antibody seen in blood. In comparability exercise, the assessment of the biological properties of a mAb is of utmost importance and to achieve this, manufacturers rely on a reference standard, most often an in-house reference standard, throughout the product’s lifespan. Bioengineered mAbs which do not have naturally occurring similars are made available commercially in mass units, although their biological activity is exclusively defined on the basis of in-house proprietary reference standards.
Because of the rising number of mAb drug candidates and drugs in the market, the World Health Organization(WHO) has realized the requirement for global standardization of mAbs, to assure their safety, quality and efficacy. WHO in collaboration with regulatory authorities, manufacturers and other stakeholders, are developing WHO international standards (IS) for mAbs, that can be used for quality control and calibration of potency bioassays, as well as in-house reference standards. Furthermore, by defining the international unit (IU) of bioactivity, WHO IS for mAbs aid the assessment and management of mAb biologics utilized by different manufacturers and jurisdictions.
Reference standards at different stages of therapeutic antibody development
- Manufacturing of mAb drugs is a tightly controlled process that requires a high level of standardization. A quality by design (QbD) approach is often adopted, where analytical assays are used to determine the quality attributes of the product; and accordingly, the formulation, upstream and downstream processes are designed to minimize deviations in quality. These critical quality attributes (CQAs) are the physical, chemical and biological properties of a mAb that define the clinical safety and efficacy. Hence, using a well-characterized reference standard is necessary to calibrate the potency and product consistency throughout its lifecycle.
- Reference standards can be created or used in various ways depending on the requirement. For technical development, separate reference materials can be used to test biological assays, physicochemical properties or product-related impurities. In this scenario, each reference standard will be specific for a certain quality attribute of the product. For example, a commercially sourced IgG mAb can be used to calibrate the Fc binding assays and Fab domain NMR structural fingerprinting. However, in general, a single reference standard is preferred throughout product development.
- In the case of the development of an innovative drug molecule, an international or national standard is not likely to be available. Here, the reference standard will be a qualified in-house reference mAb of the highest possible purity, that has been tested extensively for specificity and potency suitable for the intended use. If an international standard is available, it can be directly used as the reference or it can be used to calibrate the in-house reference standard. It is also important to store the reference drug in appropriate conditions and to periodically test lot-to-lot variability to ensure the integrity of the molecule throughout the development cycle.
- In-house reference standards evolve with the progression of drug development phases based on the results of in-vitro, in-vivo and clinical studies. However, before entering phase 3 trials, the standard that is to be used for further development and commercialization needs to be finalized. Early on, in the product development stage, an appropriately characterized lot of the mAb, termed as in-house interim reference standard, is reserved for quality control (QC) purposes. The interim standard serves as the reference for early technical development through Good Laboratory Practice-Toxicology (GLP-Tox) in order to release batches for early clinical studies. At this stage, tentative process parameters are defined and the drug formulation for Phase 1 clinical trials is confirmed. Moreover, the interim standard is used in analytical assays to characterize product properties such as primary sequence, post-translational modifications, charge and size isoforms, and potency.
Reference standards are used to evaluate various quality attributes of the drug substance and also qualify and validate the analytical methods
- After clinical trial phases 1 and 2, there can be significant modifications in formulations and upstream-downstream processes. The final approved mAb, that is representative of the commercial product, is further quality tested for its physical, chemical and biological characteristics. This batch is produced in sufficient quantity and is denoted as the in-house primary reference standard. At this point, the standard operating procedures for the manufacture of primary reference should be in place. The primary reference standard can also be obtained from a commercial source if available, known as the official reference standard. ICH Q6B recommends a two-tiered approach in reference standards where a subset of the primary reference is used as the secondary reference or the working standard. The working standard is thereafter used as the reference material for further qualitative and quantitative assays during production of clinical and commercial batches of the drug. Every new batch of the working standard is calibrated against the primary standard to ensure consistency of quality.
- Along with the evolution of reference standards, the analytical assays used in each phase of drug development also co-evolve. The reference standard is used to qualify and validate the analytical methods for their intended purposes such as screening, structural characterization, binding studies or immunogenicity tests. Reference standards further help determine the critical process parameters (CPP) for bio-pharmaceutical development.
- Overall, reference standards play a critical role in the control strategy during product development and commercial manufacturing. They ensure continuity of product quality, stability and comparability throughout its lifecycle. Thus, implementation of a reference standard program can assist successful licensure of an investigational new drug (IND) application and biologics license application (BLA) and aid entry into clinical development.
Reference standards in the development of therapeutic antibodies
Public reference standards for therapeutic antibodies
Recombinant mAbs are one of the fastest growing class of biotherapeutics and therefore WHO emphasizes on the need for establishing public or official reference standards for biopharmaceutical development. These reference materials are intended for the calibration of product potency and biological activity of in-house reference standards as well as national reference standards.
Owing to the complex nature and process-specific features of mAb drugs, it is implausible to develop a single international reference standard that can cover all the attributes of a product. Therefore, multiple attribute-specific reference standards are often required for analysis and process development.
Institutes such as NIST (US), NIBSC (UK) and regional pharmacopeial agencies are government recognized providers of metrological reference standards.
NISTmAb, an IgG1k mAb, is one of the first fully characterized reference standard that was developed recently for determining physicochemical and biophysical attributes of monoclonal antibodies.
This has been recognized by WHO as an international standard after conducting extensive bioactivity studies, including Fc-effector function and cell-based binding bioassays, in the context of Rituximab biosimilar development. Further collaborative efforts between drug manufacturers, instrument manufacturers, regulatory authorities and standards organizations are necessary to develop new metrological reference standards that can supplement best industry practices to ensure product quality of mAb drugs.
Difference between Reference listed drugs (RLDs) and Reference Standard?
The FDA industry guidance draft “Referencing Approved Drugs Products in ANDA Submissions” (link here) clearly states the difference between the reference standard and reference drugs for new generic drug applications (ANDA). According to the FDA guidance “A reference standard, which is selected by FDA, is the specific drug product that the ANDA applicant must use in conducting any in vivo bioequivalence testing”, generally this reference standard is the reference listed drug.
However, for the monoclonal antibodies and other biologic drugs, there is a
- lack of ready availability of reference standards
- the high price associated with per mg of the reference standard, while several mgs are required for an analytical experiment
Therefore, it’s very difficult to obtain the desired reference standards for methods and assay development. In such a scenario the closest alternate are the approved reference drugs available in the market.
The approved monoclonal antibody drugs or other RLDs can serve as an alternative to reference standards.
Like reference standards, reference drugs have been thoroughly characterized and this information can serve as a valuable tool in assisting analytical scientists. Therefore, you can benchmark your methods (qualifying and validating) with the help of reference drugs.
Evidentic GmbH sources mAb drug products, that are already characterized for their physico-chemical and biological properties. Hence, they can serve as attribute-specific reference standards for the development of new drugs or biosimilars.
- Schiel JE et al. Monoclonal Antibody Therapeutics: The Need for Biopharmaceutical Reference Materials. ACS Symposium Series. 2014;1176:1-34. doi: 10.1021/bk-2014-1176.ch001
- Prior S, Hufton SE, Fox B, et al. International standards for monoclonal antibodies to support pre- and post-marketing product consistency: Evaluation of a candidate international standard for the bioactivities of rituximab. MAbs. 2018;10(1):129-142. doi:10.1080/19420862.2017.1386824
- Mire-Sluis A, Ritter N, et al. Reference Standards for Therapeutic Proteins Current Regulatory and Scientific Best Practices and Remaining Needs, Part 2. BioProcess International 2014;12(5).
- https://www.nist.gov/programs-projects/nist-monoclonal-antibody-reference-material-8671. Updated September 23, 2019
- Kennett S. Regulatory Expectations for the Use of Reference Standards during Development and Beyond. CMC Strategy Forum- Reference Standards for Therapeutic Proteins 2013;15 (7).
- Tenney R. Apples to Apples Equivalency? A Bioassay Reference Material Bridging Approach for Method Lifecycle Management. CASSS Midwest Regional Forum Nov 2019