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Equal Measures

Unlike classic drugs, which have relatively simple molecular sizes and structures (typically up to 300Da), biologics are large molecules (up to 270,000Da) with highly complex, three-dimensional tertiary structures (1). To help put the magnitude of this difference into context, the chemical structures of aspirin, a small classic drug with a molecular weight (MW) of 180Da, and Humira®, a novel biologic with an MW of approximately 148,000Da, are presented side-by-side in Figure 1.

Although classic drugs, such as aspirin, are usually easily characterised with sensitive analytical methods, biologics are created using genetically modified cell lines. They often undergo significant post-translational modifications (such as glycosylation), which can give rise to considerable heterogeneity. Consequently, producing an exact replica of an existing biologic is an almost impossible task.

The Patent Cliff

The first patented biologics were recombinant versions of endogenous human proteins – such as insulin in the 1980s – followed by more complex products, such as monoclonal antibodies in the late 1990s (2). Since biologics were first introduced, sales have grown considerably year-on-year, and by 2011 global sales reached approximately $142 billion – equivalent to 19 per cent of the global biopharmaceutical market – with more than a third of this (37.6 per cent) attributed to the top ten biologics (3). However, the period of exclusivity for these top 10 biologics is fast approaching, with a patent cliff anticipated between now and 2019 (see Figure 2).

This patent cliff creates an enormous opportunity to develop generic versions of biologics. Advances in technology have led to higher production yields, while reducing production times and costs; the global recession has significantly increased the pressure on national governments to drive down the cost of health provision; and perhaps most significantly, the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) have both introduced regulatory pathways for the review and approval of ‘generic’ biologics (3-7). This paves the way to market what have become widely known as ‘biosimilars’.

Pharmacokinetic Analysis

Although the route to market for classic generics is well defined and has been successfully negotiated for many drugs over the years – typically a small number of studies in healthy volunteers are sufficient to prove physiochemical
and pharmacokinetic (PK) equivalence – the corresponding route to market for biosimilars is relatively new and considerably more complex. Here we focus on some key issues, which should be considered when performing PK analyses to prove biosimilarity:

Study Design

The most common designs associated with bioequivalence studies are crossover designs; however, as biologics tend to have much longer half-lives than classic drugs, a crossover approach is generally not practical as the wash-out period which would be required is often prohibitively long. Furthermore, the potential for biologics to elicit an immune response also limits the use of crossover studies; if a subject was to develop an immune response in the first period of a study, the subject’s ability to participate in the second treatment period would be compromised.

To negate these issues, it is common to use a parallel group design when conducting biosimilar studies. Only one treatment period is required for each subject, removing the need for a wash-out period, and the potential knock-on effect caused by a subject developing an immune response is limited. However, it is important to note that parallel designs are not without issues of their own: large sample sizes are required to ensure that there is sufficient statistical power to prove biosimilarity, and as treatment differences are estimated between subjects (rather than within subjects), it is important to account for covariates (such as age, weight and sex) in the statistical assessment.

Acceptance Limits
In classic bioequivalence studies, PK equivalence is demonstrated using bioequivalence limits of 0.80-1.25 the test and reference products are considered to be equivalent if the 90 per cent confidence interval (CI) for the ratio of geometric least squares means lies entirely within 0.80-1.25 (8).

There are currently no such limits defined for biosimilars; indeed, the EMA guidance on similar biological medicinal products currently states that: “The acceptance range to conclude clinical comparability with respect to any pharmacokinetic parameter should be based on clinical judgment, taking into consideration all available efficacy and safety information on the reference and test products. Hence, the criteria used in standard clinical comparability studies, initially developed for chemically derived, orally administered products, may not be appropriate and the clinical comparability limits should be defined and justified prior to conducting the study” (4). Therefore, it may be possible to justify wider acceptance limits; however, in our experience it is still very common for biosimilar studies to apply the same 0.80-1.25 criteria used in equivalence studies.

Anti-Drug Antibodies
Due to their nature (recombinant proteins, some with non-human origins) and a complex manufacturing process (often resulting in impurities), biologics have the potential to elicit an immune response. Indeed, nearly all biologics induce anti-drug antibodies (ADAs); but, the incidence differs widely among products and between individuals (9). In most instances, ADAs have no clinical significance, but high levels can interfere with the PK and pharmacodynamic properties of the drug – increasing clearance, for example, thus reducing the extent of systemic exposure and desired effect of the drug. It is therefore important to have an assay in place to test for the presence of ADAs at suitable intervals during the study, so that subjects who elicit an immune response can be identified.

As defined in a recent draft guideline issued by the EMA: “Differences that could have an advantage as regards to safety (for instance, lower levels of impurities or lower immunogenicity) should be explained, but may not preclude biosimilarity” (10). While differences in this area for a true biosimilar seem unlikely, equivalence in immune response cannot be assumed and is an integral part of the PK
assessment; indeed, care needs to be taken when dealing with subjects who develop an immune response.

One possible approach – which has been ratified by both the EMA and FDA in one of our recent studies – is to include subjects in the primary assessment of biosimilarity in a secondary analysis, limited only to those who did not elicit an immune response (in other words, excluding all ADA-positive subjects).

Although the statistician may find it tempting to include terms for ADA and ADA with treatment interaction in the primary analysis (so that estimates of difference can be constructed separately for each sub-population, and averaged over both), since an ADA is not a truly independent covariate, the overall estimate may be misinterpreted. In particular, an overall treatment difference would balance for similar levels of ADA response in each treatment, when it would be more appropriate to reflect the relative problems with immune response (and not correct for an imbalance).

One further point worth considering is whether the objective of equivalence could be relaxed to non-inferiority – to show that the area under the concentration-time curve (AUC) (test)/ AUC (reference) is greater than 0.8 – for the analysis on the whole population, and only show equivalence in the less variable ADA-negative population.

In the future, the exact nature of statistical assessments to be performed in terms of ADA activity may be dictated by regulators; however, until this topic has been investigated further and authorities clarify how best to deal with the PK analysis of ADA-positive subjects as standard, it is imperative that this issue is not overlooked.

Elimination Characteristics
Whereas bioequivalence studies are typically limited to AUC and maximum concentration (Cmax) as measures of the overall extent of systemic exposure and rate of absorption, respectively, elimination characteristics must also be considered when comparing biologics due to their long half-lives and potential to develop immune responses, which can significantly alter clearance. Indeed, current EMA guidelines state that: “The design of comparative PK studies should not necessarily mimic that of the standard ‘clinical comparability’ design (CHMP/ EWP/QWP/1401/98), since similarity in terms of absorption/bioavailability is not the only parameter of interest. In fact, differences in elimination characteristics between products e.g. clearance and elimination half-life should be explored” (4).

In this context, it is important to note that to fully characterise elimination kinetics in biosimilarity studies, the sampling schedule guidelines associated with equivalence (or clinical comparability) studies should still be applied: “The sampling schedule should also cover the plasma concentration time curve long enough to provide a reliable estimate of the extent of exposure which is achieved if AUC(0-t) covers at least 80 per cent of AUC(0-∞). At least three to four samples are needed during the terminal log-linear phase in order to reliably estimate the terminal rate constant, which is needed for a reliable estimate of AUC(0-∞)” (8). Thus, the last sampling time point may be many weeks, or even months, after dosing.

Relative Purity or Protein Content

Although there have been significant advances in the manufacture and production of biologics in recent years, the process is still considerably more complex than the chemical synthesis of classic drugs, and often results in impurities. Whereas “the assayed content of the batch used as test product should not differ [by] more than 5 per cent from that of the batch used as reference product” in bioequivalence studies, unless otherwise justified, it may not be possible to meet this criteria for biologics; therefore, the total protein content of the drug (both test and reference) needs to be considered when assessing biosimilarity (8). The importance of this issue is perhaps best illustrated by referencing a recent study performed to compare the PK of Retacrit® – a biosimilar – to that of Eprex® – an innovator biologic (11). This study highlighted differences in the amount and type of glycoforms between the biosimilar and the reference drug, which is to be anticipated given the different production processes. More notably, however, Eprex comprised more total protein (μg/mL) than Retacrit, which appeared to contribute to the potency (IU/mL) of Eprex, being 10 per cent higher than labelled. The EMA accepted that the PK of Eprex and Retacrit were comparable, based on the introduction of a correction factor to allow for the difference in protein content. Once this was taken into account, the comparison of PK parameters was well within the defined equivalence margins.


We have recently encountered an issue in a Phase 1 study whereby the FDA would not accept the use of an European Economic Area (EEA) licensed drug as a reference and, similarly, the EMA would not accept the sole use of a US-licensed reference. As the ultimate aim is to license the biosimilar in both the European Union (EU) and the US, both regulatory agencies have advised that it is necessary to include two reference drugs in the study and conduct a three-way comparison – test drug, US-licensed drug, EEA licensed drug. Should similarity be proven for all three treatments, subsequent Phase 2 and 3 studies may then be limited to just one reference drug.

This is in line with the recent EMA guideline on Similar Biological Medicinal Products (currently in draft form), which states: “With the aim of facilitating the global development of biosimilars and to avoid unnecessary repetition of clinical trials, it may be possible for an Applicant to compare the biosimilar in certain clinical studies and in vivo non-clinical studies (where needed) with a non-EEA authorised comparator (i.e. a non-EEA authorised version of the reference medicinal product) which will need to be authorised by a regulatory authority with similar scientific and regulatory standards as EMA (i.e. ICH countries). In addition, it will be the applicant’s responsibility to establish that the comparator authorised outside the EEA is representative of the reference product authorised in the EEA” (10).

It is difficult to believe that this will always be a requirement for biosimilar studies; perhaps the stance of the EMA and FDA will soften as more and more biosimilars come to market, and the difference between EU and US versions of the same drug are better characterised. However, until then, the inclusion of multiple reference drugs (at least in Phase 1 studies) may be essential.

These characteristics make the route to market for biosimilars more complex, with differences in study design between bioequivalence and biosimilarity studies summarised in Table 2.

Due to their long half-lives and the potential to elicit an immune response, biologics are not compatible with simple crossover designs; therefore, parallel study designs must be utilised. Acceptance limits for the 90 per cent CI of the ratio of PK parameters must be pre-defined and approved by regulators – although, in our experience, the traditional equivalence limits of 0.80-1.25 tend to remain favourable (perhaps due to familiarity). ADAs have the potential to affect PK characteristics, and should be measured so that their impact can be considered.

Unlike standard equivalence studies, the assessment of comparability must extend beyond absorption (Cmax) and overall exposure (AUC), with half-lives (t1/2) and clearance (CL) also investigated. Furthermore, the relative protein content of the test and reference drugs needs to be considered, with a suitable correction factor applied if necessary. In addition, region-specific versions of reference drugs may exist in some instances (for example, Humira); therefore, if the ultimate aim is to license the new biosimilar across multiple regions, it may be necessary to incorporate more than one reference drug in the study.


When faced with the prospect of proving PK ‘equivalence’ between a biosimilar and an innovator biologic, it may be natural to assume that standard bioequivalence study designs and analysis methods would be sufficient to meet the objectives. After all, such study designs have been successfully employed for many years to bring generic drugs to market. However, due to the various complexities of biologics, special consideration needs to be given to the study design (parallel versus cross-over), potential immunogenicity (ADAs), elimination characteristics (t1/2 and CL), and relative protein contents of the test and reference drugs before commencing the study. Furthermore, guidance should be sought from regulators to determine the most suitable acceptance limits to conclude clinical comparability, and to assess the potential benefits of using multiple region-specific reference drugs in the study. Only then, with these factors fully and adequately addressed, can an investigator be confident that a biosimilar trial will be a complete success.

1. Shellenkens H, When biotech proteins go off-patent, Trends Biotechnol (22): pp406-410, 2004
2. Calo-Fernandez B and Martinez- Hurtado JL, Biosimilars: company strategies to capture value from the biologics market, Pharmaceuticals (5): pp1,393-1,408, 2012
3. EvaluatePharma, World preview 2018 – embracing the patent cliff, Technical report, London, UK, 2012
4. EMA, Guideline on similar biological medicinal products containing biotechnology-derived proteins as active substance: non-clinical and clinical issues, 2006
5. FDA, Quality considerations in demonstrating biosimilarity to a reference protein product, 2012
6. FDA, Scientific considerations in demonstrating biosimilarity to a reference product, 2012
7. FDA, Biosimilars: questions and answers regarding implementation of the biologics price competition and innovation act of 2009, 2012
8. EMA, Guideline on the investigation of bioequivalence, 2010
9. Schellekens H, Bioequivalence and the immunogenicity of biopharmaceuticals, Nature Review Drug Discovery (1): pp457-462, 2002
10. EMA, Guideline on similar biological medicinal products (draft), 2013
11. Schellekens H, Assessing the bioequivalence of biosimilars, Drug Discovery Today (14): pp9-10, 2009

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About the author

Yvonne Moores joined AstraZeneca in 2000 as a Programming Group Manager, before moving to Quanticate in 2008 where she is the Head of Operations. In collaboration with Quanticate’s PK specialists, Yvonne and her team have authored several articles around industry topics related to biostatistics and programming. In particular, they have overseen the design and analysis of many studies to investigate the clinical comparability of biosimilars versus innovator biologics.
Yvonne Moores
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