Sales of biosimilars, or follow-on biologics, could reach $10 billion by 2015, according to The Economist. US healthcare reform legislation signed into law earlier this year created an abbreviated FDA approval pathway for biosimilars. This month, the FDA held a two-day hearing about how to implement this pathway. Also this month, the European Medicine Agency (EMEA) released guidelines for biosimilars containing monoclonal antibodies (mAbs). With these developments, as well as the pressure for lower-cost biologics, the upcoming expiration of several biologics’ patents, and the entry of several large pharmaceutical companies into the biosimilar market, biosimilar development is poised to accelerate. As with all drug development, analytical instrumentation plays a key role in the development and testing of biosimilars.

The majority of biosimilars so far has been released by Chinese and Indian firms for less regulated local markets. Since the EU’s adoption of its biosimilar approval pathway in 2005, 14 biosimilars have been approved. The EU’s pathway is based on demonstrating the biosimilar’s comparability with a reference marketed product in terms of quality, safety and efficacy. The EU guidelines require testing of physicochemical properties, biological activity and the impurity profile, as well as limited in vivo testing and Phase I and Phase III clinical testing. The amount of data required is determined on a case-by-case basis. So far, all of the biosimilars approved by the EU have been either human growth hormones, erythropoietins or granulocyte colony stimulating factors. Released last year, the World Health Organization’s biosimilar pathway guidelines specify a similar comparability exercise.

The latest EMEA guidelines, which were released November 26 for public comment, address mAbs, a more structurally and functionally complex class of biologics. MAbs currently account for 35% of the biologics market (see IBO 11/15/10). Three blockbuster mAbs will lose patent protection by 2015. In fact, the EMEA has already received request for approval advice for six mAbs biosimilars and expects to receive between two and three mAbs biosimilar applications annually, according to Reuters.

In 2009, the EMEA released guidelines for mAbs development, production, characterization and specification. The latest document addresses in vitro pharmacodynamic studies, in vivo studies and clinical studies. It states that in vivo studies are only required when necessary, but clinical trials are always required. However, clinical efficacy and safety data for one indication can be extrapolated to the reference product’s other indications.

In developing complex biosimilars, many of the same analytical challenges are faced as when developing complex biologics. “Biologics typically have complex heterogeneous molecular structures. On top of the complexity, many molecules have inherent instability,” explained Frank Moffatt, Business Development at drug developer Solvias AG. “The major shortcoming of current technology is that a wide range of different analytical tests simply cannot be avoided in order to describe the manifold features of this complex design space.”

Demonstrating comparability with the innovator product can also create additional challenges. “From an analytical perspective, the major new task is that of comparability with a reference product. This is similar to the situation where one is comparing the products of a new and an old process,” explained Dr. Moffatt. With access to only the commercialized biologic, characterization can be difficult. “This is not only technically challenging, since some formulations are almost designed to make certain analyses difficult, but also can be incredibly expensive,” he said. According to generic drug firm Sandoz, biosimilars cost an average of $100–$200 million to develop and take between seven and eight years to bring to market.

Among regulators’ concerns for mAbs biosimilar development is the characterization of post-translational modifications (PTMs), in particular, glycosylation. Subtle variations in protein structure due to PTMs can affect drug safety and efficacy. As a result, efforts to improve technologies for studying glycosylation and other changes to protein structure have received attention from regulators. In his 2009 testimony before the US Congress, Steven Kozlowski, MD, director of the FDA’s Office of Biotechnology Products, listed three properties of therapeutic proteins that “cannot be sufficiently measured at this time”: PTM, three-dimensional structure and protein aggregation.

Citing Dr. Kozlowski’s comments, at this month’s FDA hearing, Graham Jones, PhD, DSc, of Northeastern University, and Jeffrey Mazzeo, PhD, of Waters, testified about efforts at Northeastern University’s Barnett Institute, with which Waters is working, to develop robust and reliable methodology for protein measurements. Dr. Jones stated that analytical technologies can conduct the measurements listed by Dr. Kozlowski. For studying PTMs, he cited the use of LC/MS for analyzing glycosylation, as well as the use of LC-MS-NMR and micro-NMR for characterizing PTMs. Dr. Mazzeo discussed the use of amide hydrogen/deuterium exchange MS for assigning structural changes in real time. He also testified about the use of ion mobility spectrometry and MS for measuring protein aggregation.

Dr. Moffatt also highlighted the use of HPLC and MS for the development of biologics and biosimilars. “HPLC in various modes is used to examine proteins (aggregates, oxidation products), glycans and peptides (peptide mapping), and capillary electrophoresis can sometimes be highly advantageous because of the elimination of the stationary phase,” he said. “Separation in combination with MS is the dominant tool for the verification of structural features and the identification of impurities.”

Dr. Moffatt also cited NMR’s potential for biosimilar development. “Personally, I believe that NMR is a much underutilized tool principally as a result of the training of most analysts,” he stated. “Traditional chemists are much more likely to see and accept the utility of this technique that often requires no sample preparation and provides information about the whole sample, i.e., components with the option of absolute quantification.”

An area that could benefit from biosimilar development is potency testing, according to Dr. Moffatt. “A real black hole for method development may be potency assays, especially cell-based bioassays. These assays are regarded as essential in providing information that is of closest relevance to the clinic, but the high variability of these assays to some extent undermines confidence in their utility,” he told IBO. “Improved reliability therefore would be really useful. This might come from the design of the assay, including instrumentation, or in the control of experimental parameters.”

As for glycosylation, Dr. Moffatt stated, “By now there is a massive amount of experience with monoclonal antibodies, so structures can be anticipated and routine methodology applied.” However, for other types of complex biologics, glycosylation is less well understood. “For proteins in general and also when nonstandard production systems are used, we may encounter much more diverse glycan structures. In this case, plug-and-play approaches are not readily available.”

In such cases, analytical technologies are not the defining factor, according to Dr. Moffatt. “The good news is that many tools in common use are not at all esoteric, so there is no technological barrier for most analytical development laboratories. The bad news is that there is complexity; therefore, experience pays off,” he told IBO. “It is my contention, and this may be controversial, but I believe that often ‘difficulties’ arise from the current paradigm of using in-house resources, rather than turning to specialized service providers that deal with the issues on a daily basis and have built up a body of learning and standardized approaches.”

Greater standardization is also on the way in the form of standardized methodologies. “USP is currently working on developing general chapters that describe analytical approaches and critical quality attributes for some of the most common product classes of biologics, e.g., monoclonal antibodies,” said Tina S. Morris, PhD, vice president of biologics and biotechnology at the US Pharmacopeial (USP) Convention. “A USP Expert Panel will be developing General Chapter <129> ‘Therapeutic Monoclonal Antibodies—Critical Quality Attributes’ this winter.” USP recently issue Chapter <1084 >”Glycoprotein and Glycan Analysis—General Considerations.”

At the FDA hearing, Dr. Jones also discussed the need to develop proven, reliable methodologies for biosimilar characterization. In addition, he emphasized the need to broaden access to the appropriate analytical techniques and to train scientists and regulators how to use the resulting data. Dr. Moffatt noted that regulators’ needs can be expected to change with regard to analytical requirements. “In the real world, there are evolving norms or expectations on the part of the regulators concerning analytical information,” he explained. “Expectations have to be met, but do not need to be exceeded—meaning that a practical approach is needed, not one based upon everything that could in principle be measured.”

Thus, analytical approaches to characterizing biosimilars will continue to develop. “Biosimilars are a fantastic play field for the analytical scientist. Regulators want to see extensive, rigorous, thorough approaches to characterization and shelf life,” stated Dr. Moffatt. “This is exactly the type of challenge that analytical scientists warm to.” According to him, the typical volume of analytical measurements for biosimilars will not exceed that required for biologics. “Now, with the route that allows biosimilars in the USA and the growing strength of emerging markets, the resources going into biosimilar development over the next five years will increase dramatically. But until new technologies emerge, this just means more of the same. After all, comparability of biosimilars is very similar to the traditional task of assessing the impact of process changes.”

Physicochemical Characterization of mAbs

Parameter Selected Analytical Techniques

Primary structure LC-MS/MS, MS, Capillary electrophoresis (CE), SDS-PAGE

Higher order structure Circular dichroism, Differential scanning calorimetry, MS, FT-IR, NMR

Glycosylation LC/MS, CE, HPLC, Capillary gel electrophoresis