Biotherapeutics are the fastest growing drug segment today. In recent years, more than 50 biotherapeutics have been approved, and a similar number is in late stage clinical trials.
In contrast to small molecules, biotherapeutics are complex molecules with a high degree of microheterogeneity. Multiple modifications — disulfide bridges, glycosylations, oxidations, C- and N-terminal amino acid modifications —influence their biological properties and incorrect modification patterns can severely impact their safety and efficacy. Therefore, regulatory authorities demand that biotherapeutics be extensively characterized and that appropriate reference standards with safe heterogeneity patterns be described in as much detail as possible.
As first patents expire, hundreds of biosimilars and biobetters are in the drug approval pipeline. The next challenge in this development space is the validation of these “generic” versions of biotherapeutics. Recent EMA regulations allow biosimilar candidates to enter clinical phase without animal studies, provided that their properties are verified against reference standards of the original drug (read more). In theory, this is a simple mandate; but in practice, the task could not be more complicated. On one hand, biosimilars are produced in different cell lines, in different facilities and by different companies. On the other hand, characterization of biotherapeutics has relied on indirect methods that provide at best an incomplete picture of the drug. Chromatography, light scattering or spectroscopy are used to describe surface hydrophobicity and charge, apparent size and aggregation state. Edman sequencing is used to determine N-terminal amino acid sequences. And mass spectrometry (MS) methods are typically limited to so-called “bottom up” approaches that detect modifications but lose context information within the protein or between different isoforms.
In novel “top-down” MS techniques, characterization starts with the intact protein to preserve context information. Smaller entities, such as nanobodies, can be analyzed directly. Larger ones, like monoclonal antibodies, are subsequently treated with reducing agents and suitable proteases to create defined 25 kDa fragments of the light and heavy chains for a “middle-down” approach.
These new workflows for biotherapeutic characterization overcome limitations of conventional methods. Samples are first evaluated by middle-down LC-ESI-QTOF (liquid chromatography electrospray ionization quadrupole time-of-flight detection) measurements. This ultra high resolution method takes into account the effects of isotopic distribution and modelling to obtain the monoisotopic mass of the protein. At this precision level, changes in disulfide pairing or variation in deamidation of single amino acids are detected. In a second step, MALDI-ISD-TOF (matrix-assisted laser desorption/ionization with in-source decay fragmentation and time-of-flight detection) is applied to verify potential variations. This method yields simple peak patterns that are easily assigned to individual fragments. Finally, limitations of LC-ESI-QTOF in sequencing the first ten amino acids and resolving the amino acids leucine and isoleucin are overcome by adding information from MALDI-ISD MS/MS spectra (so-called T3 sequencing).
Taken together, high resolution LC-ESI-QTOF and MALDI-ISD-TOF technologies provide reliable information on molecular mass, amino acid sequence and a broad range of post-translational modifications.
In their evaluation of this integrated approach, the Suckau lab first analyzed blinded samples provided from a partner lab. They were able to determine the first de novo sequence of a 13.6 kDa nanobody with all post-translational modifications (read more). In subsequent studies (2013, 2016), the group characterized three important biotherapeutic monoclonal antibodies that were approved in the 2000s (cetuximab, panitumumab, and natalizumab). Precise molecular mass, amino acid sequence, and post translational modifications were determined for each. At a sequence verification level of nearly 100%, they discovered several disparities in two out of three available reference sequences.
Looking into the future, biosimilar validation should include a critical review of existing reference information. Novel, high precision and sensitivity, top-down MS methods surpass the power of conventional methods that were used when most biotherapeutic drugs were first characterized. State of the are mass spectroscopy technology offers new opportunities to tackle challenges in biosimilar verification and ensure efficacy and patient safety.