Perspective
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MS in the analysis of biosimilars
Biologic drugs are forming a larger and expanded part of the therapeutic drug market. The top ten best-selling drugs are currently a mix of small and large molecules, but it is expected that biologics will soon represent a large majority of the top-selling drugs. These drugs have a high degree of complexity and must be analyzed using information-rich analytical techniques to fully characterize the drug. Thus, biosimilar copies of these innovator drugs must also be intensively analyzed to ensure they have comparable analytical profiles. In this article we discuss the regulatory requirements for introducing a follow-on biologic, or biosimilar, drug on the market, how analytics in general can be used to reduce the need for comprehensive clinical trials, and how MS in particular is becoming increasingly valuable in these analyses.
Biosimilars or follow-on biologics (FOBs) are often described as the nonoriginator version of officially approved biotherapeutics, and can conceptually be thought of as highly similar to the innovator therapeutic. The demand for biosimilars is expected to grow rapidly in both the developed and the developing world, and the increased market potential has caused many traditionally originator companies such Merck [1] , AstraZeneca [2,3] Pfizer [4,5] and Boehringer Ingelheim [6] , among others, to make forays into the biosimilar development space [7] . Biotherapeutics fall in the realm of large and complex molecules in that their characteristics extend beyond the nominal molecular structure into specific arrangements and post-translational modifications (PTMs), unlike small molecules. Biotherapeutics and their nonbranded versions are complex biopolymers that are composed of modifications to their primary sequence, and these modifications include chemical modification to amino acids and also linkages that are relevant to their folding and in general to their tertiary structure. Therefore, for a biosimilar to be officially approved not only would be required to demonstrate that the primary sequence is identical to the originator biotherapeutic; but also
10.4155/BIO.14.110 © 2014 Future Science Ltd
Chris A Singleton Callen Consulting, 63 Creeley Road, Belmont, MA 02478, USA [email protected]
the type and degree of the modifications and finally the secondary and tertiary structure are also highly similar. In general, primary sequence variations are not acceptable, but some variation in PTMs and glycosylation can be tolerated. However, because they two entities are not exactly the same, some clinical trials may be required. According to the EMA, biosimilars are entities that are “developed to be similar to an existing biological medicine (the ‘reference medicine’).” Biosimilars are not the same as generics, which have simpler chemical structures and are considered to be identical to their reference medicines’ [8] . In the USA, biosimilarity is defined to mean that “the biological product is highly similar to the reference product notwithstanding minor differences in clinically inactive components,” and that “there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product” [9] . Because of the inherent complexity of biotherapeutics, identical copies have not yet been generated, even between different lots of the originator product [10] . Furthermore, originators often make manufacturing changes over the product’s life cycle. The comparability of the
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Key terms Biosimilar: Copy of an innovator biologic drug that is highly similar to the innovator drug, with acceptable differences in minor and clinically nonrelevant components. In the USA it is defined as “biological products that are demonstrated to be ‘biosimilar’ to or ‘interchangeable’ with an US FDA-licensed biological product”. New Drug Application: Vehicle through which drug sponsors formally propose that the FDA approves a new pharmaceutical for sale and marketing in the USA. Biologics Licensing Application: Request for permission to introduce, or deliver for introduction, a biologic product into interstate commerce (21 CFR 601.2).
prechange and postchange product is demonstrated by analytical studies without further clinical trials, although, in some cases, nonclinical or clinical studies are needed to demonstrate comparability (ICH Q5E) [11] . Comprehensive characterization should be performed to compare both the reference originator product and the purported biosimilar product as to establish that these two entities are comparable in terms of safety and efficacy, such as in the case of filgrastim [12] . The in-depth analysis allows the innovator product and biosimilar to be compared so as to discern any differences, which could have potential clinical relevance. In this perspective, we stress the need for cutting-edge analytical technologies in the development and implementation of information intensive techniques to fully characterize (to the furthest ability of current technology) the originator and biosimilar. The authors realize that as technology advances, more extensive characterization will become available that was not accessible when the original comparison was done. Therefore we define ‘fully characterize’ to mean using all current mass spec techniques that can be used to evaluate primary, secondary and tertiary protein structure, as well as an evaluation of drug product and excipients. MS is ideally situated to provide the depth of knowledge needed to compare and evaluate the similarity of a putative biosimilar and the innovator drug [13] , as comparative case studies have shown [14] . As regulations are becoming increasingly harmonized globally, we will discuss regulatory issues in general terms and mention regulatory bodies specifically only when necessary for the purposes of differentiation. Biosimilars are meant to act as substitutes for an originator biologic drug once patent and regulatory protection has expired. Because of the level of heterogeneity of biologic drugs they are many times more complex than small molecule drugs; small differences in glycosylation for example may alter receptor binding. A small molecule drug may require content uniformity, water content, contaminants and excipient tests, among other tests, to show that it is analytically
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equivalent to the originator small molecule drug. In contrast a biologic drug has many more potential variations, such as PTMs, folding differences in the tertiary structure that may lead into aggregation, and glycosylation patterns. Because of this complexity, an exact match to every parameter will be difficult to achieve when it comes to the development of biosimilars [15] . Since biosimilar molecules are a more recent innovation than generic small molecules, there is less guidance on the development and characterization for biologics. The EMA approved biosimilar drugs without a specific framework in place, with the EMA deciding on what nonclinical trials are necessary on a ‘case-by-case’ basis [16] . While the US FDA has not formally approved a biosimilar, it has approved versions of biologic drugs such as insulin under a different framework, and plans to evaluate biosimilars using a ‘totality of the evidence’-based approach [17] to approval. The authors realize the difficulty in trying to promulgate guidelines for such a diverse and varied type of medicine. However this approach, while necessary in the short term as guidelines are being developed, can be harmful in the long term. Both originator and generic manufacturers suffer from a lack of clear guidance as to what does and does not constitute biosimilarity and interchangeabililty. Interchangeability allows pharmacists to substitute a nonbranded drug for a branded drug. A major biosimilar manufacturer has gone on to state that as biosimiliars are designed to “...behave the same as reference products,” one cannot assume they are dissimilar or inferior, and biosimilars in general should not be held to ‘a higher standard of interchangeabililty’ [18] . If biosimilar products cannot be designated as biologically interchangeable, biosimilar manufacturers would have to develop an independent sales force to market the FOB [19] . This would increase the overall cost and negate some of the financial benefits of a noninnovator alternative. One issue in particular is what degree of analytical characterization is necessary to suggest bioequivalence and what degree of similarity this implies, which could impact the number and size of clinical studies required. In its draft guidance on scientific considerations in biosimilars, the FDA states “The stepwise approach should start with extensive structural and functional characterization of both the proposed product and the reference product, which serves as the foundation of a biosimilar development program” [20] . Analytical characterization is the sine qua non of proving analytical similarity, as quality, safety, efficacy and immunogenicity cannot be considered similar if the analytical data do not demonstrate that the biosimilar product is highly similar to the reference product to warrant consideration. In addition others have suggested
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going beyond typical analytical comparability when determining similarity [21] . Current opinion ranges widely, from declaring that there is no need for clinical studies [22] to believing that is an absolute requirement both legally and for ensuring the safety of the patient [23] . Some regulatory agencies require clinical trials, but will waive the requirement if the manufacturer of the biosimilar product can prove close similarity to the reference product [24] . For a typical small molecule generic drug in the USA, the company submitting the Abbreviated New Drug Application (ANDA) is allowed to reference results from the original New Drug Application (NDA) filed by the originator. Most protein biologic drugs fall under a different federal act that small molecules do, and are treated differently in terms of approval applications. This is more of an artifact of different types of drugs (biologic vs small molecule) falling under different regulatory systems in the USA, a difference that is not always recognized by regulatory agencies in other countries [25] . Currently, biosimilar applications cannot reference clinical results from originator applications, leading some to state ‘Nothing authorizes access to trade secrets of originator products to biosimilar companies [18] . However, if biosimilar manufacturers are required to perform the extensive clinical trials required of an originator product the cost of the biosimilar would be increase substantially. In that case biosimilar manufacturers would be encouraged to file a new Biologics Licensing Application (BLA) and promote a drug as novel or a biobetter, as opposed to a biosimilar. Such an approach does not hold the potential for lowering the cost of biologic drugs that are off-patent, as there will be less of an incentive to promote biosimilars. Regardless of the particular regulatory pathway that must be followed, it is clear that information-rich analytical data are becoming more of a necessity, either to obviate the need for clinical trials or to strengthen the case made from a clinical trial that the two drugs are close enough to demonstrate similar efficacy, safety and immunogenicity. Opinion varies widely within the scientific community as to the necessary extent of data. While we agree with some authors when they state, “Scientists are of the opinion that the use of biosimilars is an opportunity for us to use cutting-edge technology to solve health problems and guide clinical processes,” we disagree when they go on to state that “…the analytical tests currently available are not sophisticated enough to detect the slight but important structural differences between originator and biosimilar products” [26] . Indeed, the FDA has stated that a direct comparison of products was possible in certain cases because of “...improvements in the availability and sophistication of analytical techniques” [27] .
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Furthermore, in some cases such as the recently released guidelines for biosimilar development in India, comprehensive analytical data may reduce the need for some in vivo studies, stating that “Based on demonstration of similarity in the comparative assessment, a similar biologic may require reduced preclinical and clinical data package as part of submission for market authorization” [28] . The FDA has also stated the same in the draft guidance on scientific considerations for evaluating biosimilars, stating that when demonstrating biosimilarity, the agency has the discretion to determine if any analytical, animal or clinical studies are unnecessary [20] . We anticipate this trend to continue, especially in the interim as regulatory agencies look at biosimilar regulatory submission on a case-by-case basis and where strong analytical data are crucial to proving the similarity of two products and render some in vivo studies unnecessary. Other researchers have noted that there have been significant differences in the glycosylation patterns and amino acid sequence between a candidate biosimilar and the innovator product when analyzed using high-resolution LC–MS [29] . However, it must be noted that the putative biosimilar in question was not yet approved, and the authors specify that the primary sequence must be the same for a biosimilar to be approved in Europe and the USA [30] . Exactly how close the biosimilar must be to receive that designation is not well defined at this time. Although drug complexity will increase over time, so will the ability of analytical techniques to analyze and characterize these complex formulations. Hence there is an increasing need for more complex and information rich analysis, which will only become more necessary as biologic drugs become more complex. In that vein we discuss the necessity of advanced analytics in general with a focus on MS in particular for the analysis of biosimilars. The interested reader is encouraged to study other excellent reviews on the topic of MS for therapeutic antibodies [31] and biosimilar analysis and comparability [13] . Techniques for characterization Chemical characterization of molecular entities has traditionally included techniques such as HPLC, size exclusion, fluorescence spectroscopy, circular dichroism, Fourier-transform infrared spectroscopy, NMR and MS. In the recent years with the fast advancement in the instrumentation of mass spectrometers, this technique has been widely accepted and adopted to the extent of being considered among the front row techniques for the characterization of these complex biomolecules. These instruments can aid in the characterization of the primary sequence and its PTMs as well as inferences of the tertiary structure through indirect measurements.
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Key term Ion mobility MS: Type of mass analysis where an analyte can be separated according to its size, shape, charge and conformation. Ions are separated due to differing mobility in an inert carrier gas.
Most instruments have the capability of measuring changes in concentration that range between 100- and 1000-fold and the sensitivity for the new instruments usually borders the high attomole, low femtomole for small molecules. Sensitivity though is inversely proportional to the molecular weight of the analyte. The analysis of large molecules is usually conducted in the tens of picomoles to obtain data that can be deconvoluted and analyzed. The advent of new Orbitrap instruments promises the delivery of even isotopic distribution of large molecules of the size of antibodies based on improvements of the architecture of the analyzer as well as the electronics [32] . Commercial instruments that include ion mobility add an extra dimension for the analysis of small and large molecules. The basis of the ion mobility is that molecules will be able to traverse an ion mobility chamber which is filled with an inert gas. The molecules will separate and resolve at a rate proportional to the charge and cross-sectional area given by its shape and size. Based on this property structural isomers, isobars and conformers that have identical elemental composition but different structural arrangement can be separated in the gas phase [33] . Traveling-wave ion mobility MS has been applied for the analysis of intact proteins or complexed proteins [34] , as well as aggregates [26] . Figure 1 shows the application of ion mobility in analyzing a biotherapeutic proteins, with the native MS illustrating the detection of dimeric monoclonal antibody. Although the analysis of intact proteins is still in its infancy it has a lot of potential to offer with regards to the relationship between tertiary structure, cross-sectional area and charge state in the gas phase. Care should be taken, however, as researchers have determined that solution phase structure is not always transferred to the gas phase, and so ion mobility may not represent the structure of the protein in solution [35] . Protein sequencing and identification of PTMs are the most classical use of mass spectrometers for biological analytes. Figure 2 shows a mirror plot of the LC–MS run from a tryptic digest of trastuzumab compared with a potential biosimilar. There is an additional peak of m/z 1872.97 Da in the biosimilar chromatogram that is not present in the innovator, and a chromatographic peak near 32.2 min of m/z 1904.94 Da that is present in the innovator but not the putative biosimilar. The authors of that work found that the
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discrepancy correlated with a difference in the published sequence versus one found in DrugBank. Thus the combination of precursor mass identification with the fragmentation information acquired from MSMS scans can yield a valuable and confident identification of the peptide sequencing. Traditional quadrupole time-of-flight (QqTOF) instruments and newly developed hybrid instruments that can deliver high mass resolution and mass accuracy have increased the confidence in the assignment. Addition of different types of fragmentation to the general collision induced dissociation added to the repertoire of useful techniques for elucidating not only the identity but also the position of modifications that are highly labile or dissociation energies that are distant from the backbone of peptides. The analysis of intact and reduced antibodies is another critical area of biosimilar analysis. Top-down analysis and intact characterization are relatively fast analysis [36] and so are amenable to the faster throughput needs of comparative analysis between innovator and biosimilar molecules, or between different reactor runs of biologic drug manufacture. Intact analysis is a useful tool in degradation studies as well, monitoring changes such as pyroglutamate formation or changes in glycosylation patterns [37] . Reduction and alkylation or papain digestion break down the antibody into constituent parts, enabling more granular analysis of the biologic drug in question. The characterization of the glycans on the antibody are also of critical importance in ensuring that the biosimilar candidate is indeed similar to the innovator material, and research has shown that there are several different ways to use LC–MS to study glycosylation of therapeutic proteins [38,39] . While intact and reduced antibody analysis can provide information about the nature of the glycans pendant on the drug, releasing the glyans either enzymatically or chemically, labeling with a fluorescent tag (e.g., anthranilic acid, 2-amino benzamide [40] or procainamide) and then analyzing by LC–MS with an inline fluorescence detector provides much more abundant information about the glycoprofile and its qualitative and quantitative properties [41] . The large number of potential glycans means that MS is especially suited to their identification [42] and can aid in quantitation when numerous peaks co-elute in LC applications. MALDI is also a powerful ionization technique for analyzing glycans [43] and is commonly used in glycan analysis. Sialic acids are present on the terminus of many glycans and are an example of a how glycan composition can have an effect on immunogenicity [44] . These sialic acids can be profiled by chemically releasing them and labeling with 1,2-diamino-4,5-methylenedioxybenzene (DMB), although this analysis is usually done
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Drift time (milli/s) Figure 1. MS and ion mobility–MS analysis of intact trastuzumab. (A & C) Intact MS analysis of trastuzumab. ESI–TOF mass spectra of trastuzumab in (A) denaturing or (C) native conditions. The inserts shows an extended view of the (A) 44+ and (C) 25+ charge states with resolution of the different glycoforms: (a) 147 917.1 ± 1.1 Da (G0/G0F), (b) 148 061.7 ± 0.8 Da (G0F/G0F); (c) 148 222.4 ± 0.9 Da (G0F/G1F); (d) 148 383.8 ± 0.8 Da (G1F/G1F); and (e) 148 544.3 ± 1.0 Da (G1F/G2F). (B & D) ion mobility–MS analysis of trastuzumab. Ion mobility mobilograms of trastuzumab in (B) denaturing or (D) native conditions. Ion mobility–MS data obtained in native conditions (D) reveal small amounts of dimeric mAb. Reproduced with permission from [14] © American Chemical Society (2013).
with fluorescence detection on LC and analysis by LC–MS is not strictly necessary for routine analysis. Sialic acids have also been analyzed in biological matrices by LC–MS [45–47] and MALDI [48] , and while typical comparability studies would not need to be done in biofluids, the additional selectivity and information of the mass spectrometer can be a significant advantage. Structural analysis can be performed as well [49] , for structure elucidation of less common sialic acids that are not in a typical reference panel. Fucosylation is another glycan phenomenon that can affect antibody binding and cytotoxicity [50] . Some hypofucosylated versions of IgGs have been shown to mediate enhanced killing of tumor cells in whole blood assays [51] , and while this may normally be desirable it could still constitute a significant difference between an innovator and a biosimilar. In recent years cell lines
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have been engineered to produce different amounts of fucose, galactose, bisecting GlcNAc and sialic acid to alter the cytotoxicity of the antibody [52] , so measuring the levels and types of glycans in the innovator material is crucial in order to match efficacy and immunogenicity. Intact antibody analysis can be performed after treatment with an endoglycosidase, which cleaves the glycan between the N-acetyl glucosamine (GlcNAc) residues of the core N-linked glycan structure, leaving the fucose-attached GlcNac pendant on the antibody. Host cell protein (HCP) analysis is performed to analyze the residual protein left over from the purification process. For example, therapeutic proteins produced in host cells can co-purify with residual proteins native to the host cell, which may cause immunogenicity issues and are thus required to be measured by regulatory agencies [53] . HCP assays may use different types
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Figure 2. LC–MS plot of a tryptic digest of trastuzumab versus a potential biosimilar. The mirror plots show differences between the innovator drug and the putative biosimilar, with significant differences near 32.2 and 34.9 min. (A) LC–MS chromatograms; (B) am expanded view of the charge-reduced, isotope-deconvoluted LC–MS chromatograms from 32.0–35.0 min. Reproduced with permission from [29] .
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of mass spectrometers for qualitative and quantitative analysis [54] , but modern instruments are often capable of performing both functions [55,56] . MS has the ability to be nonspecific and so can detect HCPs in a more holistic manner than an immunospecific method [57] , although affinity enrichment can increase detection levels [58] . Application of the hydrogen–deuterium exchange (HDX) technique to MS has provided the ability to provide a rapid insight into the tertiary structure conformation of proteins and conjugated proteins. HDX can aid in elucidation of the structure and dynamics of proteins on the basis of the rate of exchange of amide protons by their deuterium isotope [59] . This technique was successfully coupled to MS in the early 1990s by Katta and Chait [60] and since then it has been applied to numerous structural studies [61] , conformational changes [62] and physiological processes [63] . Furthermore, it was successfully used as part of a comparison study between rituximab and a candidate biosimilar [64] and has shown promise in biopharmaceutical comparability studies [65] . Although some technical details may need to be resolved beforehand, HDX is a powerful technique that provides a rapid and sensitive way of addressing structural conformations. Hydroxyl radical footprinting is another technique that provides valuable structural information of proteins. The technique is based on the mapping of the surface of solvent-exposed regions of proteins using radical reagents such as hydrogen peroxide. The hydroxyl radicals introduce fast, somewhat nonspecific and covalent labels on the protein backbone. As long as the secondary reaction of the unfolding of the protein upon oxidation is controlled, the labeling of the surface exposed residues is likely to provide insight into the structural conformation of the native protein [66] . It has been shown that higher order structure of therapeutic monoclonal antibodies can be characterized by this approach [67] . Research has demonstrated that this technique can be successfully applied to comparative analysis of biologic drugs [68] and so would be useful in the evaluation of biosimilars. Top-down proteomics is a MS technique that complements the primary sequence information from the bottom-up approach, since the fragmentation of the intact molecule is carried out in the gas phase. Upon selection of the proper charge state and protein isoform, it provides not only primary sequence information but also PTMs associated with the selected isoform. Traditionally for this kind of analysis, high resolution instruments such as Fourier transform-ion cyclotron resonance mass spectrometers were utilized due to their resolving power and the ability to include electron capture dissociation for efficient fragmentation of
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Key terms Hydrogen–deuterium exchange: LC technique in which protons on a protein are exchanged with deuterium. More accessible protons exchange faster and more readily, and conformation can be elucidated in this manner. Typically paired with MS. Hydroxyl radical footprinting: Using radical reagents to map the solvent-exposed region of a protein, by incorporating covalent labels on the backbone of the protein.
large molecules. The new development in high resolution OrbitrapTM Fourier transform MS instruments that have the electron transfer dissociation (ETD) capabilities allow the analysis of large molecules such as antibodies [69] . ETD is also used for characterization of the disulfide linkages in proteins [70] and antibodies [71] . However, biopharmaceutical companies using cutting edge MS ought to be aware that the robustness of the measurements using MS instruments is highly dependent on the ability to replicate the measurement of the standards that define the system suitability and performance of the instrument [72] . On the software end, MS manufacturers are mostly focusing on software that are amenable for peptide analysis, but still significant advances are needed for the automated analysis of intact molecules, complex posttranslational modifiers such as glycans. Several inroads into MS analysis of glycans have been made, such as SysBioWare [73] , Glykoextractor [74] and SimGlycanTM [75] , and we hope this trend toward effective and automated analysis continues. For intact mass analysis there are only a few reliable algorithms for mass deconvolution, and the next steps should be to reliably automate the deconvolution process for large sample numbers and provide effective data visualization. With the large and complex data sets that are routinely being analyzed today, we feel that software will be as important a factor in instrument and vendor choice as traditional factors such as sensitivity and resolution. For all complex mass spectrometric analyses, variability becomes a critical issue. The issue of instrument type is highly relevant since not only does each instrument manufacturer have minor differences in their geometry, source design, and so on. there are also vast differences in some analyzer designs (e.g., orbital trap vs TOF) and large molecules can behave very differently in these different instruments. Furthermore the software used for processing may not be consistent either, for example deconvolution software used for reconstructing the intact neutral mass of a protein may have different algorithms from different manufacturers. The best solution for this is the use of a consistent innovator reference standard with each analysis (or pooled stan-
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Perspective Singleton dard to give an ‘average’ set of values), so that it can be seen if changes in response are due to true changes or instrument variability. However, with so many parameters to measure, ensuring the data are comparable between innovator and biosimilar is not just a question of the raw numbers but also one of instrument capability. Suppose the biosimilar manufacturer may use an instrument that was not as sensitive as the latest generation, and thus the two entities may appear to be highly similar on a superficial level, but in reality may be different in ways that are beyond the capabilities of that mass spectrometer to measure. In conclusion, we acknowledge the role that analytics plays in biosimilar development in general, but would like to highlight the increasing importance that information-rich MS plays. In the view of the author, the most important tool will be the development of software that provides a rapid quantitative comparison between samples, while reducing the back-end data processing needed to extract meaningful information. Critically, such software must not only allow for the fast comparison and analysis but also allow the user to easily modify the relevant attributes of the processing, as the author has had experience with software that was too automated and did not allow for the flexibility needed in research and development. While we have discussed many new hardware tools to aid in the analysis, utilizing these requires software that can rapidly assess the differ-
ences among analytes. The advent of new tools and processing for MS ensures that it will become increasingly indispensable in future biosimilar development. Future perspective As biologic drugs become a larger part of our ability to combat diseases, biosimilars will become an important tool for ensuring long-term affordability and patient access to medicines. Biologic drugs and their manufacture are complex and require analytical techniques that can exhaustively characterize them. MS in general and specific MS techniques in particular have the ability to perform this characterization and deliver the quality and quantity of information necessary to ensure analytical equivalence. We see this trend continuing in the future and MS gaining even more importance in the field, as current MS technologies are refined and novel techniques are developed. Financial & competing interests disclosure The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Executive summary • Biologic drugs are incredibly complex molecules whose full characterization requires information-rich analytical techniques. • Biosimilar drugs are not exact copies of innovator biologic drugs but are highly similar and have no clinically significant differences. • MS is uniquely positioned to provide a wealth of high-quality data when evaluating and comparing biosimilar therapeutics to innovator biologic drugs. • Various specialized techniques in MS, such as hydrogen–deuterium exchange, ion mobility and hydroxyl radical footprinting provide information that is difficult for other non-MS techniques to obtain in terms of speed, information and sensitivity of analysis.
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Pfizer starts biosimilar rituximab Phase I/II trial (2012). www.biosimilarnews.com/pfizer-starts-biosimilar-rituximabphase-iii-trial
Papers of special note have been highlighted as: • of interest; •• of considerable interest
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Engen JR. Analysis of protein conformation and dynamics by hydrogen/deuterium exchange MS. Anal. Chem. 81(19), 7870–7875 (2009).
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Houde D, Peng Y, Berkowitz SA, Engen JR et al. Post-translational modifications differentially affect IgG1 conformation and receptor binding. Mol. Cell. Proteomics 9(8), 1716–1728 (2010).
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Lodowski DT, Palczewski K, Miyagi M. Conformational changes in the G protein-coupled receptor rhodopsin revealed by histidine hydrogen–deuterium exchange. Biochemistry 49(44), 9425–9427 (2010).
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MS in the analysis of biosimilars
Biologic drugs are forming a larger and expanded part of the therapeutic drug market. The top ten best-selling drugs are currently a mix of small and large molecules, but it is expected that biologics will soon represent a large majority of the top-selling drugs. These drugs have a high degree of complexity and must be analyzed using information-rich analytical techniques to fully characterize the drug. Thus, biosimilar copies of these innovator drugs must also be intensively analyzed to ensure they have comparable analytical profiles. In this article we discuss the regulatory requirements for introducing a follow-on biologic, or biosimilar, drug on the market, how analytics in general can be used to reduce the need for comprehensive clinical trials, and how MS in particular is becoming increasingly valuable in these analyses.
Biosimilars or follow-on biologics (FOBs) are often described as the nonoriginator version of officially approved biotherapeutics, and can conceptually be thought of as highly similar to the innovator therapeutic. The demand for biosimilars is expected to grow rapidly in both the developed and the developing world, and the increased market potential has caused many traditionally originator companies such Merck [1] , AstraZeneca [2,3] Pfizer [4,5] and Boehringer Ingelheim [6] , among others, to make forays into the biosimilar development space [7] . Biotherapeutics fall in the realm of large and complex molecules in that their characteristics extend beyond the nominal molecular structure into specific arrangements and post-translational modifications (PTMs), unlike small molecules. Biotherapeutics and their nonbranded versions are complex biopolymers that are composed of modifications to their primary sequence, and these modifications include chemical modification to amino acids and also linkages that are relevant to their folding and in general to their tertiary structure. Therefore, for a biosimilar to be officially approved not only would be required to demonstrate that the primary sequence is identical to the originator biotherapeutic; but also
10.4155/BIO.14.110 © 2014 Future Science Ltd
Chris A Singleton Callen Consulting, 63 Creeley Road, Belmont, MA 02478, USA [email protected]
the type and degree of the modifications and finally the secondary and tertiary structure are also highly similar. In general, primary sequence variations are not acceptable, but some variation in PTMs and glycosylation can be tolerated. However, because they two entities are not exactly the same, some clinical trials may be required. According to the EMA, biosimilars are entities that are “developed to be similar to an existing biological medicine (the ‘reference medicine’).” Biosimilars are not the same as generics, which have simpler chemical structures and are considered to be identical to their reference medicines’ [8] . In the USA, biosimilarity is defined to mean that “the biological product is highly similar to the reference product notwithstanding minor differences in clinically inactive components,” and that “there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product” [9] . Because of the inherent complexity of biotherapeutics, identical copies have not yet been generated, even between different lots of the originator product [10] . Furthermore, originators often make manufacturing changes over the product’s life cycle. The comparability of the
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Key terms Biosimilar: Copy of an innovator biologic drug that is highly similar to the innovator drug, with acceptable differences in minor and clinically nonrelevant components. In the USA it is defined as “biological products that are demonstrated to be ‘biosimilar’ to or ‘interchangeable’ with an US FDA-licensed biological product”. New Drug Application: Vehicle through which drug sponsors formally propose that the FDA approves a new pharmaceutical for sale and marketing in the USA. Biologics Licensing Application: Request for permission to introduce, or deliver for introduction, a biologic product into interstate commerce (21 CFR 601.2).
prechange and postchange product is demonstrated by analytical studies without further clinical trials, although, in some cases, nonclinical or clinical studies are needed to demonstrate comparability (ICH Q5E) [11] . Comprehensive characterization should be performed to compare both the reference originator product and the purported biosimilar product as to establish that these two entities are comparable in terms of safety and efficacy, such as in the case of filgrastim [12] . The in-depth analysis allows the innovator product and biosimilar to be compared so as to discern any differences, which could have potential clinical relevance. In this perspective, we stress the need for cutting-edge analytical technologies in the development and implementation of information intensive techniques to fully characterize (to the furthest ability of current technology) the originator and biosimilar. The authors realize that as technology advances, more extensive characterization will become available that was not accessible when the original comparison was done. Therefore we define ‘fully characterize’ to mean using all current mass spec techniques that can be used to evaluate primary, secondary and tertiary protein structure, as well as an evaluation of drug product and excipients. MS is ideally situated to provide the depth of knowledge needed to compare and evaluate the similarity of a putative biosimilar and the innovator drug [13] , as comparative case studies have shown [14] . As regulations are becoming increasingly harmonized globally, we will discuss regulatory issues in general terms and mention regulatory bodies specifically only when necessary for the purposes of differentiation. Biosimilars are meant to act as substitutes for an originator biologic drug once patent and regulatory protection has expired. Because of the level of heterogeneity of biologic drugs they are many times more complex than small molecule drugs; small differences in glycosylation for example may alter receptor binding. A small molecule drug may require content uniformity, water content, contaminants and excipient tests, among other tests, to show that it is analytically
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equivalent to the originator small molecule drug. In contrast a biologic drug has many more potential variations, such as PTMs, folding differences in the tertiary structure that may lead into aggregation, and glycosylation patterns. Because of this complexity, an exact match to every parameter will be difficult to achieve when it comes to the development of biosimilars [15] . Since biosimilar molecules are a more recent innovation than generic small molecules, there is less guidance on the development and characterization for biologics. The EMA approved biosimilar drugs without a specific framework in place, with the EMA deciding on what nonclinical trials are necessary on a ‘case-by-case’ basis [16] . While the US FDA has not formally approved a biosimilar, it has approved versions of biologic drugs such as insulin under a different framework, and plans to evaluate biosimilars using a ‘totality of the evidence’-based approach [17] to approval. The authors realize the difficulty in trying to promulgate guidelines for such a diverse and varied type of medicine. However this approach, while necessary in the short term as guidelines are being developed, can be harmful in the long term. Both originator and generic manufacturers suffer from a lack of clear guidance as to what does and does not constitute biosimilarity and interchangeabililty. Interchangeability allows pharmacists to substitute a nonbranded drug for a branded drug. A major biosimilar manufacturer has gone on to state that as biosimiliars are designed to “...behave the same as reference products,” one cannot assume they are dissimilar or inferior, and biosimilars in general should not be held to ‘a higher standard of interchangeabililty’ [18] . If biosimilar products cannot be designated as biologically interchangeable, biosimilar manufacturers would have to develop an independent sales force to market the FOB [19] . This would increase the overall cost and negate some of the financial benefits of a noninnovator alternative. One issue in particular is what degree of analytical characterization is necessary to suggest bioequivalence and what degree of similarity this implies, which could impact the number and size of clinical studies required. In its draft guidance on scientific considerations in biosimilars, the FDA states “The stepwise approach should start with extensive structural and functional characterization of both the proposed product and the reference product, which serves as the foundation of a biosimilar development program” [20] . Analytical characterization is the sine qua non of proving analytical similarity, as quality, safety, efficacy and immunogenicity cannot be considered similar if the analytical data do not demonstrate that the biosimilar product is highly similar to the reference product to warrant consideration. In addition others have suggested
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going beyond typical analytical comparability when determining similarity [21] . Current opinion ranges widely, from declaring that there is no need for clinical studies [22] to believing that is an absolute requirement both legally and for ensuring the safety of the patient [23] . Some regulatory agencies require clinical trials, but will waive the requirement if the manufacturer of the biosimilar product can prove close similarity to the reference product [24] . For a typical small molecule generic drug in the USA, the company submitting the Abbreviated New Drug Application (ANDA) is allowed to reference results from the original New Drug Application (NDA) filed by the originator. Most protein biologic drugs fall under a different federal act that small molecules do, and are treated differently in terms of approval applications. This is more of an artifact of different types of drugs (biologic vs small molecule) falling under different regulatory systems in the USA, a difference that is not always recognized by regulatory agencies in other countries [25] . Currently, biosimilar applications cannot reference clinical results from originator applications, leading some to state ‘Nothing authorizes access to trade secrets of originator products to biosimilar companies [18] . However, if biosimilar manufacturers are required to perform the extensive clinical trials required of an originator product the cost of the biosimilar would be increase substantially. In that case biosimilar manufacturers would be encouraged to file a new Biologics Licensing Application (BLA) and promote a drug as novel or a biobetter, as opposed to a biosimilar. Such an approach does not hold the potential for lowering the cost of biologic drugs that are off-patent, as there will be less of an incentive to promote biosimilars. Regardless of the particular regulatory pathway that must be followed, it is clear that information-rich analytical data are becoming more of a necessity, either to obviate the need for clinical trials or to strengthen the case made from a clinical trial that the two drugs are close enough to demonstrate similar efficacy, safety and immunogenicity. Opinion varies widely within the scientific community as to the necessary extent of data. While we agree with some authors when they state, “Scientists are of the opinion that the use of biosimilars is an opportunity for us to use cutting-edge technology to solve health problems and guide clinical processes,” we disagree when they go on to state that “…the analytical tests currently available are not sophisticated enough to detect the slight but important structural differences between originator and biosimilar products” [26] . Indeed, the FDA has stated that a direct comparison of products was possible in certain cases because of “...improvements in the availability and sophistication of analytical techniques” [27] .
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Furthermore, in some cases such as the recently released guidelines for biosimilar development in India, comprehensive analytical data may reduce the need for some in vivo studies, stating that “Based on demonstration of similarity in the comparative assessment, a similar biologic may require reduced preclinical and clinical data package as part of submission for market authorization” [28] . The FDA has also stated the same in the draft guidance on scientific considerations for evaluating biosimilars, stating that when demonstrating biosimilarity, the agency has the discretion to determine if any analytical, animal or clinical studies are unnecessary [20] . We anticipate this trend to continue, especially in the interim as regulatory agencies look at biosimilar regulatory submission on a case-by-case basis and where strong analytical data are crucial to proving the similarity of two products and render some in vivo studies unnecessary. Other researchers have noted that there have been significant differences in the glycosylation patterns and amino acid sequence between a candidate biosimilar and the innovator product when analyzed using high-resolution LC–MS [29] . However, it must be noted that the putative biosimilar in question was not yet approved, and the authors specify that the primary sequence must be the same for a biosimilar to be approved in Europe and the USA [30] . Exactly how close the biosimilar must be to receive that designation is not well defined at this time. Although drug complexity will increase over time, so will the ability of analytical techniques to analyze and characterize these complex formulations. Hence there is an increasing need for more complex and information rich analysis, which will only become more necessary as biologic drugs become more complex. In that vein we discuss the necessity of advanced analytics in general with a focus on MS in particular for the analysis of biosimilars. The interested reader is encouraged to study other excellent reviews on the topic of MS for therapeutic antibodies [31] and biosimilar analysis and comparability [13] . Techniques for characterization Chemical characterization of molecular entities has traditionally included techniques such as HPLC, size exclusion, fluorescence spectroscopy, circular dichroism, Fourier-transform infrared spectroscopy, NMR and MS. In the recent years with the fast advancement in the instrumentation of mass spectrometers, this technique has been widely accepted and adopted to the extent of being considered among the front row techniques for the characterization of these complex biomolecules. These instruments can aid in the characterization of the primary sequence and its PTMs as well as inferences of the tertiary structure through indirect measurements.
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Key term Ion mobility MS: Type of mass analysis where an analyte can be separated according to its size, shape, charge and conformation. Ions are separated due to differing mobility in an inert carrier gas.
Most instruments have the capability of measuring changes in concentration that range between 100- and 1000-fold and the sensitivity for the new instruments usually borders the high attomole, low femtomole for small molecules. Sensitivity though is inversely proportional to the molecular weight of the analyte. The analysis of large molecules is usually conducted in the tens of picomoles to obtain data that can be deconvoluted and analyzed. The advent of new Orbitrap instruments promises the delivery of even isotopic distribution of large molecules of the size of antibodies based on improvements of the architecture of the analyzer as well as the electronics [32] . Commercial instruments that include ion mobility add an extra dimension for the analysis of small and large molecules. The basis of the ion mobility is that molecules will be able to traverse an ion mobility chamber which is filled with an inert gas. The molecules will separate and resolve at a rate proportional to the charge and cross-sectional area given by its shape and size. Based on this property structural isomers, isobars and conformers that have identical elemental composition but different structural arrangement can be separated in the gas phase [33] . Traveling-wave ion mobility MS has been applied for the analysis of intact proteins or complexed proteins [34] , as well as aggregates [26] . Figure 1 shows the application of ion mobility in analyzing a biotherapeutic proteins, with the native MS illustrating the detection of dimeric monoclonal antibody. Although the analysis of intact proteins is still in its infancy it has a lot of potential to offer with regards to the relationship between tertiary structure, cross-sectional area and charge state in the gas phase. Care should be taken, however, as researchers have determined that solution phase structure is not always transferred to the gas phase, and so ion mobility may not represent the structure of the protein in solution [35] . Protein sequencing and identification of PTMs are the most classical use of mass spectrometers for biological analytes. Figure 2 shows a mirror plot of the LC–MS run from a tryptic digest of trastuzumab compared with a potential biosimilar. There is an additional peak of m/z 1872.97 Da in the biosimilar chromatogram that is not present in the innovator, and a chromatographic peak near 32.2 min of m/z 1904.94 Da that is present in the innovator but not the putative biosimilar. The authors of that work found that the
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discrepancy correlated with a difference in the published sequence versus one found in DrugBank. Thus the combination of precursor mass identification with the fragmentation information acquired from MSMS scans can yield a valuable and confident identification of the peptide sequencing. Traditional quadrupole time-of-flight (QqTOF) instruments and newly developed hybrid instruments that can deliver high mass resolution and mass accuracy have increased the confidence in the assignment. Addition of different types of fragmentation to the general collision induced dissociation added to the repertoire of useful techniques for elucidating not only the identity but also the position of modifications that are highly labile or dissociation energies that are distant from the backbone of peptides. The analysis of intact and reduced antibodies is another critical area of biosimilar analysis. Top-down analysis and intact characterization are relatively fast analysis [36] and so are amenable to the faster throughput needs of comparative analysis between innovator and biosimilar molecules, or between different reactor runs of biologic drug manufacture. Intact analysis is a useful tool in degradation studies as well, monitoring changes such as pyroglutamate formation or changes in glycosylation patterns [37] . Reduction and alkylation or papain digestion break down the antibody into constituent parts, enabling more granular analysis of the biologic drug in question. The characterization of the glycans on the antibody are also of critical importance in ensuring that the biosimilar candidate is indeed similar to the innovator material, and research has shown that there are several different ways to use LC–MS to study glycosylation of therapeutic proteins [38,39] . While intact and reduced antibody analysis can provide information about the nature of the glycans pendant on the drug, releasing the glyans either enzymatically or chemically, labeling with a fluorescent tag (e.g., anthranilic acid, 2-amino benzamide [40] or procainamide) and then analyzing by LC–MS with an inline fluorescence detector provides much more abundant information about the glycoprofile and its qualitative and quantitative properties [41] . The large number of potential glycans means that MS is especially suited to their identification [42] and can aid in quantitation when numerous peaks co-elute in LC applications. MALDI is also a powerful ionization technique for analyzing glycans [43] and is commonly used in glycan analysis. Sialic acids are present on the terminus of many glycans and are an example of a how glycan composition can have an effect on immunogenicity [44] . These sialic acids can be profiled by chemically releasing them and labeling with 1,2-diamino-4,5-methylenedioxybenzene (DMB), although this analysis is usually done
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Drift time (milli/s) Figure 1. MS and ion mobility–MS analysis of intact trastuzumab. (A & C) Intact MS analysis of trastuzumab. ESI–TOF mass spectra of trastuzumab in (A) denaturing or (C) native conditions. The inserts shows an extended view of the (A) 44+ and (C) 25+ charge states with resolution of the different glycoforms: (a) 147 917.1 ± 1.1 Da (G0/G0F), (b) 148 061.7 ± 0.8 Da (G0F/G0F); (c) 148 222.4 ± 0.9 Da (G0F/G1F); (d) 148 383.8 ± 0.8 Da (G1F/G1F); and (e) 148 544.3 ± 1.0 Da (G1F/G2F). (B & D) ion mobility–MS analysis of trastuzumab. Ion mobility mobilograms of trastuzumab in (B) denaturing or (D) native conditions. Ion mobility–MS data obtained in native conditions (D) reveal small amounts of dimeric mAb. Reproduced with permission from [14] © American Chemical Society (2013).
with fluorescence detection on LC and analysis by LC–MS is not strictly necessary for routine analysis. Sialic acids have also been analyzed in biological matrices by LC–MS [45–47] and MALDI [48] , and while typical comparability studies would not need to be done in biofluids, the additional selectivity and information of the mass spectrometer can be a significant advantage. Structural analysis can be performed as well [49] , for structure elucidation of less common sialic acids that are not in a typical reference panel. Fucosylation is another glycan phenomenon that can affect antibody binding and cytotoxicity [50] . Some hypofucosylated versions of IgGs have been shown to mediate enhanced killing of tumor cells in whole blood assays [51] , and while this may normally be desirable it could still constitute a significant difference between an innovator and a biosimilar. In recent years cell lines
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have been engineered to produce different amounts of fucose, galactose, bisecting GlcNAc and sialic acid to alter the cytotoxicity of the antibody [52] , so measuring the levels and types of glycans in the innovator material is crucial in order to match efficacy and immunogenicity. Intact antibody analysis can be performed after treatment with an endoglycosidase, which cleaves the glycan between the N-acetyl glucosamine (GlcNAc) residues of the core N-linked glycan structure, leaving the fucose-attached GlcNac pendant on the antibody. Host cell protein (HCP) analysis is performed to analyze the residual protein left over from the purification process. For example, therapeutic proteins produced in host cells can co-purify with residual proteins native to the host cell, which may cause immunogenicity issues and are thus required to be measured by regulatory agencies [53] . HCP assays may use different types
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HT42 660.35 Da
Innovator HT10 1310.65 Da
HT3 1089.55 Da
Deamidated LT3* 1991.98 Da
Deamidated HT10* 1311.64 Da 0
1311.65 Da Deamidated HT10*
1991.98 Da 660.35 Da Deamidated LT3* HT42
1089.55 Da HT3
50
1310.66 Da HT10 Biosimilar
100 32.0
33.0
1872.97 Da (EPQVYTLPPSRDELTK) HT34-35 34.0 35.0
Retention time (min)
Figure 2. LC–MS plot of a tryptic digest of trastuzumab versus a potential biosimilar. The mirror plots show differences between the innovator drug and the putative biosimilar, with significant differences near 32.2 and 34.9 min. (A) LC–MS chromatograms; (B) am expanded view of the charge-reduced, isotope-deconvoluted LC–MS chromatograms from 32.0–35.0 min. Reproduced with permission from [29] .
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of mass spectrometers for qualitative and quantitative analysis [54] , but modern instruments are often capable of performing both functions [55,56] . MS has the ability to be nonspecific and so can detect HCPs in a more holistic manner than an immunospecific method [57] , although affinity enrichment can increase detection levels [58] . Application of the hydrogen–deuterium exchange (HDX) technique to MS has provided the ability to provide a rapid insight into the tertiary structure conformation of proteins and conjugated proteins. HDX can aid in elucidation of the structure and dynamics of proteins on the basis of the rate of exchange of amide protons by their deuterium isotope [59] . This technique was successfully coupled to MS in the early 1990s by Katta and Chait [60] and since then it has been applied to numerous structural studies [61] , conformational changes [62] and physiological processes [63] . Furthermore, it was successfully used as part of a comparison study between rituximab and a candidate biosimilar [64] and has shown promise in biopharmaceutical comparability studies [65] . Although some technical details may need to be resolved beforehand, HDX is a powerful technique that provides a rapid and sensitive way of addressing structural conformations. Hydroxyl radical footprinting is another technique that provides valuable structural information of proteins. The technique is based on the mapping of the surface of solvent-exposed regions of proteins using radical reagents such as hydrogen peroxide. The hydroxyl radicals introduce fast, somewhat nonspecific and covalent labels on the protein backbone. As long as the secondary reaction of the unfolding of the protein upon oxidation is controlled, the labeling of the surface exposed residues is likely to provide insight into the structural conformation of the native protein [66] . It has been shown that higher order structure of therapeutic monoclonal antibodies can be characterized by this approach [67] . Research has demonstrated that this technique can be successfully applied to comparative analysis of biologic drugs [68] and so would be useful in the evaluation of biosimilars. Top-down proteomics is a MS technique that complements the primary sequence information from the bottom-up approach, since the fragmentation of the intact molecule is carried out in the gas phase. Upon selection of the proper charge state and protein isoform, it provides not only primary sequence information but also PTMs associated with the selected isoform. Traditionally for this kind of analysis, high resolution instruments such as Fourier transform-ion cyclotron resonance mass spectrometers were utilized due to their resolving power and the ability to include electron capture dissociation for efficient fragmentation of
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Perspective
Key terms Hydrogen–deuterium exchange: LC technique in which protons on a protein are exchanged with deuterium. More accessible protons exchange faster and more readily, and conformation can be elucidated in this manner. Typically paired with MS. Hydroxyl radical footprinting: Using radical reagents to map the solvent-exposed region of a protein, by incorporating covalent labels on the backbone of the protein.
large molecules. The new development in high resolution OrbitrapTM Fourier transform MS instruments that have the electron transfer dissociation (ETD) capabilities allow the analysis of large molecules such as antibodies [69] . ETD is also used for characterization of the disulfide linkages in proteins [70] and antibodies [71] . However, biopharmaceutical companies using cutting edge MS ought to be aware that the robustness of the measurements using MS instruments is highly dependent on the ability to replicate the measurement of the standards that define the system suitability and performance of the instrument [72] . On the software end, MS manufacturers are mostly focusing on software that are amenable for peptide analysis, but still significant advances are needed for the automated analysis of intact molecules, complex posttranslational modifiers such as glycans. Several inroads into MS analysis of glycans have been made, such as SysBioWare [73] , Glykoextractor [74] and SimGlycanTM [75] , and we hope this trend toward effective and automated analysis continues. For intact mass analysis there are only a few reliable algorithms for mass deconvolution, and the next steps should be to reliably automate the deconvolution process for large sample numbers and provide effective data visualization. With the large and complex data sets that are routinely being analyzed today, we feel that software will be as important a factor in instrument and vendor choice as traditional factors such as sensitivity and resolution. For all complex mass spectrometric analyses, variability becomes a critical issue. The issue of instrument type is highly relevant since not only does each instrument manufacturer have minor differences in their geometry, source design, and so on. there are also vast differences in some analyzer designs (e.g., orbital trap vs TOF) and large molecules can behave very differently in these different instruments. Furthermore the software used for processing may not be consistent either, for example deconvolution software used for reconstructing the intact neutral mass of a protein may have different algorithms from different manufacturers. The best solution for this is the use of a consistent innovator reference standard with each analysis (or pooled stan-
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Perspective Singleton dard to give an ‘average’ set of values), so that it can be seen if changes in response are due to true changes or instrument variability. However, with so many parameters to measure, ensuring the data are comparable between innovator and biosimilar is not just a question of the raw numbers but also one of instrument capability. Suppose the biosimilar manufacturer may use an instrument that was not as sensitive as the latest generation, and thus the two entities may appear to be highly similar on a superficial level, but in reality may be different in ways that are beyond the capabilities of that mass spectrometer to measure. In conclusion, we acknowledge the role that analytics plays in biosimilar development in general, but would like to highlight the increasing importance that information-rich MS plays. In the view of the author, the most important tool will be the development of software that provides a rapid quantitative comparison between samples, while reducing the back-end data processing needed to extract meaningful information. Critically, such software must not only allow for the fast comparison and analysis but also allow the user to easily modify the relevant attributes of the processing, as the author has had experience with software that was too automated and did not allow for the flexibility needed in research and development. While we have discussed many new hardware tools to aid in the analysis, utilizing these requires software that can rapidly assess the differ-
ences among analytes. The advent of new tools and processing for MS ensures that it will become increasingly indispensable in future biosimilar development. Future perspective As biologic drugs become a larger part of our ability to combat diseases, biosimilars will become an important tool for ensuring long-term affordability and patient access to medicines. Biologic drugs and their manufacture are complex and require analytical techniques that can exhaustively characterize them. MS in general and specific MS techniques in particular have the ability to perform this characterization and deliver the quality and quantity of information necessary to ensure analytical equivalence. We see this trend continuing in the future and MS gaining even more importance in the field, as current MS technologies are refined and novel techniques are developed. Financial & competing interests disclosure The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
Executive summary • Biologic drugs are incredibly complex molecules whose full characterization requires information-rich analytical techniques. • Biosimilar drugs are not exact copies of innovator biologic drugs but are highly similar and have no clinically significant differences. • MS is uniquely positioned to provide a wealth of high-quality data when evaluating and comparing biosimilar therapeutics to innovator biologic drugs. • Various specialized techniques in MS, such as hydrogen–deuterium exchange, ion mobility and hydroxyl radical footprinting provide information that is difficult for other non-MS techniques to obtain in terms of speed, information and sensitivity of analysis.
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