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Biophysical Characterization
C H A P T E R
14 Biophysical Characterization: An Integral Part of the “Totality of the Evidence” Concept Damian J. Houde, Steven A. Berkowitz Department of Protein Pharmaceutical Development, Biogen Idec, Inc., Cambridge, MA, USA
14.1 BIOPHARMACEUTICAL DEVELOPMENT The process of developing a protein biopharmaceutical is a long, precarious, complex, and an expensive process, which unfortunately has a very low probability of success (at least at present). This is testified by the fact that the biopharmaceutical landscape is littered with unsuccessful drug products [1e4]. This outcome is not surprising if we consider the characteristics of these drugs (as pointed out in Chapters 1e3). The most notable of which includes the following: (1) the large, heterogeneous, complex, and fragile nature of these molecules, (2) the mechanism by which they are made, using living cells, which we have limited knowledge on and ability to control in detail, (3) our incomplete understanding about the interactions of these therapeutic compounds within a living system and how the living system works, and (4) the limitation in the ability of the analytical tools we currently have to characterize them [5]. As a result, at best, we can only attempt to minimize the risk of making uninformed and thus poor decisions that lead to failure by taking a holistic approach and acquire as much (useful) information as possible under the constraints of economic and financial limitations (remembering that developing biopharmaceutical is not a philanthropic endeavordit’s a business!). It is in just such an environment that the manufacturer who attempts to develop a biopharmaceutical and the regulators how will approve or reject that biopharmaceutical must operate in. Consequently, setting standards for development and approval too high can lead to miss opportunities that could have favorably impacted the lives of many. However, setting the standards for development and approval too low can on the other hand do great harm.
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14.1.1 “One-size-fit-all” versus “Case-by-case” The idea that the process of developing biopharmaceuticals follows the same exact path, where one only needs to successfully execute the same predetermined number of sequential activities to realize a successful commercial drug is misleading. Similarly, a “one-size-fit-all” approach for assessing biopharmaceuticals to determine their ability to be approved as a commercial drug is just as misleading (at least at present) [6]. The individual personalities or behavior of each disease and each biopharmaceutical, and the accessory components required for its development (formulations, container closures, raw material, cell lines, etc.) conspire to contribute a unique variability to outcomes that leads to different issues, which must be resolved for each individual biopharmaceutical. Consequently, the only rational scientific approach to deal with this situation is to evaluate each drugedisease combination on its own merit, via the all too frequent and infamous phrase “case-by-case” assessment.
14.2 AN INTRODUCTION TO THE “TOTALITY OF THE EVIDENCE” AND ITS MORE GLOBAL MEANING IN DEVELOPING BIOPHARMACEUTICALS On February 9, 2012 the US Food and Drug Administration, FDA, formalized its approach concerning how it would evaluate and approve biosimilars as generic-like copies of biopharmaceuticals that have lost or will be losing patent protection due to expiry [7ae7d]. Although certainly lacking in detail, the approach was officially captured in the simple and brief terminology, “Totality of the Evidence.” Essentially, this simple phrase encapsulated the FDA’s thoughts concerning how it would evaluate and approve biosimilars as genericlike copies of biopharmaceuticals. In principle, this phrase constitutes the FDA’s plan to migrate risk, which may exist in trying to assess any difference(s) or residual gaps in knowledge between the innovator and biosimilar in assessing the approval or rejection of biosimilar drugs. By critically evaluating the total data package of a biosimilar’s filing, the FDA hopes to determine whether a biosimilar is an adequate copy of an existing innovator biopharmaceutical. As a result, the concept of “Totality of the Evidence” has now become completely and uniquely synonymous with the area of biosimilar development and approval. In coining this phrase, one can trace its origin back to at least two articles published by the FDA, one in 2007 another in 2011. In both of these articles the phrase “Totality of the Evidence” was used with a much broader intent of encompassing all biopharmaceuticals (and pharmaceuticals) in general. In the 2011 article in The New England Journal of Medicine [6], the FDA stated the following: The FDA has traditionally relied on integrating various kinds of evidence in making regulatory decisions. Such a “totality of the evidence” approach..
And in the 2007 article in Nature Reviews: Drug Discovery [8] the FDA stated: On the basis of these data, the FDA concluded that the totality of the evidence indicated that the Bioferon product and Avonex were sufficiently comparable to rely on the data from major efficacy study using Bioferon’s product to support licensure of Avonex.
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In this statement, the FDA specifically made reference to the landmark Bioferone Avonex case that lead to the 1996 approval of Avonex. Specifically, in this case the FDA approved a biopharmaceutical in which a major change in the final drug product was made (a change in the cell line used to make the drug) after conducting pivotal efficacy trials. This change was allowed based on a scientific data package containing biophysical, biochemical, biological, and minimum clinical data that demonstrated adequate comparability between Bioferon’s Interferon beta-1a (IFN-b-1a) and Biogen’s (which is today called Biogen Idec) IFN-b-1a molecule (which was approved as Avonex). By agreeing to the scientific soundness of the comparability package, the agency allowed Biogen to use the phase III data obtained with the Bioferon’s IFN-b-1a material to support its drug filing with its new IFN-b-1a (derived from a new cell line) without the need to undertake major new series of phase III clinical trials. Although the formal introduction of the “Totality of the Evidence” concept on February 9, 2012 was centered on its association with developing biosimilars, on a much broader level, one should not lose track of its more important association with all forms of drug development. This concept, as illustrated in Figure 14.1, rests on the underpinning that in trying to assess whether the filing of any drug merits approval, it is the integration and evaluation of all four sources of information: biological, biophysical, biochemical, and clinical, that is the determining factor.
FIGURE 14.1 Pictorial representation of the “Totality of the Evidence” concept or approach of integrating information from biological, biochemical, biophysical, and clinical areas in assessing the merit for approving a biopharmaceutical for commercial use.
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14.3 BIOPHYSICAL CHARACTERIZATION IN DEVELOPING PROTEIN BIOPHARMACEUTICALS In Chapter 2, we considered the areas where biophysical characterization contributes to the developmental process of biopharmaceuticals. These areas are summarized and depicted in Figure 14.2. 1. In the case of “drug candidate selection,” biophysical characterization is focused on identifying a molecule that can best tolerate the widest range of experimental conditions that the molecule may encounter during its production, e.g., in terms of pH, salt, temperature, etc. Assessing the behavior of a drug candidate’s ability to be taken to high protein concentration (in terms of aggregation and increase in viscosity) is also important, especially if there is a plan or need to deliver a high concentrated liquid form of the drug via a patient self-administered subcutaneous syringe injection. On conducting these assessments, additional stresses may also be applied, with the purpose of trying to discern differences via robustness and ruggedness among the remaining short list of candidate molecules left for consideration. In general, this approach is used as a “tie breaker” in an attempt to filter through the best drug candidates with the best attribute of stability. These properties are evaluated with the hope that they will most likely translate into an increased ease in developability and manufacturability. 2. In the case of “formulation selection,” biophysical characterization is focused on establishing a suitable formulation buffer (including excipients) that will provide adequate stability to enable the drug product to achieve an expiry that is measured in years. In general, the greater the stability and the greater the range of extreme experimental parameters the drug can tolerate in a given formulation buffer, the better the formulation. A good formulation will maintain a biopharmaceutical’s biophysical fingerprint in stability studies for periods of time in excess of two years.
FIGURE 14.2 The key areas in process development where biophysical characterization plays a role in the development of a biopharmaceutical.
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3. In the case of “drug variant analysis,” two routes exist for generating these variants, one involves a chemical change (e.g., post-translational modification) and the other involves a physical change. In the case of a chemical change, biophysical characterization is focused on assessing what, if any, differences might exist in the biophysical fingerprint of the chemically altered forms of the biopharmaceutical. However, in the case when a variant arises from a physical change, since their detection requires a change in the biophysical fingerprint to begin with, hence no further biophysical characterization work is needed. 4. For “drug consistency, comparability, and compatibility,” biophysical characterization is focused on how well the biophysical fingerprint of the target biopharmaceutical is maintained on a lot-to-lot basis, after introducing a change(s) in the manufacturing of a biopharmaceutical or on exposing the biopharmaceutical to new materials without incurring any meaningful differences.
14.4 BUILDING A BIOPHARMACEUTICAL’S BIOPHYSICAL FINGERPRINT Much of the biophysical characterization activities outlined in Figure 14.2 and discussed briefly in the previous section are dependent on building and utilizing a biopharmaceutical’s biophysical fingerprint. This fingerprint is a key element in assessing the higher-order structure (HOS) information on these biopharmaceuticals and is employed in these activities as the master comparator for determining if there are observable differences in test sample(s). Although no one biophysical tool can adequately provide a complete fingerprint (as mentioning in Chapter 2, Section 2.3.3), its development is thus achieved via a composite of three general approaches summarized in Figure 14.3 and discussed in more detail in the following subsections.
FIGURE 14.3 The underlining components of biophysical characterization that leads to the biophysical fingerprint of a biopharmaceutical.
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14.4.1 Building a Biopharmaceutical’s Biophysical Fingerprint (Part I): Using the Standard (Basic or Core) Biophysical Characterization Tools (Direct Low-Resolution Assessment of a Biopharmaceutical’s HOS) The inherent ability of a protein to perform its function is tied to its structuredwhich has given rise to the principle of structureefunction [9e11], see Chapter 1. Unfortunately our inability to assess, on a routine basis, the complete spatial and temporal structure of a protein in its given environment is a major limitation when developing a biopharmaceutical drug. Although a protein’s primary structure plays a major role in determining its overall HOS, direct and independent approaches are needed to confirm this HOS structure and to detect changes in this structure when they occur. This is especially true when trying to detecting small changes in HOS, including changes that are noncovalent in nature. It is in this area where biophysical characterization plays a useful role. In Chapter 4, we outlined the standard (basic or core) biophysical package for characterizing a biopharmaceutical. This package consists of a composite of biophysical techniques that make up what we refer to as tier 1 and 2 level biophysical tools (see Chapter 4). They include the following: (1) UV, fluorescence, circular dichroism (CD) or/and Fourier transform infrared spectroscopy to provide spectral fingerprint, (2) size exclusion chromatography, static light scattering, or/and dynamic light scattering, particle analysis to provide solution size and mass distribution information (concerning aggregate and fragmentation), and (3) differential scanning calorimetry (DSC) and analytical ultracentrifugation (AUC) to provide domain level information and more accurate global solution behavior information. This general collection of biophysical tools provides a reasonable resource for acquiring direct biophysical characterization information on a biopharmaceutical. Although this is not high-resolution information, which makes it difficult to detect small changes, such data collectively serves as a strong foundation from which a more detailed biophysical fingerprint can be developed, see Figure 14.3.
14.4.2 Building a Biopharmaceutical’s Biophysical Fingerprint (Part II): Via Biophysical Properties While the information provided by the “low-resolution” biophysical tools, mentioned in the last section, is often very limited, an indirect approach (as discussed in Chapter 2) can be taken to supplement and enhance this low-resolution HOS information. This indirect approach involves analyzing the biophysical properties of a biopharmaceutical over a range of conditions (particularly stressed conditions, which pushes the molecule to its limit of stability) using lowresolution techniques. The acquisition of information concerning the biophysical properties of a protein drug are used to proxy for the inability to directly access more detailed biophysical HOS information on the drug. What links this indirect approach of studying a biopharmaceutical’s biophysical properties, to revealing additional information about its HOS, is that the spatial and temporal HOS of a therapeutic protein is in fact the major factor responsible for determining its biophysical properties. As a result, changes in the HOS of a therapeutic protein are very likely to perturb that protein’s biophysical properties in some way. Hence, how well (in terms of precision and accuracy) we measure these properties, coupled with measuring a sufficient range of different properties, determines how well we can detect changes in a biopharmaceutical’s HOS. Clearly, the more information we obtain on different or orthogonal
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properties of a biopharmaceutical the better, most likely, will be our ability to characterize its biophysical fingerprint. Nevertheless, we must realize that in the “real world,” limitations will exist and we will need to identify and focus on measuring only a limited number of biophysical properties that yield useful information. Biophysical properties associated with the following areas are of particular interest in the biopharmaceutical industry: 1. 2. 3. 4. 5. 6.
pH Ionic strength Temperature Freeze/thaw cycling Agitation High concentration
On probing the biophysical properties of a protein drug by exposing it to a range of values in each of the above mentions areas, significant detailed information about the biophysical fingerprint of a drug can be extracted.
14.4.3 Building a Biopharmaceutical’s Biophysical Fingerprint (Part III): Via Advanced (High-Resolution) Biophysical Tools The final step in developing a biopharmaceutical’s biophysical fingerprint involves the use of biophysical tools that can provide information with much higher spatial and temporal resolution. In general, we placed these tools into tiers levels 3 and 4 (see Chapter 4). The biophysical tools that we included in these specific tier levels were hydrogen/deuterium exchange mass spectrometry, small-angle X-ray scattering, and nuclear magnetic resonance. Presently (i.e., 2014), we feel that these tools offer the most significant and practical capability for generating high-resolution information that will also provide much more opportunities for providing high-resolution biophysical characterization in the very near future. In general, the informational level fingerprint that these biophysical tools are capable of providing brings us closer to the “ideal biophysical tool” outlined in the beginning of Chapter 3. At present, these tools will most likely not be employed in routine characterization, rather they will be used for high priority assessments and investigations (e.g., in key lot-to-lot comparisons, when significant changes in drug manufacturing are made or in key biosimilar vs innovator comparisons). Once these technologies improve and their robustness is realized, these tools will likely see more routine use in the future. However, there are two pressing issues that will arise, especially in the early stages of their use. The first concerns the high cost and high level of expertise required to run and interpret the data from these tools (which certainly presents problems for small biotech companies) and second, the eventual uncovering of small differences in HOS, due to their high resolution. In the case of the latter, the big question then becomes ‘whether these differences are significant or not?’ 14.4.3.1 Getting Access to Advanced Biophysical Instruments/Expertise In dealing with advanced analytical instruments, whether it is for biophysical characterization or any other areas of science, there will be a high need to justify the acquisition of the instrument. This is going to be particularly true when the instrument or technology has not been well established or has not been established to be useful in a given area where it is being
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planned to be used for the first time. Consequently, significant resources and effort will be required in terms of staff, time, and money by the company interested in acquiring a particular advanced biophysical instrument. Even if a benefit is realized, assessing whether the data it provides is robust, reproducible, and accurate enough so that a herculean effort is not required to realize its benefit is another important factor in assessing its utility. In addition, other factors must be weighed in, including the associated risk of not realizing any useful benefit in acquiring the instrument or technology against the other beckoning needs of the company. For these types of investigations, it is hard to imagine that small biopharmaceutical companies will have the capability to take on such activities. Thus, it is likely that medium-to-large biopharmaceutical companies will be the key players capable of initiating such work. However, even for these companies trying to determine whether it is worthwhile to bring in-house advanced biophysical tools is a real issue. Even in the case where an advance biophysical instrument is already realized to be a benefit, the problem of getting access to these instruments for the many small biotechnology companies is still a reality. A possible approach for overcoming these initial problems for getting access to advance biophysical instruments might be realized through collaborations with willing academic investigators (who actually have the instrument or are working on developing them) and/ or scientific instrumentation vendors who are trying to commercialize the instrument. Such an approach could offer a more effective and efficient way to deal with the problems of cost and expertise. In addition, these collaborations could also constitute a key way to accelerate the development of these instruments. The realization of such an accelerating effect would be achieved via the interplay of those who are at the forefront of the development and commercialization of these instruments with those who could best put these instruments to use in ways the developers were not even aware of. Another practical route for getting access to these advanced instruments is of course to work with contract laboratories that have the instrumentation and expertise. However, in the beginning even these labs will face the same problem of trying to assess the utility of purchasing such advanced instrumentation, from a business prospective, in terms of attracting enough customers to cover its acquisition and associated costs of running it, in addition to providing a profit. Overall, it should be apparent that getting access to advanced instrumentation and expertise to assess them is a real problem; a problem that unfortunately can stand in the way or at least slow scientific progress that can truly provide more meaningful insight into a drug’s overall development success, quality, and cost. Consequently, we need to find and embrace creative ways to overcome this problem. 14.4.3.2 Better Resolution is Likely to Reveal the Presence of Small Differences: Are These Differences Important? The second issue, mentioned in Section 14.4.3 that can arise when investigating and using advanced biophysical characterization tools is their potential for uncovering new and subtle differences between protein drug molecules that were previously unknown. Such findings could be very enlightening. However, until these differences are understood, they could also introduce unnecessary diversion of time, resources, and money by slowing the progress of a biopharmaceutical program, if their importance is of no consequence or if the difference turns out to be false. Indeed, when applying new methodology, especially more advance and complex methodology to new areas of investigation, the true intrinsic variation in the method
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could take a while to assess and may differ from what was initially thought to be the case. For example, in using AUC to quantifying the level of aggregation with the computer program SEDFIT, initial estimates as to the level of aggregation that could be measured were about 0.2% [12,13], however, present estimates appear closer to about 2%, see Chapter 9, Section 9.5.6.1. The reality of these scenarios can make management uneasy and unwilling to invest in resources to assess and use these advanced analytical tools. Consequently, advanced biophysical tools that offer the potential to improve resolution, by providing more detailed information pose the question, “is this new knowledge useful or is it just a distraction?”
14.5 DETECTING SMALL DIFFERENCES IN BIOPHARMACEUTICALS VIA BIOPHYSICAL CHARACTERIZATION MEASUREMENTS When discussing the topic of detecting small differences in biophysical measurements between biopharmaceutical samples, two important pieces of information will need to be carefully assessed. The first concerns whether the difference is real (in terms of the uncertainty of the measurement itself) and the second is whether it is within the accepted limits of variability for producing the drug product. In the following subsections we will deal with these two pieces of information separately.
14.5.1 DifferencedOutside the Uncertainty Level (Precision or Reproducibility) of a Biophysical Characterization Measurement A key element in trying to assess the importance of any observed difference revealed by any biophysical characterization measurement (or any measurement for that matter) is to determine whether that difference is real. Critical to this assessment is the element of understanding the uncertainty of the recorded data. If the data are not reproducible, then no matter what parameter is being measured or information generated, the end result will be of little or no value to the end user. For the biopharmaceutical scientist conducting comparability studies (e.g., to assess whether any significant lot-to-lot difference exists), the ability to detect a meaningful difference hinges on how precise the measurements can be made, in addition to how good the resolution and sensitivity of these measurements are. This point is illustrated in Figure 14.4 where lot-to-lot comparisons are made on a biopharmaceutical using far-UV CD and DSC. In Figure 14.4 (A) and (C), there is some indication that there may be a small difference in some of the lots. However, on including error bars (equal to 1 standard deviation) from repeat measurements on the same sample, we can immediately see that these apparent small differences are not significant. As a result, it is of great importance in extracting useful information from biophysical characterization measurements that some level of assessment or qualification be conducted to assess the precision and sensitivity of the measurements [14] and understand what factors influence their variability that needs to be tightly controlled.
14.5.2 DifferencedWhat’s Important and What’s Not Important within the Concept of Biopharmaceutical Consistency and Comparability (or Similarity) After establishing that a detected difference(s) in a measured biophysical parameter is real (due to the difference exceeding the established uncertainty inherent in the
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FIGURE 14.4 Lot-to-lot comparability of a biopharmaceutical to assess drug product consistency using (A) farUV circular dichroism, (B) same as (A), but 1 standard deviation (SD) error bars are included and (C) differential scanning calorimetry, and (D) same as (C), but 1 SD error bars are included.
measurement), one is faced with the final problem of determining whether this difference is important or not important. In making this assessment we are involved with the tightly coupled critical concepts of drug product consistency and comparability that must be met as part of drug approval. Consistency and comparability of each measured biophysical parameter is linked and operates within an established boundary (or limit) of allowable variability determined for each parameter (which is equal or greater than the uncertainty determined for each parameter). Thus, if a difference is detected in a measured biophysical parameter that exceeds this allowable boundary value of variability, then the drug product’s consistency and therefore its comparability are violated, making the observed difference important. Since biopharmaceuticals cannot be made so they are identical on a lot-to-lot basis, due to the inherent nature in which these drugs are made, it becomes clear that the proper setting of these boundaries of variability for each parameter (which are a function of the impact of the parameter on the drug and level of control of manufacturing that needs to be demonstrated) are of great of importance. Thus, although a difference(s) in a measured biophysical parameter could be detected, that difference will be of no importance if that difference falls within the accepted level of variability allowed for that parameter for that particular drug product.
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14.6 CONCLUSION In the begin of this chapter, we introduced the contemporary phrase “Totality of the Evidence” and briefly explained its present association with the issues surrounding the approach the FDA has taken to deal with the uncertainties in assessing the approval of biosimilars. However, we are confident that the much broader intent of this phrase was intended as a global process of considering all the data in weighting the risk against the benefits in assessing the approval of any drug is what the FDA had intended. As a result, the application of this concept for evaluating biosimilars, is just a natural extension of its original broader intended purpose. In developing this approach or mind set, the FDA highlighted a number of situations in its past (see their 2007 article [8]), where this approach was used. One such example concerned the landmark event that allowed Biogen to file its IFN-b-1a (Avonex) drug for approval after using its former partner’s (Bioferon) phase III trials data obtained using Bioferon’s IFN-b-1a that was made from a different cell line. The key for allowing this to take place was the successful demonstration by Biogen, to the FDA, that adequate comparability existed between both IFN-b-1a molecules using a “Totality of the Evidence” approach of biological, biochemical, biophysical, and limited clinical data. With the success of this event, the biopharmaceutical industry moved from the “Process is the Product” to “Well-characterized” biopharmaceutical era. An era that now allows the sciences, including the biophysical sciences, to now play a far more productive role in enabling changes to biopharmaceuticals products to be made without the need for extensive clinical trials (depending on the outcome of comparability studies). This change in thinking opened a significant opportunity for improving the process of developing biopharmaceuticals, for bringing new innovative drugs to the marketplace, and to encourage and expedite the ability of biopharmaceutical companies to make changes in the existing manufacturing processes (to improve drug quality and manufacturing efficiency with the hope of passing these benefits on to the patient). In putting this book together, we and our co-authors have tried to show where the present state of biophysical characterization stands and have in some cases offered suggestions as to where opportunities may exist for improvements in the near future. Nevertheless, in the context of the totality of the evidence we would like to briefly remind the reader that even with our best intentions and more importantly with our best science, we still cannot avoid surprises in drug development and approval outcomes [15e21], given the underlining complexity of these molecules and the existing gaps in our knowledge. As a result, we must take solace in the realization that at present there is wisdom in our awareness of our ignorance [22] as to how biological systems work and the only path to remove this ignorance, is to do (better) science.
References [1] Hay M, Thomas DW, Craighead JL, Economides C. Clinical development success rates for investigational drugs. Rosenthal J Nat Biotechnol January 2014;32(1):40e51. [2] Samanen J. The structure and business of biopharmaceutical companies including the management of risk and resources. In: Ganellin CR, Jefferis R, Roberts S, editors. Introduction to biological and small molecule drug research and development: theory and case studies. Waltham (MA): Academic Press; 2013. p. 235e6.
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[3] Rydzewski RM. Real world drug discovery: a chemist’s guide to biotech and pharmaceutical research. Oxford (UK): Elsevier; 2008. pp. 147e148. [4] Brown D, Superti-Furga G. Rediscovering the sweet spot in drug discovery. Drug Discov Today December 1, 2003;8(23):1067e77. [5] Berkowitz SA, Engen JR, Mazzeo JR, Jones GB. Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars. Nat Rev Drug Discov June 29, 2012;11(7):527e40. [6] Kozlowski S, Woodcock J, Midthun K, Sherman RB. Developing the nation’s biosimilars program. N Engl J Med August 4, 2011;365(5):385e8. [7] [a] http://www.fda.gov/newsevents/newsroom/pressannouncements/ucm291232.htm. [b] http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM291128. pdf. [c] http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM291134. pdf. [d] http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM273001. pdf. [8] Woodcock J, Griffin J, Behrman R, Cherney B, Crescenzi T, Fraser B, et al. The FDA’s assessment of follow-on protein products: a historical perspective. Nat Rev Drug Discov June 2007;6(6):437e42. [9] Anfinsen CB. Principles that govern the folding of protein chains. Science July 20, 1973;181(4096):223e30. [10] Hegyi H, Gerstein M. The relationship between protein structure and function: a comprehensive survey with application to the yeast genome. J Mol Biol April 23, 1999;288(1):147e64. [11] Sadowski MI, Jones DT. The sequence-structure relationship and protein function prediction. Curr Opin Struct Biol June 2009;19(3):357e62. [12] Dam J, Schuck P. Calculating sedimentation coefficient distributions by direct modeling of sedimentation velocity concentration profiles. Methods Enzymol 2004;384:185e212. [13] Shuck P. Diffusion-deconvoluted sedimentation coefficient distributions for the analysis of interacting and non-interacting protein mixtures. In: Scott DJ, Harding SE, Rowe AJ, editors. Analytical ultracentrifugation: techniques and methods. The Royal Society of Chemistry; 2005. pp. 26e50. [14] Jiang Y, Li C, Gabrielson J. Qualification of biophysical methods for the analysis of protein therapeutics. In: Narhi LO, editor. Biophysics for therapeutic protein development. NY: Springer; 2013. pp. 99e126. [15] http://www.fda.gov/downloads/Drugs/DevelopmentApprovalProcess/HowDrugsareDevelopedandApproved/ ApprovalApplications/TherapeuticBiologicApplications/ucm094510.pdf. [16] http://www.ema.europa.eu/docs/en_GB/document_library/Application_withdrawal_assessment_report/ 2010/01/WC500061394.pdf. [17] Mack G. FDA balks at myozyme scale-up. Nat Biotechnol June 2008;26(6):592. [18] http://www.fda.gov/ohrms/dockets/ac/08/briefing/2008-4389b1-04-Genzyme.pdf. [19] Trysabri Rydzewski RM. Real world drug discovery: a chemist’s guide to biotech and pharmaceutical research. Oxford (UK): Elsevier; 2008. p. 19. [20] http://www.fda.gov/Drugs/DrugSafety/PostmarketDrugSafetyInformationforPatientsandProviders/ ucm107198.htm. [21] http://www.fda.gov/Drugs/DrugSafety/PostmarketDrugSafetyInformationforPatientsandProviders/ ucm199872.htm. [22] Firesrtein S. Ignorance: How It Drives Science. NY: Oxford University Press, NY; 2012.
14 Biophysical Characterization: An Integral Part of the “Totality of the Evidence” Concept Damian J. Houde, Steven A. Berkowitz Department of Protein Pharmaceutical Development, Biogen Idec, Inc., Cambridge, MA, USA
14.1 BIOPHARMACEUTICAL DEVELOPMENT The process of developing a protein biopharmaceutical is a long, precarious, complex, and an expensive process, which unfortunately has a very low probability of success (at least at present). This is testified by the fact that the biopharmaceutical landscape is littered with unsuccessful drug products [1e4]. This outcome is not surprising if we consider the characteristics of these drugs (as pointed out in Chapters 1e3). The most notable of which includes the following: (1) the large, heterogeneous, complex, and fragile nature of these molecules, (2) the mechanism by which they are made, using living cells, which we have limited knowledge on and ability to control in detail, (3) our incomplete understanding about the interactions of these therapeutic compounds within a living system and how the living system works, and (4) the limitation in the ability of the analytical tools we currently have to characterize them [5]. As a result, at best, we can only attempt to minimize the risk of making uninformed and thus poor decisions that lead to failure by taking a holistic approach and acquire as much (useful) information as possible under the constraints of economic and financial limitations (remembering that developing biopharmaceutical is not a philanthropic endeavordit’s a business!). It is in just such an environment that the manufacturer who attempts to develop a biopharmaceutical and the regulators how will approve or reject that biopharmaceutical must operate in. Consequently, setting standards for development and approval too high can lead to miss opportunities that could have favorably impacted the lives of many. However, setting the standards for development and approval too low can on the other hand do great harm.
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14.1.1 “One-size-fit-all” versus “Case-by-case” The idea that the process of developing biopharmaceuticals follows the same exact path, where one only needs to successfully execute the same predetermined number of sequential activities to realize a successful commercial drug is misleading. Similarly, a “one-size-fit-all” approach for assessing biopharmaceuticals to determine their ability to be approved as a commercial drug is just as misleading (at least at present) [6]. The individual personalities or behavior of each disease and each biopharmaceutical, and the accessory components required for its development (formulations, container closures, raw material, cell lines, etc.) conspire to contribute a unique variability to outcomes that leads to different issues, which must be resolved for each individual biopharmaceutical. Consequently, the only rational scientific approach to deal with this situation is to evaluate each drugedisease combination on its own merit, via the all too frequent and infamous phrase “case-by-case” assessment.
14.2 AN INTRODUCTION TO THE “TOTALITY OF THE EVIDENCE” AND ITS MORE GLOBAL MEANING IN DEVELOPING BIOPHARMACEUTICALS On February 9, 2012 the US Food and Drug Administration, FDA, formalized its approach concerning how it would evaluate and approve biosimilars as generic-like copies of biopharmaceuticals that have lost or will be losing patent protection due to expiry [7ae7d]. Although certainly lacking in detail, the approach was officially captured in the simple and brief terminology, “Totality of the Evidence.” Essentially, this simple phrase encapsulated the FDA’s thoughts concerning how it would evaluate and approve biosimilars as genericlike copies of biopharmaceuticals. In principle, this phrase constitutes the FDA’s plan to migrate risk, which may exist in trying to assess any difference(s) or residual gaps in knowledge between the innovator and biosimilar in assessing the approval or rejection of biosimilar drugs. By critically evaluating the total data package of a biosimilar’s filing, the FDA hopes to determine whether a biosimilar is an adequate copy of an existing innovator biopharmaceutical. As a result, the concept of “Totality of the Evidence” has now become completely and uniquely synonymous with the area of biosimilar development and approval. In coining this phrase, one can trace its origin back to at least two articles published by the FDA, one in 2007 another in 2011. In both of these articles the phrase “Totality of the Evidence” was used with a much broader intent of encompassing all biopharmaceuticals (and pharmaceuticals) in general. In the 2011 article in The New England Journal of Medicine [6], the FDA stated the following: The FDA has traditionally relied on integrating various kinds of evidence in making regulatory decisions. Such a “totality of the evidence” approach..
And in the 2007 article in Nature Reviews: Drug Discovery [8] the FDA stated: On the basis of these data, the FDA concluded that the totality of the evidence indicated that the Bioferon product and Avonex were sufficiently comparable to rely on the data from major efficacy study using Bioferon’s product to support licensure of Avonex.
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In this statement, the FDA specifically made reference to the landmark Bioferone Avonex case that lead to the 1996 approval of Avonex. Specifically, in this case the FDA approved a biopharmaceutical in which a major change in the final drug product was made (a change in the cell line used to make the drug) after conducting pivotal efficacy trials. This change was allowed based on a scientific data package containing biophysical, biochemical, biological, and minimum clinical data that demonstrated adequate comparability between Bioferon’s Interferon beta-1a (IFN-b-1a) and Biogen’s (which is today called Biogen Idec) IFN-b-1a molecule (which was approved as Avonex). By agreeing to the scientific soundness of the comparability package, the agency allowed Biogen to use the phase III data obtained with the Bioferon’s IFN-b-1a material to support its drug filing with its new IFN-b-1a (derived from a new cell line) without the need to undertake major new series of phase III clinical trials. Although the formal introduction of the “Totality of the Evidence” concept on February 9, 2012 was centered on its association with developing biosimilars, on a much broader level, one should not lose track of its more important association with all forms of drug development. This concept, as illustrated in Figure 14.1, rests on the underpinning that in trying to assess whether the filing of any drug merits approval, it is the integration and evaluation of all four sources of information: biological, biophysical, biochemical, and clinical, that is the determining factor.
FIGURE 14.1 Pictorial representation of the “Totality of the Evidence” concept or approach of integrating information from biological, biochemical, biophysical, and clinical areas in assessing the merit for approving a biopharmaceutical for commercial use.
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14.3 BIOPHYSICAL CHARACTERIZATION IN DEVELOPING PROTEIN BIOPHARMACEUTICALS In Chapter 2, we considered the areas where biophysical characterization contributes to the developmental process of biopharmaceuticals. These areas are summarized and depicted in Figure 14.2. 1. In the case of “drug candidate selection,” biophysical characterization is focused on identifying a molecule that can best tolerate the widest range of experimental conditions that the molecule may encounter during its production, e.g., in terms of pH, salt, temperature, etc. Assessing the behavior of a drug candidate’s ability to be taken to high protein concentration (in terms of aggregation and increase in viscosity) is also important, especially if there is a plan or need to deliver a high concentrated liquid form of the drug via a patient self-administered subcutaneous syringe injection. On conducting these assessments, additional stresses may also be applied, with the purpose of trying to discern differences via robustness and ruggedness among the remaining short list of candidate molecules left for consideration. In general, this approach is used as a “tie breaker” in an attempt to filter through the best drug candidates with the best attribute of stability. These properties are evaluated with the hope that they will most likely translate into an increased ease in developability and manufacturability. 2. In the case of “formulation selection,” biophysical characterization is focused on establishing a suitable formulation buffer (including excipients) that will provide adequate stability to enable the drug product to achieve an expiry that is measured in years. In general, the greater the stability and the greater the range of extreme experimental parameters the drug can tolerate in a given formulation buffer, the better the formulation. A good formulation will maintain a biopharmaceutical’s biophysical fingerprint in stability studies for periods of time in excess of two years.
FIGURE 14.2 The key areas in process development where biophysical characterization plays a role in the development of a biopharmaceutical.
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3. In the case of “drug variant analysis,” two routes exist for generating these variants, one involves a chemical change (e.g., post-translational modification) and the other involves a physical change. In the case of a chemical change, biophysical characterization is focused on assessing what, if any, differences might exist in the biophysical fingerprint of the chemically altered forms of the biopharmaceutical. However, in the case when a variant arises from a physical change, since their detection requires a change in the biophysical fingerprint to begin with, hence no further biophysical characterization work is needed. 4. For “drug consistency, comparability, and compatibility,” biophysical characterization is focused on how well the biophysical fingerprint of the target biopharmaceutical is maintained on a lot-to-lot basis, after introducing a change(s) in the manufacturing of a biopharmaceutical or on exposing the biopharmaceutical to new materials without incurring any meaningful differences.
14.4 BUILDING A BIOPHARMACEUTICAL’S BIOPHYSICAL FINGERPRINT Much of the biophysical characterization activities outlined in Figure 14.2 and discussed briefly in the previous section are dependent on building and utilizing a biopharmaceutical’s biophysical fingerprint. This fingerprint is a key element in assessing the higher-order structure (HOS) information on these biopharmaceuticals and is employed in these activities as the master comparator for determining if there are observable differences in test sample(s). Although no one biophysical tool can adequately provide a complete fingerprint (as mentioning in Chapter 2, Section 2.3.3), its development is thus achieved via a composite of three general approaches summarized in Figure 14.3 and discussed in more detail in the following subsections.
FIGURE 14.3 The underlining components of biophysical characterization that leads to the biophysical fingerprint of a biopharmaceutical.
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14.4.1 Building a Biopharmaceutical’s Biophysical Fingerprint (Part I): Using the Standard (Basic or Core) Biophysical Characterization Tools (Direct Low-Resolution Assessment of a Biopharmaceutical’s HOS) The inherent ability of a protein to perform its function is tied to its structuredwhich has given rise to the principle of structureefunction [9e11], see Chapter 1. Unfortunately our inability to assess, on a routine basis, the complete spatial and temporal structure of a protein in its given environment is a major limitation when developing a biopharmaceutical drug. Although a protein’s primary structure plays a major role in determining its overall HOS, direct and independent approaches are needed to confirm this HOS structure and to detect changes in this structure when they occur. This is especially true when trying to detecting small changes in HOS, including changes that are noncovalent in nature. It is in this area where biophysical characterization plays a useful role. In Chapter 4, we outlined the standard (basic or core) biophysical package for characterizing a biopharmaceutical. This package consists of a composite of biophysical techniques that make up what we refer to as tier 1 and 2 level biophysical tools (see Chapter 4). They include the following: (1) UV, fluorescence, circular dichroism (CD) or/and Fourier transform infrared spectroscopy to provide spectral fingerprint, (2) size exclusion chromatography, static light scattering, or/and dynamic light scattering, particle analysis to provide solution size and mass distribution information (concerning aggregate and fragmentation), and (3) differential scanning calorimetry (DSC) and analytical ultracentrifugation (AUC) to provide domain level information and more accurate global solution behavior information. This general collection of biophysical tools provides a reasonable resource for acquiring direct biophysical characterization information on a biopharmaceutical. Although this is not high-resolution information, which makes it difficult to detect small changes, such data collectively serves as a strong foundation from which a more detailed biophysical fingerprint can be developed, see Figure 14.3.
14.4.2 Building a Biopharmaceutical’s Biophysical Fingerprint (Part II): Via Biophysical Properties While the information provided by the “low-resolution” biophysical tools, mentioned in the last section, is often very limited, an indirect approach (as discussed in Chapter 2) can be taken to supplement and enhance this low-resolution HOS information. This indirect approach involves analyzing the biophysical properties of a biopharmaceutical over a range of conditions (particularly stressed conditions, which pushes the molecule to its limit of stability) using lowresolution techniques. The acquisition of information concerning the biophysical properties of a protein drug are used to proxy for the inability to directly access more detailed biophysical HOS information on the drug. What links this indirect approach of studying a biopharmaceutical’s biophysical properties, to revealing additional information about its HOS, is that the spatial and temporal HOS of a therapeutic protein is in fact the major factor responsible for determining its biophysical properties. As a result, changes in the HOS of a therapeutic protein are very likely to perturb that protein’s biophysical properties in some way. Hence, how well (in terms of precision and accuracy) we measure these properties, coupled with measuring a sufficient range of different properties, determines how well we can detect changes in a biopharmaceutical’s HOS. Clearly, the more information we obtain on different or orthogonal
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properties of a biopharmaceutical the better, most likely, will be our ability to characterize its biophysical fingerprint. Nevertheless, we must realize that in the “real world,” limitations will exist and we will need to identify and focus on measuring only a limited number of biophysical properties that yield useful information. Biophysical properties associated with the following areas are of particular interest in the biopharmaceutical industry: 1. 2. 3. 4. 5. 6.
pH Ionic strength Temperature Freeze/thaw cycling Agitation High concentration
On probing the biophysical properties of a protein drug by exposing it to a range of values in each of the above mentions areas, significant detailed information about the biophysical fingerprint of a drug can be extracted.
14.4.3 Building a Biopharmaceutical’s Biophysical Fingerprint (Part III): Via Advanced (High-Resolution) Biophysical Tools The final step in developing a biopharmaceutical’s biophysical fingerprint involves the use of biophysical tools that can provide information with much higher spatial and temporal resolution. In general, we placed these tools into tiers levels 3 and 4 (see Chapter 4). The biophysical tools that we included in these specific tier levels were hydrogen/deuterium exchange mass spectrometry, small-angle X-ray scattering, and nuclear magnetic resonance. Presently (i.e., 2014), we feel that these tools offer the most significant and practical capability for generating high-resolution information that will also provide much more opportunities for providing high-resolution biophysical characterization in the very near future. In general, the informational level fingerprint that these biophysical tools are capable of providing brings us closer to the “ideal biophysical tool” outlined in the beginning of Chapter 3. At present, these tools will most likely not be employed in routine characterization, rather they will be used for high priority assessments and investigations (e.g., in key lot-to-lot comparisons, when significant changes in drug manufacturing are made or in key biosimilar vs innovator comparisons). Once these technologies improve and their robustness is realized, these tools will likely see more routine use in the future. However, there are two pressing issues that will arise, especially in the early stages of their use. The first concerns the high cost and high level of expertise required to run and interpret the data from these tools (which certainly presents problems for small biotech companies) and second, the eventual uncovering of small differences in HOS, due to their high resolution. In the case of the latter, the big question then becomes ‘whether these differences are significant or not?’ 14.4.3.1 Getting Access to Advanced Biophysical Instruments/Expertise In dealing with advanced analytical instruments, whether it is for biophysical characterization or any other areas of science, there will be a high need to justify the acquisition of the instrument. This is going to be particularly true when the instrument or technology has not been well established or has not been established to be useful in a given area where it is being
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planned to be used for the first time. Consequently, significant resources and effort will be required in terms of staff, time, and money by the company interested in acquiring a particular advanced biophysical instrument. Even if a benefit is realized, assessing whether the data it provides is robust, reproducible, and accurate enough so that a herculean effort is not required to realize its benefit is another important factor in assessing its utility. In addition, other factors must be weighed in, including the associated risk of not realizing any useful benefit in acquiring the instrument or technology against the other beckoning needs of the company. For these types of investigations, it is hard to imagine that small biopharmaceutical companies will have the capability to take on such activities. Thus, it is likely that medium-to-large biopharmaceutical companies will be the key players capable of initiating such work. However, even for these companies trying to determine whether it is worthwhile to bring in-house advanced biophysical tools is a real issue. Even in the case where an advance biophysical instrument is already realized to be a benefit, the problem of getting access to these instruments for the many small biotechnology companies is still a reality. A possible approach for overcoming these initial problems for getting access to advance biophysical instruments might be realized through collaborations with willing academic investigators (who actually have the instrument or are working on developing them) and/ or scientific instrumentation vendors who are trying to commercialize the instrument. Such an approach could offer a more effective and efficient way to deal with the problems of cost and expertise. In addition, these collaborations could also constitute a key way to accelerate the development of these instruments. The realization of such an accelerating effect would be achieved via the interplay of those who are at the forefront of the development and commercialization of these instruments with those who could best put these instruments to use in ways the developers were not even aware of. Another practical route for getting access to these advanced instruments is of course to work with contract laboratories that have the instrumentation and expertise. However, in the beginning even these labs will face the same problem of trying to assess the utility of purchasing such advanced instrumentation, from a business prospective, in terms of attracting enough customers to cover its acquisition and associated costs of running it, in addition to providing a profit. Overall, it should be apparent that getting access to advanced instrumentation and expertise to assess them is a real problem; a problem that unfortunately can stand in the way or at least slow scientific progress that can truly provide more meaningful insight into a drug’s overall development success, quality, and cost. Consequently, we need to find and embrace creative ways to overcome this problem. 14.4.3.2 Better Resolution is Likely to Reveal the Presence of Small Differences: Are These Differences Important? The second issue, mentioned in Section 14.4.3 that can arise when investigating and using advanced biophysical characterization tools is their potential for uncovering new and subtle differences between protein drug molecules that were previously unknown. Such findings could be very enlightening. However, until these differences are understood, they could also introduce unnecessary diversion of time, resources, and money by slowing the progress of a biopharmaceutical program, if their importance is of no consequence or if the difference turns out to be false. Indeed, when applying new methodology, especially more advance and complex methodology to new areas of investigation, the true intrinsic variation in the method
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could take a while to assess and may differ from what was initially thought to be the case. For example, in using AUC to quantifying the level of aggregation with the computer program SEDFIT, initial estimates as to the level of aggregation that could be measured were about 0.2% [12,13], however, present estimates appear closer to about 2%, see Chapter 9, Section 9.5.6.1. The reality of these scenarios can make management uneasy and unwilling to invest in resources to assess and use these advanced analytical tools. Consequently, advanced biophysical tools that offer the potential to improve resolution, by providing more detailed information pose the question, “is this new knowledge useful or is it just a distraction?”
14.5 DETECTING SMALL DIFFERENCES IN BIOPHARMACEUTICALS VIA BIOPHYSICAL CHARACTERIZATION MEASUREMENTS When discussing the topic of detecting small differences in biophysical measurements between biopharmaceutical samples, two important pieces of information will need to be carefully assessed. The first concerns whether the difference is real (in terms of the uncertainty of the measurement itself) and the second is whether it is within the accepted limits of variability for producing the drug product. In the following subsections we will deal with these two pieces of information separately.
14.5.1 DifferencedOutside the Uncertainty Level (Precision or Reproducibility) of a Biophysical Characterization Measurement A key element in trying to assess the importance of any observed difference revealed by any biophysical characterization measurement (or any measurement for that matter) is to determine whether that difference is real. Critical to this assessment is the element of understanding the uncertainty of the recorded data. If the data are not reproducible, then no matter what parameter is being measured or information generated, the end result will be of little or no value to the end user. For the biopharmaceutical scientist conducting comparability studies (e.g., to assess whether any significant lot-to-lot difference exists), the ability to detect a meaningful difference hinges on how precise the measurements can be made, in addition to how good the resolution and sensitivity of these measurements are. This point is illustrated in Figure 14.4 where lot-to-lot comparisons are made on a biopharmaceutical using far-UV CD and DSC. In Figure 14.4 (A) and (C), there is some indication that there may be a small difference in some of the lots. However, on including error bars (equal to 1 standard deviation) from repeat measurements on the same sample, we can immediately see that these apparent small differences are not significant. As a result, it is of great importance in extracting useful information from biophysical characterization measurements that some level of assessment or qualification be conducted to assess the precision and sensitivity of the measurements [14] and understand what factors influence their variability that needs to be tightly controlled.
14.5.2 DifferencedWhat’s Important and What’s Not Important within the Concept of Biopharmaceutical Consistency and Comparability (or Similarity) After establishing that a detected difference(s) in a measured biophysical parameter is real (due to the difference exceeding the established uncertainty inherent in the
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FIGURE 14.4 Lot-to-lot comparability of a biopharmaceutical to assess drug product consistency using (A) farUV circular dichroism, (B) same as (A), but 1 standard deviation (SD) error bars are included and (C) differential scanning calorimetry, and (D) same as (C), but 1 SD error bars are included.
measurement), one is faced with the final problem of determining whether this difference is important or not important. In making this assessment we are involved with the tightly coupled critical concepts of drug product consistency and comparability that must be met as part of drug approval. Consistency and comparability of each measured biophysical parameter is linked and operates within an established boundary (or limit) of allowable variability determined for each parameter (which is equal or greater than the uncertainty determined for each parameter). Thus, if a difference is detected in a measured biophysical parameter that exceeds this allowable boundary value of variability, then the drug product’s consistency and therefore its comparability are violated, making the observed difference important. Since biopharmaceuticals cannot be made so they are identical on a lot-to-lot basis, due to the inherent nature in which these drugs are made, it becomes clear that the proper setting of these boundaries of variability for each parameter (which are a function of the impact of the parameter on the drug and level of control of manufacturing that needs to be demonstrated) are of great of importance. Thus, although a difference(s) in a measured biophysical parameter could be detected, that difference will be of no importance if that difference falls within the accepted level of variability allowed for that parameter for that particular drug product.
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14.6 CONCLUSION In the begin of this chapter, we introduced the contemporary phrase “Totality of the Evidence” and briefly explained its present association with the issues surrounding the approach the FDA has taken to deal with the uncertainties in assessing the approval of biosimilars. However, we are confident that the much broader intent of this phrase was intended as a global process of considering all the data in weighting the risk against the benefits in assessing the approval of any drug is what the FDA had intended. As a result, the application of this concept for evaluating biosimilars, is just a natural extension of its original broader intended purpose. In developing this approach or mind set, the FDA highlighted a number of situations in its past (see their 2007 article [8]), where this approach was used. One such example concerned the landmark event that allowed Biogen to file its IFN-b-1a (Avonex) drug for approval after using its former partner’s (Bioferon) phase III trials data obtained using Bioferon’s IFN-b-1a that was made from a different cell line. The key for allowing this to take place was the successful demonstration by Biogen, to the FDA, that adequate comparability existed between both IFN-b-1a molecules using a “Totality of the Evidence” approach of biological, biochemical, biophysical, and limited clinical data. With the success of this event, the biopharmaceutical industry moved from the “Process is the Product” to “Well-characterized” biopharmaceutical era. An era that now allows the sciences, including the biophysical sciences, to now play a far more productive role in enabling changes to biopharmaceuticals products to be made without the need for extensive clinical trials (depending on the outcome of comparability studies). This change in thinking opened a significant opportunity for improving the process of developing biopharmaceuticals, for bringing new innovative drugs to the marketplace, and to encourage and expedite the ability of biopharmaceutical companies to make changes in the existing manufacturing processes (to improve drug quality and manufacturing efficiency with the hope of passing these benefits on to the patient). In putting this book together, we and our co-authors have tried to show where the present state of biophysical characterization stands and have in some cases offered suggestions as to where opportunities may exist for improvements in the near future. Nevertheless, in the context of the totality of the evidence we would like to briefly remind the reader that even with our best intentions and more importantly with our best science, we still cannot avoid surprises in drug development and approval outcomes [15e21], given the underlining complexity of these molecules and the existing gaps in our knowledge. As a result, we must take solace in the realization that at present there is wisdom in our awareness of our ignorance [22] as to how biological systems work and the only path to remove this ignorance, is to do (better) science.
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