Biopharmaceutical industry's biophysical toolbox

C H A P T E R

3 Biopharmaceutical industry’s biophysical toolbox Damian J. Houdea, Steven A. Berkowitzb a

Biomolecular Discovery, Relay Therapeutics, Cambridge, MA, United States; bConsultant, Sudbury, MA, United States

3.1 Attributes of a single biophysical tool to characterize and detect changes in the higher order structure of a biopharmaceutical Although it is unlikely that any single biophysical tool will ever provide all the necessary information needed to characterize the higher order structure (HOS) of a protein drug, it is a worthy idea to contemplate the characteristics as to just what such a hypothetical biophysical tool might look like, in terms of its key attributes. Such characteristics are, in part, coarsely illustrated in Fig. 3.1. Preferably, the tool should be capable of providing a unique quantitative signal read out for each basic structural element that makes up the drug that has good sensitivity with high spatial resolution resulting no or minimum overlapping of each of these unique signals. These characteristics can be broken down into four essential parts. First is the ability to measure signals from as small a unit of the protein as possible (e.g., atomic resolution would be ideal, but amino acid level might be acceptable, see Fig. 3.2). Second is the ability to detect all such signals emitted from a single molecule; essentially, we want to detect and interrogate as much of the protein’s structure (hopefully the entire structure) as possible. Third is the ability to separate or resolve these signals spatially in a manner using some parameter space (e.g., relative atomic distances, wavelength, temperature, time, etc.) with minimal signal overlap. The fourth and final characteristic would be the ability to quantitatively record all these signals with the highest precision (and accuracy) possible. The highest resolution of a protein’s HOS would amount to knowing all the relative positions of the atoms in the entire protein molecule (again see Fig. 3.2). Having accurate data on every atom in a protein, in terms of their spatial coordinates, as well as their temporal behavior (i.e., dynamics) would correspond to the ultimate situation in detecting a difference in the smallest element of the protein drug. Such direct information (as mentioned in Chapter 2) can presently be approached by only two techniques, X-ray crystallography and nuclear

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FIG. 3.1 The impact of overlapping (A) versus nonoverlapping (B) signals from a biopharmaceutical on the ability of a biophysical measurement to detect changes in the HOS between different drug samples. In the example given in this figure, the biophysical measurement on a protein drug is derived from only six different structural elements on the drug molecule. Each structural elements outputs a signal, S(1)eS(6) in terms of this particular biophysical measurement. In this specific example, two drug samples (referred to as sample 1 and sample 2) are being compared in which the only difference that exists between them is due to a 20% reduction in the intensity of S(5) in sample 2. This difference is indicated by the same difference in the vertical spacing value between the two dotted black lines in Parts A and B (that corresponds to the intensity of level of S(5) in samples 1 and 2) and whose difference is represented in both parts by the symbol D5. However, in Part A, there is extensive overlapping of the six signals, which in the area where S(5) appears along the x-axis (of the data output), S(5) now only accounts for about 25% of the total output signal. Given this reduction in the contribution from S(5), the original difference (D5) in signal S(5) between samples 1 and 2 is also reduced to 0.25D5 as indicated by the blue (gray in print version) dotted lines (which now amounts to only a 5% reduction in the total signal where the S(5) appears in the data output). With the same 99% confidence limit, indicated by the error bars in Parts A and B, the biophysical measurement difference value of 0.25D5 in S(5) between samples 1 and 2 in Part A will go undetected in the case where the signal outputs overlap, due to the level of statistical noise in the data, while in the case of Part B where signal outputs are non-overlapping, the original D5 difference is maintained and can be detected.

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FIG. 3.2 A schematic representation of the hierarchy of spatial resolution of a biopharmaceutical’s HOS is shown. The different structural elements and their relationship to the three basic forms of chemical and physical changes (which include post-transtrantional modifications (PTMs) and fragmentation, non-covalent changes (silent changes) and aggregation) that can impact the HOS and biophysical properties of a biopharmaceutical are also illustrated.

magnetic resonance (NMR). However, the application of these tools are beyond the scope of this book and will not be discussed (also see Chapter 2, Section 2.3.3.1).

3.2 Studying the biophysical properties of a biopharmaceutical as an indirect approach for characterizing changes in its HOS In the last decade, the ability of more advanced biophysical tools to provide direct information on the HOS of proteins has greatly improved [1e3]. However, the impracticality or present limitations in utilizing these tools in the process development area of today’s biopharmaceutical industry still remains. As a result, many biophysical tools and their associated methods have evolved to support an alternative approach for extracting indirect information about a molecule’s HOS. This approach uses biophysical characterization studies to gather information on the “biophysical properties” of these molecules. The success of using biophysical properties to proxy for the direct determination of the HOS of a biopharmaceutical rests on the idea that the combination of a molecule’s chemical composition and its HOS give rise to unique biophysical properties (Fig. 3.3). Through this critical linkage, information

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FIG. 3.3 The impact of chemical and physical changes on the HOS (as indicated by the objects shape, rectangle ¼ native HOS, hexagon ¼ altered HOS) and biophysical properties of the monomeric form of a biopharmaceutical after its primary structure is synthesized and folded to attain its normal HOS. (A) An example where a large specific chemical addition is made to a biopharmaceutical to give a new form of the drug that displays different biophysical properties and an altered HOS relative to the original biopharmaceutical. (B) The same as for “A” but the chemical modification does not alter the HOS of the original drug. However, the resulting drug still displays different biophysical properties because it is chemically modified. (C) An example where a small specific chemical addition (post-translational modifications, PTMs) occurs on a biopharmaceutical that does not alter the HOS, but may or may not alter biopharmaceutical’s biophysical properties. (D) The same example as in “C” but the original biopharmaceutical’s HOS and biophysical properties are altered. (E) A physical change that alters the HOS and biophysical properties of the biopharmaceutical. (F) The same as in “E” but the physical change does not alter the HOS or biophysical properties of the biopharmaceutical.

about changes in a biopharmaceutical’s biophysical properties should indirectly shed light on changes to its HOS. Although this information does not delineate the spatial coordinates of the protein drug’s atomic structure, it is hoped that by measuring a collection of simple parameters derived from the molecule’s various biophysical properties an indirect fingerprint of the biopharmaceutical’s HOS is obtained. Using such information, constraints on the variation of these biophysical properties can be put into place such that if the value for these parameters falls outside a predetermined, established, and agreed-upon range, they would imply a likely change in the biopharmaceutical’s HOS.

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In general, in the biopharmaceutical process development area, it is the simple biophysical tools that monitor the biophysical properties of a biopharmaceutical that forms the backbone of most of the biophysical studies conducted during the development of these drugs. In most cases, these simple biophysical tools often yield straightforward information, that is represented by a single numerical parameter (e.g., melting temperature, Tm, sedimentation coefficient, s) or by a two-dimensional graphical profile (e.g., in the case of circular dichroism, by a CD spectrum or in the case of chromatography by a chromatogram). Changes that can be introduced into a biopharmaceutical’s HOS during its production that can affect its efficacy, can be categorized into the following two areas: (1) those caused by chemical (covalent) changes, e.g., due to post-translational modifications, PTMs, and fragmentation, and (2) those caused by physical (noncovalent) changes, e.g., due to temperature and agitation. In most cases, these chemical and physical changes are typically confined to small areas of the biopharmaceutical molecule that may not lead to any significant change in the drug’s HOS (Fig. 3.3C and F). These changes are often so small that they can be very difficult to detect and therefore lead to no measurable change in the biophysical properties of the biopharmaceutical. On the other hand, there are cases where a small chemical or physical change could lead to a significant change in the HOS of the biopharmaceutical and therefore, lead to measurable changes in its biophysical properties [4e6] (Fig. 3.3D and E). Thus, there is significant variation from one protein to another in the ability of a protein to respond to the same chemical or physical change. Even within the same protein the same modification in one area of a biopharmaceutical can lead to no significant change in HOS, while the same type of modification in another part of the same protein molecule can lead to very significant HOS changes [7]. As a result, to minimize the inability of detecting a change in a protein drug’s biophysical properties (and therefore its HOS), we are left with the risk mitigating task of applying multiple biophysical tools that probe different biophysical properties of these drugs. In the case of chemical changes (PTMs), it should be noted, however, that it is also possible that a chemical change (especially on the surface of the biopharmaceutical) may lead to a very measurable change in the biophysical property of a biopharmaceutical but result in no measurable change in its HOS (Fig. 3.3B) [7]. The assessment of such situations in fact is an important development activity in making certain that novel classes of protein drugs that require direct chemical modification are not altered in terms of their HOS. Such protein drugs include those that involve the direct coupling of a second protein (in forming fusion proteins), a large chemical polymer (e.g., pegylation), or a small toxic compound to make a conjugated biopharmaceuticals, (antibody drug conjugate, ADC) [8e13]. While this indirect HOS approach may be more straightforward, there are still important considerations, such as “how well do the measured biophysical properties proxy for assessing information on a biopharmaceutical's HOS”? The answer to this question hinges on finding and using an adequate number of biophysical tools that can collectively provide information about the various parts of the protein’s HOS that regulate different physical properties of the protein drug. By measuring several different biophysical properties that are dependent on different attributes of the biopharmaceutical’s HOS, a better opportunity is created for generating a more effective fingerprint of the biopharmaceutical’s HOS. Hence, the success of this approach ultimately rests on understanding what attribute of a biopharmaceutical’s

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HOS is being probed, how sensitive the technique is at assessing a change, and are they important. As an example, consider the use of global hydrogen exchange (HX) with mass spectrometry detection for probing the conformational properties of backbone amide hydrogens on an intact biopharmaceutical (Chapter 12). When a biopharmaceutical is placed into a formulation buffer made with deuterated water instead normal water, many amide hydrogens present in a biopharmaceutical will start to exchange with deuterium in the deuterated water (H/DX). However, a change in the HOS of a biopharmaceutical can increase or decrease the H/DX of some amide hydrogens. In some cases, the result (change in the total number of exchanged hydrogens) may not show any change, indicating no change in HOS. Another example concerns intrinsic fluorescence measurements (Chapter 5). In this case, the signal recorded is from a limited number of reporter elements (i.e., aromatic amino acid residues only). If the change in a protein drug’s HOS is not located within a region where the reporter element is present (i.e., an aromatic residue), the change is likely to go undetected. Similarly, if there is a change in the drug’s conformation that contains more than one reporter element (e.g., two tryptophan amino acids), it is possible that the change in the HOS could cause one tryptophan to increase in fluorescence while the other may decrease in fluorescence. In the latter situation, unless the fluorescence signals from each tryptophan can be uniquely resolved (possibly via a large-enough wavelength shift), the overall fluorescence spectrum will look like that of the original unaltered protein drug. Clearly, having uniquely resolved readouts from as many parts of the protein drug as possible will increase our ability to better characterize and detect a change in the HOS of a biopharmaceutical. In addition, we will also see that, in some cases, the ability to have more than one biophysical technique to probe the same biophysical attribute using different physical principles (e.g., size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC) in assessing protein aggregation) can also be beneficial in securing confidence in the data and the conclusions that are drawn [14,15].

3.3 General considerations in analyzing the biophysical properties of biopharmaceuticals Given the number of commercial biopharmaceuticals that are on the market where the active pharmaceutical ingredient (API) is a protein, the word “biopharmaceutical” has commonly been found to mean protein drug (or protein therapeutic). In 2012, the Food and Drug Administration (FDA) defined any polypeptide chain longer than 40 amino acids [16] as a protein. Although other chemical classes of biopharmaceuticals exist, where the API is not a protein (e.g., RNAi, DNA (in gene therapy) [17,18], peptides (polypeptide chain <40 amino acids), or polysaccharides) [19,20], their contribution to the commercial landscape of the biopharmaceutical industry is at present still very small. As indicated in the previous two chapters, our focus in this book is on protein drugs (or protein biopharmaceuticals). From Chapters 1 and 2 the words “complex,” “folded,” and “fragile,” come close to providing a quick and coarse summary of a protein drug’s biophysical structure. However, there is another attribute that has not been given as much attention until recently, which concerns their “dynamic” or “temporal” properties. The relatively large range of motion, in terms of time, amplitude, and the extent of the physical structures involved in these

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movements far exceeds those encountered by simpler and smaller drug molecules referred to as pharmaceuticals, which for all practical purposes have static structures. Thus, it should be no surprise that protein drugs cannot be treated in the same manner as their pharmaceutical counterparts. A critical element in developing a successful protein drug requires a clear understanding of the stability boundaries of its HOS. Such knowledge is of great importance, as indicated in Chapter 2, not only for developing formulations that will meet long-term stability requirements of the final form of the commercial drug, but also in ensuring that the HOS of the biopharmaceutical is maintained in all areas where the biopharmaceutical undergoes processing (which includes purification, formulation compounding, preparation of dosing solution, and an array of testing that includes biophysical measurements). In some cases, however, the processing of these protein drugs can lead to structural alterations in the protein molecule (e.g., during low pH exposure during viral inactivation steps). The extent and duration of these structural changes must be carefully considered and understood to assure the protein drug’s HOS successfully returns to its efficacious, native, and stable structure as it moves through the various manufacturing steps during its production into its final drug product form. It should be noted that in some areas of biophysical testing, the measurement process itself utilizes the systematic disruption of the biopharmaceutical’s HOS as the basis for detecting a change in the HOS of these molecules. In using this approach, the disruption process must be applied in a very controlled environment so that it can be done reproducibly (e.g., differential scanning calorimetry, DSC, see Chapter 11). In conducting such biophysical measurements, some basic properties (which in the case of DSC is monitored by carefully recording the uptake or output of heat as a function of temperature, see Chapter 11) and insightful information can be collected that reveal subtle changes in the biopharmaceutical’s HOS (see Sections 3.4 and 3.5.2 for a further discussion on this topic in this chapter).

3.3.1 Dealing with the physical and chemical state of a biopharmaceutical in conducting biophysical measurements (sample processing) In Chapter 2 the problems associated with handling and processing biosimilar biopharmaceuticals and the accompanying innovator biopharmaceuticals was discussed in some detail. In the case of developing an original biopharmaceutical, many of the same challenges exist when conducting biophysical measurements. The simplest way to deal with the sample handling issue is to minimize it. To achieve this, the most straightforward approach is to analyze the sample “as it exists” in the drug container closure (vial or whatever container the sample is provided in) by placing a sample of the drug directly into the biophysical instrument and making the measurement as quickly as possible. There is no other way to do less to a sample in order to make the biophysical measurement. The issue is now a question of whether the measurement can be successfully performed on the sample, given its concentration and solution matrix (i.e., formulation buffer). If this is not possible, for example because the biopharmaceutical’s concentration is too high, the next logical step is to dilute the sample using its formulation buffer. Here, not much of a change will be introduced into the protein, but even this simple step may have removed information about what might be going on at its vialed higher concentration, for example, reversible concentration-dependent aggregation [21,22]. If there is aggregation and it is reversible one

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may suppose that it is not critical or important to understand. However, such aggregation could transition into a permanent change over time [23]. In this case, it is very likely that the key mechanism for detecting this situation would be during long-term stability testing. If this knowledge can be gained at a much earlier time during the development process, via appropriate biophysical studies, it would be a great benefit to the drug developer, since it would significantly reduce the amount of time and money relative to the problem surfacing later during these long-term stability studies. If high concentration is not an issue, but rather it is the sample’s very low concentration that is the problem, the sample may require a concentration step before the analysis is performed. This can be easily accomplished with centrifugation spin cartridges, but as will be discussed below, this can also introduce issues. A more difficult and potentially challenging problem in sample processing is the presence of certain formulation components or excipients that can interfere with biophysical measurements [24,25]. In this situation, diluting the drug may still be an answer, but in this case, a different buffer will be needed that does not interfere or hopefully perturb the physicochemical state of the drug. The success of this approach is controlled by a combination of factors. The first is the need to dilute the initial level of the interfering substance to an acceptable level, which will allow the biophysical measurement to be successfully carried out. The second is the need to make sure that the level of dilution does not dilute the biopharmaceutical so that it has a negative impact on the accuracy of the measurement. If the lowest level of dilution cannot satisfy these criteria, the protein drug will need to undergo passive dialysis or an active buffer exchange via centrifugation spin cartridges [26]. The use of the latter comes with the potential for issues (in addition to those indicated in Chapter 2) that includes the following: 1. During the buffer exchange process, a portion of the protein drug will be concentrated to a very high concentration at the liquidemembrane interface. This could introduce problems associated with aggregation that might lead to artifacts. 2. Depending on the porosity of the membrane, two different outcomes can be encountered. (1) If the porosity is too low, some excipient components may be concentrated in the protein solution, e.g., nonionic detergent that have formed micelles might not pass through the membrane easily [27] and (2) some buffer component may be selectively removed from solution due to preferential binding to the membrane surface, for example, again detergents [28,29]. 3. The possibility of selectively removing low molecular weight (MW) impurities (e.g., fragmented protein drug material). Additionally, it should be noted that passive dialysis has its own limitations as well. For example, the time required to adequately remove interfering solution components may take more than a day and require several exchanges of buffer or the sample may non-specifically interact with the membranes surface. Such time requirements may not be compatible with the stability of the biopharmaceutical. For biophysical characterization measurements, those methods that minimize sample handling would be highly preferable. However, those approaches that do require sample processing steps should not necessarily be avoided. Rather, what we are emphasizing here is that important consideration and care should be given to the potential impact of sample handling and processing steps [30] to ensure that whatever potential stress or change influencing the physicochemical environment during sample processing can be tolerated and I. Proteins and biophysical characterization in the biopharmaceutical industry

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at the same time be applied reproducibly to all samples. By making these critical assessments one is better assured that the observed differences or even comparability between samples are real.

3.3.2 Biophysical properties of a biopharmaceutical that can be probed to assess information about its HOS Fig. 3.2 outlines the hierarchy of spatial resolution as it pertains to a protein’s HOS. For biophysical analysis, as size increases, resolution generally decreases, and the level of change and the significance of that change become more difficult to recognize. In this section, a brief discussion concerning the main areas covered by the analysis of protein HOS is given. 3.3.2.1 Changes in quaternary structure It should be realized that one of the most troubling properties of a protein drug is its propensity to form aggregates. This property is specifically tied to the molecule’s quaternary HOS, which also corresponds to the lowest level of resolution of a protein’s HOS (Fig. 3.2). At this structural level, the property of MW and size are good attributes that can be used to monitor the formation of aggregates. Although aggregates can be covalent in nature, which makes their detection via biophysical tools in some ways easier, more often protein drug aggregates arise through a very wide range of weak noncovalent interactions, making their assessment more challenging. This challenge arises because the noncovalent bonds that hold the aggregates together are fragile and can be easily altered. Such alteration could cause aggregates to fall apart or to continue to aggregate into higher MW aggregates (e.g., dimers could further aggregate to form a tetramer). The latter outcome (i.e., nucleation-controlled aggregation [23,31]) is more of a concern and more likely to occur in cases where aggregation initially proceeds through conformational instability, where less stable forms of the protein drug can become trapped, exposing surfaces with a greater propensity to form aggregates. Furthermore, the aggregates present in a given protein drug sample may, in some cases, possess both a covalent and noncovalent nature [32e34]. In fact, protein drug aggregates can display heterogeneous forms [32,35], as well as some interesting properties, for example, reversible concentration-dependent behavior [21e23,36,37]. In assessing protein drug aggregation, the use of multiple biophysical methods (e.g., AUC, SEC, and light scattering, LS) is likely required to adequately evaluate and characterize the aggregates overall complexity, properties [14,15,38], and to deal with the inherent and unique limitations of each method. Simply knowing the total amount of aggregation present at any one time may not be enough to forecast the potential for aggregation problems that may arise later in development, particularly during long-term stability studies. Hence, studying the aggregation process and the aggregates themselves is important to uncover and avoid problems later on in the drug development process. In addition, given the complexity, diversity, instability, and the range of sizes of the aggregates that can be encountered (dimers to subvisible particles), along with the unique limitations of the biophysical methods only further points to the need for using more than one biophysical technique to detect and characterize these materials. While aggregation results in a material having an increase in mass, there is another attribute that is important to consider. This attribute concerns the interface created between two or more monomeric drug molecules, which facilitate the binding between the proteins

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that normally sequesters surface-exposed chemical groups from bulk solvent interactions. Characterizing the nature of the sequestered chemical groups could serve as useful surrogate markers for aggregation and help to further characterize where and how these drug molecules are interacting. Ultimately, such studies could help provide information on how to ultimately suppress or even eliminate the aggregation [39]. 3.3.2.2 Changes in secondary and tertiary structures Changes in a protein drug that affect their secondary and/or their tertiary structure can be very difficult to detect, especially if they involve only a very small part of the protein. However, those changes that lead to alterations in surface properties offer a possible route for being detected via chromatographic techniques. This use of chromatography is due to the importance of protein surface interactions with the surface of the chromatographic material. Such changes in chromatography may occur directly through alterations in protein-chromatography interactions or indirectly through changes in bulk solvent-protein preferential interaction (see Chapter 14) [40,41]. Thus, any change in the exposed chemical groups on the protein surface can cause different interactions to occur with the chromatographic particle's surface, which can translate into a different chromatographic elution profile. In addition, changes in the secondary/tertiary structure of the biopharmaceutical could also affect the overall global size and shape of the protein drug molecule, allowing hydrodynamic techniques such as AUC and viscosity to be used to detect changes [21,38]. Differences in the secondary and tertiary structure can also alter the number of weak noncovalent bonds present and/or their configuration, which could weaken or strengthen the global or local structure of a protein drug. Such changes could be detected via analysis of structural stability (i.e., DSC and chemical denaturants) or perhaps by spectroscopic techniques [32,42].

3.4 The utility of using stress to monitor changes in the HOS profile of a protein drug As part of assessing a protein drug candidate’s developability and/or manufacturability, and in searching for an optimum formulation, the monitoring of a protein drug’s HOS plays an important role (see Chapter 2). This is reflected in the many studies that expose these drugs to an array of stresses that are aimed at assessing how robust they are at withstanding various conditions. These stresses can include different temperatures, agitation, freeze/thaw cycles, and exposure to various contact surfaces, to name just a few examples. The key reason for applying stress is to learn how and/or whether the protein drug’s HOS is impacted. The longer and harsher the conditions that a protein drug can withstand a given stress, the more favorably, in general, the drug candidate and/or its formulation will be looked upon as being appropriate. Detecting the onset or presence of a HOS change resulting from a change in the manufacturing process, a less-than optimal formulation or a potential drug candidate with a less-than optimal drug developability profile may take months or years to uncover using normal stability studies. In such studies, the protein drug is incubated under different

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conditions for various amounts of time (e.g., 3, 6, 9, 12 months or longer), which are dictated in a stability study protocol. In these studies, at specific and predetermined time points, aliquots of the protein drug are removed and analyzed using an array of different biochemical, biological, and biophysical methods to assess the stability, integrity, and activity of the drug. If the first negative result is not observed until the 12-month time point, a year may have been wasted. Hence, tools capable of detecting small differences in a drug or a small difference in a small population (variant forms of the protein drug) to sound early alarms about potential weaknesses in the drug rather than wait 1 or 2 years are highly desirable. One approach for achieving this is to apply some form of stress to a biopharmaceutical over much shorter time periods. That the goal is to achieve a situation where the stressing process can help accelerate and reveal subtle differences that may exist in the native state of the biopharmaceutical. Without the use of such stress, existing differences may be too small to detect under stable conditions or take too long to see using normal stability studies. Fig. 3.4 graphically illustrates the benefit of such an approach. Here, a stress is applied in a wellcontrolled repeatable way to two biopharmaceutical samples that only slightly differ in the native state, but the difference is too small to detect. On applying and then removing the stress, the resulting states may now more easily reveal the subtle underlining difference that initially existed (Fig. 3.4A). Hence, the basis for introducing stress studies is to leverage the process of

FIG. 3.4

The utility of using stress studies to facilitate the process of detecting differences in the HOS of protein drugs. (A) In this example, two protein drug samples, in their native state, differ slightly in their HOS, but the difference is too small to detect in terms of the data read out from any biophysical method (difference is within the uncertainty of the statistical variability of the method). However, if an appropriate stress is applied and subsequently removed at some later time point (t ¼ n), the resulting difference between the original proteins may be revealed (note: depending on the circumstance the stress may need to be in place while the biophysical measurement is being made to reveal the difference between the two samples. (B) The same situation as in “A,” but a biophysical method is used to study the kinetic process of the change induced by the stress to detect the initial difference in the HOS between both drug samples. (C) The same situation as in “B,” but in the final state that both samples show no difference in HOS. In this latter case, a difference can only be detected by monitoring the kinetic process leading to the change.

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controlled stressing to create a situation that leads to an amplified difference in a drug’s altered state(s) that allows for the small native state difference to be detected (again see Fig. 3.4A). In addition, if the stress can be initiated and then monitored using a biophysical method, it is also possible that the kinetic process or path leading to these states may serve as a useful tool to help detect subtle differences between biopharmaceutical drug samples (Fig. 3.4B and C). However, it should be noted that care needs to be taken in applying this approach because the level of stress required to reveal a difference may represent phenomena that are unrealistic and of no real concern. Thus, the difference that may exist between two or more drug samples could have no realistic bearing in terms of meaningful instability that are relevant to what the drug will encounter in the field (i.e., heating a protein to 90  C).

3.5 Present biophysical toolbox There are many different biophysical tools available for characterizing a protein drug’s HOS. Table 3.1 summarizes a number of the more commonly employed tools with their indicated basic strengths and brief comment concerning what features of a protein drug’s HOS they best probe. Most of these tools typically focus on exploiting one major physiochemical property of a protein such as size, secondary structure content, thermodynamic stability, or aggregation properties. However, it is not uncommon to find that some of these tools can also probe other properties of a biopharmaceutical at the same time. As a result, multiple pieces of information can at times be extracted from a given measurement. In addition, some biophysical methods can overlap in their ability to study the same biophysical property of a protein. For example, far-ultraviolet (UV) CD and Fourier transform infrared spectroscopy (FTIR) both monitor physical properties of a polypeptide chain backbone, while SEC and AUC can both provide information on a protein’s total aggregation. In these paired examples, each method provides information on a similar or even the same property but does so through very different physical phenomena. Hence, the methods used in each paired example are orthogonal to each other in their approach to assess the same or similar biophysical parameter. Such redundancy may seem wasteful, leading one to ponder on why one would use different methods to get the same data? This turns out to be a rather critical and necessary component in the successful characterization of a biopharmaceutical. For example, SEC is almost always chosen to access a biopharmaceutical’s aggregation because it is fast (typically takes several minutes), relatively easy to run, and requires little sample (a few micrograms) [2,14,15]. These attributes allow SEC to fit very well into the process development area as a useful and productive tool. However, once the sample is injected on to a column, the protein is diluted, and its physicochemical environment changes significantly due to the difference in the sample’s formulation buffer and the mobile phase used in the SEC method [14]. As a result, information concerning concentration-dependent effects may be lost and aggregates initially present may disappear or in some rare cases be induced due to these changes. Additionally, SEC is performed with a column packed with porous chromatography particles which exposes the proteins to very high surface area that is chemically unique. While this chemical surface is theoretically inert in an appropriately functioning SEC method, secondary interactions are possible and have been reported [15,43,44] that may remove the aggregate material from solution. Consequently, AUC is often needed to confirm the validity of the SEC results at critical

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TABLE 3.1

The current biophysical toolbox: attributes associated with selected tools.

AA, amino acid; AF4, asymmetric flow field flow fractionation; B2, second viral coefficient; Cryo-EM, cyro-electron microscopy; CZE, capillary zone electrophoresis; D, diffusion; ET, electron tomography; FFF, field flow fractionation; HIC, hydrophobic interaction chromatography; HF5, hollow fiber flow field flow fractionation; icIEF, capillary imaging isoelectric focusing; IEF, isoelectric focusing; ITC, isothermal titration calorimetry; kD, interaction parameter; MW, molecular weight; SANS, small angle neutron scattering; SAXS, small angle x-ray scattering; SIC & CIC, self-interaction & cross-interaction chromatography; WAXS, wide angle x-ray scattering; h, viscosity

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points in the development cycle because it does not suffer these limitations. Nevertheless, AUC has its own set of limitation that make its use as a routine biophysical aggregation tool difficult. This includes higher labor and intensive data processing. AUC is also a low sample throughput method and suffers from an inability to measure very low levels of aggregation (level below w2%) in comparison to a well-functioning SEC method (see Chapter 9, Section 9.5.6.1). It should be apparent that each biophysical tool has its own limitations. Thus, the only way to detect and verify differences or assure comparability when limitations are being encountered is to take advantage of orthogonal methods that measure the same or similar parameters. While there are many different techniques and classifications, for the purposes of this book, we have chosen to discuss and categorize only the more commonly used methods. Our classification includes nine main categories: 1. 2. 3. 4. 5. 6. 7. 8.

Hydrodynamic (transport) methods (e.g., AUC, see Chapter 9). Thermodynamic methods (e.g., DSC, isothermal calorimetry (ITC), see Chapter 11). Chromatographic and Electrophoretic methods (see Chapter 14). Spectroscopic methods (e.g., CD, FTIR, UV, fluorescence, and NMR; see Chapters 5, 6, and 13). Mass spectrometry (H/DX-MS, covalent labeling, etc.; see Chapter 12). Scattering methods (e.g., SLS, DLS, SAXS and WAXS; see Chapter 8). Particle analysis (e.g., light obscuration and flow imaging; see Chapter 10). Other methods (e.g., affinity, kineticechemical reaction, digestions, and denaturation).

This categorization is by no means definitive and as will be seen in subsequent sections of this chapter and in the subsequent chapters of this book, some biophysical methods could be categorized in more than one grouping. Again, it is not the aim of this book to describe and discuss every biophysical method in use, but rather to describe those techniques that can be more universally used with less difficulty, offer the most information, and add the most value toward the characterization of a biopharmaceutical’s HOS.

3.5.1 Methods for studying the hydrodynamic properties Methods that study the hydrodynamic properties of biopharmaceuticals essentially probe the molecule’s global HOS properties. These techniques are primarily concerned with the biopharmaceutical’s quaternary structure (the assembly of those biopharmaceuticals that are composed of more than one polypeptide to form a final unique structure) that are reflect by their ability to characterize mass, size, and shape. However, in some cases, additional information concerning the size and shape of the monomeric biopharmaceutical can also provide information about changes in their secondary and tertiary structure. These global HOS properties are probed by monitoring the molecule’ movement or transport through a liquid medium in response to some driving force. Such driving forces include. 1. Movement driven by the thermal kinetic energy content of the protein as exploited in translational diffusion measurements as carried out in DLS. 2. Movement in response to a high centrifugal field as exploited in sedimentation velocity AUC (SV-AUC). 3. Resistance to a controlled flow force as exploited in viscosity measurements.

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4. Movement in response to two perpendicular liquid flow fields as used in Asymmetric Flow Field Flow Fractionation (AF4) or more recently Hollow Fiber Flow Field Flow Fractionation (HF5). 5. Partitioning between a mobile and stationary phase via thermal kinetic energy content of the protein (diffusion) in combination with steric constraints as exploited in size-exclusion chromatograms (SECs). In general, data derived from these methods can yield a simple numerical parameter such as a diffusion coefficient (D), hydrodynamic radius of an equivalent sphere (Rh), sedimentation coefficient (s), and total aggregation, with the latter being one of the chief reasons that these methods are employed in the biopharmaceutical industry. On the other hand, some of these methods can also provide a more detailed picture or profile of the biopharmaceutical sample’s heterogeneity or microheterogeneity via distribution plots. The latter information is by far more useful since this type of information becomes a unique fingerprint of the biopharmaceutical. In comparison providing only a single value for parameters such as s and D mentioned above is not unique. These single value parameters can correspond to a homogeneous sample or an infinite number of different combinations of mixtures that provide the same parameter value [45,46]. The ability to generate distribution plots is far more useful and highly sought after, since they effectively provide a more unique and higher resolution picture (fingerprint) that characterizes the protein drug. These distribution plots show the concentration of the biopharmaceutical material with a value for the physical parameter “x” being measured, c(x). For example, the concentration of material with a specific s value, c(s), as a function of s in the case of the SV-AUC or the concentration of material with a specific Rh value, c(Rh), as a function of Rh in the case of DLS. Plotting such c(x) versus x values yields a distribution plot, as shown in Fig. 3.5. Given the uniqueness of these distribution plots a far better assessment of similarity or lack of similarity between biopharmaceutical samples can be achieved in comparison to those biophysical techniques that only yield a single value parameter.

FIG. 3.5 A simple example illustrating a distribution plot of a biophysical parameter "x", for example, distribution of sedimentation coefficients, s, measured in Svedberg units (S), or hydrodynamic radius of an equivalent sphere, Rh, measured in nanometers. In these plots, the value of the biophysical parameter, for example, s or Rh, is plotted on the x-axis, while the amount or concentration of the protein drug material with a specific value for s or Rh is plotted on the y-axis.

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An interesting area where hydrodynamic measurements are finding significant use is in the characterization of high-concentration biopharmaceuticals. Here, the key area of concern is the induction of aggregates over time due to the common driving force of high concentration. This turns out to be a very difficult task to assess since any reversible aggregation that might be present can be easily masked or hidden by the strong opposing effects of solution nonideality, which also increase with high protein concentrations [47e49], also see Chapter 15 for further discussion on this topic. However, progress in using these types of biophysical tools in combination with computer modeling to deconvolute biophysical data acquired over a range of concentrations are beginning to appear that may help extract useful information [39].

3.5.2 Methods for studying the thermodynamic properties The two most dominant methods used in this area are DSC and ITC. Of these, the former has found greater use in assessing information on the HOS of biopharmaceuticals (see Chapter 11 for more details). In this approach, the HOS of the biopharmaceutical is examined by studying the flow of heat as a protein is exposed to a continuous increase in temperature. The utility of these measurements in studying the HOS of protein drugs is nicely described by Creighton, “The expansion of a macromolecule with increasing temperature reflects its atomic packing and flexibility [50].” Simply put, there is a certain amount of energy associated with the folded conformation of a unique linear arrangement of specific amino acids and its dynamic structure. In addition, the higher order packing and interaction of more than one polypeptide chains to form the quaternary structure of a functioning drug or an aggregated form of the drug also contribute to the resulting thermal signature of the biopharmaceutical. Therefore, it becomes possible that perturbations in a biopharmaceutical’s HOS may be revealed by monitoring changes in its melting profile. Thus, the energy released or taken up as a function of increasing temperature provides a unique fingerprint of a biopharmaceutical’s HOS. In conducting this type of experiment, the investigator must realize that as a protein unfolds, a multitude of reactions are ongoing, including the release and uptake of heat from the disruption of secondary bonds, changes in solvation, and preferential interactions of chemical elements of the protein with various components of the formulation buffer. In addition, interactions between the unfolded, partially unfolded, and even intact (still native) protein drug molecules are also possible, which can cause aggregated material to form that might yield precipitates (i.e., nucleation events) [23,51]. As far as DSC measurements are concerned, the thermal output recorded at any temperature is the sum of “all reactions” that are going on at any given time. Since the objective of thermodynamic analysis is to interrogate the sum spectrum of heat released or taken up as a function of increasing temperature, DSC provides a significant capability to distinguish differences between samples of the same protein drug. This is particularly useful for protein drugs that are multidomain proteins. Since domain structures are often unique and independent structural elements of a multidomain protein (see Chapter 1) that can melt at different temperatures. The ability to resolve the melting of different domains yields characteristic plots, called thermograms (which effectively correspond to thermal distribution plots of the change in heat, or more accurately heat capacity, as a function of temperature plotted vs. temperature). Such plots can provide the melting temperature and the associated heat of the denaturation of each domain in the biopharmaceutical molecule.

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The second thermodynamic technique mentioned is ITC. In this method, a known fixed amount of a biopharmaceutical is titrated with known amounts of another material that will act as a ligand and uniquely bind to the biopharmaceutical, such as the drug target (e.g., membrane receptor) or important cofactor (e.g., calcium [10]. In carrying out this titration, a fixed amount of heat is either given off or taken up. The titration of a specific binding partner continues until the binding sites on the fixed amount of biopharmaceutical placed in the instrument are saturated. Given that the binding reaction requires the biopharmaceutical to maintain a necessary and unique HOS to facilitate the binding, any changes in the HOS could influence the binding and thus the amount of heat change recorded per unit binding site. Hence, ITC could be useful to detect changes in HOS by taking advantage of the unique and specific binding reactions that take place between the biopharmaceutical and its binding partner that is employed in these measurements.

3.5.3 Methods for studying chromatographic properties In using chromatographic methods for characterizing the HOS properties of biopharmaceuticals, we are typically concerned with methods that function through direct enthalpic interactions involving secondary bonding interactions between the chemical groups on the surface of the biopharmaceutical and the stationary phase of the chromatographic material under specific solution conditions. However, there is one form of chromatography that is of great importance to the biopharmaceutical industry whose mode of separation is not based on enthalpic interactions, SEC. In SEC, chromatographic separation is achieved through entropic properties in combination with the pore structure of the chromatography particles. The presence of enthalpic interactions is detrimental to SEC (see Chapter 7). Notwithstanding SEC, chromatography is a methodology in which one probes the unique surface chemical topology of a biopharmaceutical in terms of its interactions with the chromatographic particle's surface chemical topology in combination with the mobile phase solution conditions used during chromatography [52,53]. Since a biopharmaceutical’s unique surface chemical composition and topology are dictated by several attributes (which includes a protein’s primary structure, HOS and dynamics), chromatographic methods offer unique opportunities to detect subtle alterations in a protein’s HOS that arise due to changes in the protein drug’s chemical surface topology (see Chapter 14 for more on this topic). A change in HOS that affects the spatial arrangement of chemical groups on the surface of a protein drug, either directly or indirectly (via allosteric effects), could be detected as an altered elution profile, (e.g., shift in retention time, peak shape or presents or disappearance of peaks). Using an array of chromatographic materials with different surface chemistries, many different aspects of a biopharmaceutical’s HOS can be investigated. However, as indicated in Fig. 3.3C, chemical changes can occur to a biopharmaceutical (e.g., PTMs), which have no significant impact to its overall HOS. These chemical changes, while insignificant to a biopharmaceuticals HOS, they can nevertheless result in a change in the biopharmaceutical’s biophysical properties that in this case just correspond to changes in chromatographic properties. Even in cases where large chemical molecules are coupled to the target drug for various reasons, e.g., through the fusion of two proteins, as in the creation of a fusion protein or the conjugation of a large chemical entity, such as polyethylene glycol (PEG) via pegylation (i.e., 10 or 20 kDa PEG), the HOS of the target drug portion of a biopharmaceutical

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can be unaffected, but its chromatographic properties can be greatly changed [54] (Fig. 3.3B). Thus, chromatographic data should be evaluated carefully, especially when detected chromatographic differences between biopharmaceutical samples are uncovered. In many cases, such differences should incorporate follow-up investigation work with other biophysical methods to fully understand the significance of the difference. Given the high diversity of different chemical surfaces commercially available, chromatography can be a powerful tool for detecting small changes in the HOS of a biopharmaceutical by monitoring specific chemical interactions with chromatography materials with a different surface chemistry. This capability is further enhanced by the ability to couple chromatography with biophysical and biochemical detectors (e.g., MS, and LS detectors), which extends our ability to further characterize both the biophysical and chemical properties of the different fractionated components (peaks) present in a given biopharmaceutical that is revealed during a given chromatographic separation. In addition, the ability to make highly specialized chemical surfaces is also available. One particularly interesting example is self-interaction chromatography (SIC) [55e57]. This form of chromatography involves the immobilization of a biopharmaceutical to a chromatography particle’s surface to make a highly unique chromatographic stationary phase. Using this chromatography material, the same biopharmaceutical is then injected onto the column, and the elution behavior of the biopharmaceutical is monitored to assess its ability to interact with itself (via the same biopharmaceutical bound on the chromatography particle’s surface) under different environmental conditions. Such studies could be of great importance in terms of understanding the aggregating propensity of these drugs, especially at high concentrations, see chapter 15 for further discussion on this topic. For more detail information concerning the use of chromatography for detecting changes in a biopharmaceuticals HOS, see Chapter 14.

3.5.4 Methods for studying electrophoretic properties Electrophoretic methods probe the HOS of biopharmaceuticals by predominantly exploiting their charge. Since the ability of various amino acids to bear a charge is dependent on its local physicochemical environment, most important of which is the pH, the charge on a biopharmaceutical is highly dependent on the drug’s formulation buffer and its local HOS conformational environment. In the biopharmaceutical industry, there are three major forms of electrophoresis, which includes sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native electrophoresis and isoelectric focusing (IEF), which are all today commonly performed in a capillary format. In the case of SDS-PAGE, the use of SDS in the method effectively destroys any opportunity to obtain useful information about a biopharmaceutical’s HOS other than the information concerning the covalent nature of aggregates that might be present in the sample. Hence, we will have no further discussion concerning SDS-PAGE. In the case of native electrophorsis, samples are run under nondenaturing conditions, which enables the HOS of a biopharmaceutical to be maintained giving this technique the opportunity to provide information about changes in the HOS of the sample being characterized. In native electrophoresis,

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biopharmaceutical separation is dependent on the molecule’s shape, MW and intrinsic charge (through a gel support network that introduces some sieving effect). It should be noted, however, that native electrophoretic bands tend to be much broader than electrophoretic bands observed via SDS-PAGE and, in many cases, these bands can be significantly smeared. Nevertheless, when good separation conditions are found native electrophoresis can be a quick and easy way to probe a sample's size and heterogeneity via charge. In IEF, electrophoresis is conducted under either native or denatured conditions, where the influence of an electric field on a solution containing an appropriate mixture of ampholytes (which provides the pH gradient necessary for separation) cause the biopharmaceutical to migrate to a pH where the net charge on the protein drug molecules is zero. The pH at which this occurs is referred to as isoelectric point, or pI. Although the pI is dependent on the amino acid composition of the biopharmaceutical, when IEF is conducted under conditions where the native state of the biopharmaceutical is maintained, the measured pI will also be a function of the HOS of the biopharmaceutical. Under such native conditions, the folded state of the protein drug will significantly influence the ability of different amino acids to ionize. As a result, changes in the HOS of the biopharmaceutical may be detected via changes in the resulting electropherogram or/and measured pI. It is important to remember that not all HOS changes will necessarily induce a change in charge and conversely not all changes in charge reflect to a change in HOS. In the case of the latter, a PTM may add a fixed charge to the surface of a biopharmaceutical by chemical modifications, i.e., sialylation of attached carbohydrates or phosphorylation of several different amino acids, without changing the biopharmaceuticals HOS (see Fig. 3.3D as well as further discussions on native IEF (nIEF) given in chapter 14). A more modern form of native electrophoresis that is conducted in a capillary format in the absence of any gel, which also offers the opportunities to characterize a biopharmaceutical’s hydrodynamic properties is capillary (free) zone electrophoresis (CZE). The movement of a biopharmaceutical in CZE under the influence of an electric field (under native conditions) is dependent on two key features of the biopharmaceutical; (1) its net charge and (2) its hydrodynamic properties (i.e., global shape). Both these features are affected by the HOS of the biopharmaceutical. A unique attribute of CZE is its very high resolution, which is due to a phenomenon called plug-flow. The plug-flow model eliminates the normal peak broadening effects of sample movement through an open tube, due to the unique electrophoretic movement (flow) of the sample molecules and bulk buffer matrix resulting from the potential presence of electroosmotic flow (which could enhance or oppose the normal electrophoretic mobility of the protein drug and its various variant forms). The resulting high resolution and very sharp peaks that CZE can provide can result in very impressive fingerprint of a biopharmaceutical’s microheterogeneity (e.g., see Figs. 2.3 and 2.4 in Chapter 2). For more detail information concerning the use of electrophoretic separation sciences for detecting changes in the HOS of biopharmaceuticals, see Chapter 14. In addition, we also refer the interested reader to the following books for a more thorough discussion of this topic: Capillary Electrophoresis of Proteins and Peptides by Strege and Lagu [58] and to Characterization and Analysis of Biopharmaceutical Proteins by Lou et al. [59].

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3.5.5 Methods for studying spectroscopic properties Spectroscopy encompasses a group of biophysical methods that are concerned with the initial absorbance of electromagnetic radiation by specific structural groupings of chemical elements or atoms on a molecule, called a chromophore. Of the distinguishing features between each of these various methods, the property concerning the limited range of wavelengths of the electromagnetic spectrum that a given spectroscopic method can use is the most important. Given this uniqueness, each spectroscopic method only probes certain chromophores on the protein drug, e.g., aromatic groups and polypeptide bonds. In general, these interactions proceed by the initial absorbance of some proportion of the incident electromagnetic radiation; however, in some forms of spectroscopy (fluorescence), this absorbed electromagnetic radiation can be reemitted at different wavelengths. Although spectroscopy is a diverse field and includes many methods, those methods that make measurements in the UV, visible (VIS), infrared, and radio-wave region of the electromagnetic spectrum are generally found to be of the most interest to the biopharmaceutical scientist. What links these spectroscopic methods to their ability to provide information about the HOS of a biopharmaceutical is that the physicochemical environment of each chromophore influences their ability to interact with the impinging electromagnetic radiation. Since the physicochemical environment of these chromophores is determined by the folded state or HOS of the biopharmaceutical, spectroscopic methods can provide us with indirect information when the HOS of a biopharmaceutical has changed. In addition, information concerning the general nature of the physical and chemical environment of these chromophores on the drug molecule can also be obtained from the details concerning the amount of absorbance or emission of electromagnetic radiation and the specific wavelength at which these interactions occur. Thus, changes in the HOS of a biopharmaceutical may perturb or alter the physicochemical environment of some chromophores resulting in a change in the way a chromophore normally interacts with the specific range of electromagnetic radiation causing a change(s) in the readout (spectra) from a form of spectroscopy. It should be noted that the extent of the change in a biopharmaceutical necessary to detect the change in its HOS is by no means consistent from one spectroscopic method to another or from one biopharmaceutical to another. Factors such as what chromophores are perturbed and to what extent their local environment are changed as a result of a change(s) in HOS, the resolution, sensitivity, and selectivity of each spectroscopy method along with what fraction of the drug population has this change all play very important roles in determining whether these changes in HOS can be detected. In UVeVIS, fluorescence, and CD spectroscopy, the electromagnetic radiation that is utilized occurs in the UVeVIS region (w190e700 nm), whereas in FTIR, the wavelength of electromagnetic radiation utilized ranges from approximately 750 nm to 1 mm. In fluorescence emission spectroscopy, light of a certain wavelength is absorbed causing an electron to jump from a ground state to an excited state. As the electron relaxes back to its ground state, energy is lost and the system emits light with a different (longer) wavelength and lower energy. Molecules that exhibit fluorescence have aromatic groups, which in the case of protein drugs concern the amino acids Trp, Phe and Tyr with Trp being the dominate source of fluorescence in protein drug. In fluorescence, the physicochemical behavior of the fluorophore can be extremely sensitive to its physicochemical environment. Changes in polarity;

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exposure or exclusion from water will affect the fluorescent signal and inform the investigator whether these amino acids may be located predominately on the surface or in the internal folded region of biopharmaceutical. Thus, fluorescence is often used to investigate a protein’s tertiary structure. In contrast to UVeVIS and fluorescence, CD measures differences in how a molecule absorbs left and right circularly polarized light. The technique is sensitive to changes in the secondary structure of the protein’s polypeptide backbone (in the far UV, below w260 nm), as well as its tertiary structure (in the near UV, in the wavelength between w260 and 300 nm). As a result, CD is used widely in characterizing a protein’s HOS. In FTIR, the absorbance of electromagnetic radiation (in the wavelength range from w5500 to 8300 nm) is associated with the vibrational properties of the amide bond in the polypeptide backbone. Here, the stretching and bending characteristics of the protein’s amide groups in the polypeptide chain are measured. Since the peptide bond is directly involved in a protein’s HOS, in terms of its folding and secondary interactions that are involved in its stabilization, FTIR analysis is a useful tool for analyzing and assessing changes in a protein’s secondary structure. In general, these three forms of spectroscopic analysis occur in all stages of process development, from cell-line selection to final product characterization and lot-to-lot comparability. The details associated with the general theory, practice, and applications of UV, fluorescence, and FTIR will be discussed further in Chapter 5, while those associated with CD will be examined in Chapter 6. In NMR spectroscopy, the electromagnetic radiation that is utilized occurs in the radiowave region of the electromagnetic spectrum. While most forms of spectroscopy rely on the interaction of radiation with electrons, NMR spectroscopy is concerned with how the protons in the atomic nuclei of atoms absorb radiofrequency electromagnetic radiation in a magnetic field [60]. However, not all nuclei can provide an NMR signal. Only those that have appropriate spin properties will be active, which includes 1H, 13C, and 15N, being the ones that are of greatest interest in the case of biopharmaceuticals. Since each nucleus in a protein drug will be in a different spatially unique position, its ability to interact with the probing electromagnetic radiation will vary and be dependent on its local physicochemical environment. Hence, the resonant frequency of each nuclei will be different and will result in an observable unique shift (referred to as a chemical shift) in the NMR spectrum, which can eventually be linked to a nucleus’s relative position within the molecular structure of the biopharmaceutical. However, it is important to note that to use NMR for the purpose of detecting a change in the HOS of a biopharmaceutical, a knowledge of the location of these nuclei in the protein is not required. The simple generation of the NMR spectrum can serve as a useable fingerprint for the characterization of the HOS [61]. Since many biopharmaceutical proteins are large, often corresponding to 150 kDa or larger, the active nuclei that they contain will result in an NMR spectrum that will consist of overlapping NMR bands that will likely be too difficult to deconvolute. Even in NMR instruments with powerful magnetic fields (>800 MHz), which control the sensitivity and resolution of the resulting NMR spectrum, the challenge of deconvoluting such complex NMR spectra can be overwhelming. Hence, more complex approaches will be required. The details associated with NMR theory, practice, and applications, as well as the practicality for use in biopharmaceutical development will be addressed in Chapter 13.

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For NMR and fluorescence, various forms of these spectroscopies exist that provide an opportunity to extract dynamic information about the molecules being studied [62,63]. These methods constitute more advanced methodologies and, in some cases, involve more sophisticated hardware and software data analysis, which at present have been little explored in the biopharmaceutical industry. Nevertheless, these forms of NMR and fluorescence may warrant interest soon in attempts to increase our capability to detect a small unique difference in the HOS of biopharmaceuticals.

3.5.6 Methods for studying the mass spectrometry properties MS has distinguished itself as one of the most preeminent analytical techniques for studying protein’s primary structure. However, in the last decade, MS has extended its footprint beyond primary structure characterization and is now being used more regularly in structural biology to study and characterize protein conformation, dynamics, and proteine protein interactions [64e67]. Work in this area is also finding its way into the normal work flow of the biopharmaceutical industry [5,7e9,32,65e73]. MS is showing great promise for providing support in understanding biopharmaceutical/target interactions to help and improve the design of more effective protein drugs [5,7,8,32,67e71] and in the improvement of our ability to biophysically characterize the HOS of biopharmaceuticals with a much greater resolution [64e72]. In HOS structural analysis using MS, three main areas have evolved: native, covalent labeling, and a form of HX, called hydrogen/deuterium exchange MS (H/DX-MS). While both native MS and covalent labeling can be very informative in providing details pertaining to protein structure-function, we feel that H/DX-MS offers the most potential for the biopharmaceutical industry. H/DX-MS provides detailed information about a biopharmaceutical’s HOS in its formulation matrix and can reach spatial resolution levels down to one amino acid using very small amounts of material. HX is a phenomenon where hydrogen atoms that are covalently bound to certain elements (i.e., nitrogen, oxygen, and/or sulfur), spontaneously undergo exchange reaction with hydrogen atoms in the surrounding solvent. In general, it has been found that those hydrogen atoms associated with the amide bond in the polypeptide chain of protein drugs are of the greatest interest. This interest is due to their enormous range in the rate of HX, which can span eight orders of magnitude [68], and their strategic location along the polypeptide chain backbone, which serves as an excellent source of information about changes in a protein's HOS. This large dynamic range in amide hydrogen HX is associated with their physicochemical environment and interactions with other atoms via predominately secondary interactions (hydrogen bonding), which in turn is determined by the folding of the polypeptide chain to form the HOS of the protein drug. Hence, it is this “inherent and natural built-in reporting property” of HX of the amide hydrogen in the polypeptide chain backbone that depends on the HOS of the molecule that makes H/DX-MS such a useful biophysical tool to assess changes in a biopharmaceutical. Although several analytical tools have found use as detectors to analytically follow the H/DX process, the specific use of MS offers several useful benefits. These benefits include

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low sample requirements (few picomoles), the ability to perform automated measurements under native or formulated buffer conditions in a day or less, with high spatial resolution information about a biopharmaceutical’s structure. In fact, recent experiments have combined H/DX-MS with electron transfer dissociation (ETD) fragmentation to achieve single amino acid resolution [69e71]. As a result, H/DX-MS has attracted a great deal of interest in the biopharmaceutical industry, where it appears to be a very powerful biophysical characterization tool. Besides its wide use in the biopharmaceutical research, the technique is also gaining popularity in the process development area where it is being used in formulation studies [72,74], characterization investigations [5,8], and comparability and biosimilar assessments [9]. Further details associated with the theory, practice, and applications of biophysical MS for developing protein biopharmaceuticals will be addressed in Chapter 12.

3.5.7 Methods for studying the LS properties Exposing a solution of macromolecules to electromagnetic radiation at a wavelength where this radiation is not absorbed by the macromolecule can provide very useful information concerning the macromolecule’s mass, size, and shape via a process called scattering. There are two general areas in the electromagnetic spectrum where scattering methods are conducted. In the visible range where the method is referred to more generally as LS and in the short wavelength range of electromagnetic spectrum involving X-rays where it is referred to as solution X-ray scattering. In the area referred to as LS, it turns out that there are two totally different approaches that are used. In the more classical approach it is the intensity of the time-averaged amount of scattered light that is measured; while in the more modern form of scattering (which required the development of the laser before it could be employed practically as a commonly general laboratory tool), the basis of the method is the time-dependent variation in the intensity of the detected scattered light that is important. This latter scattering method has been referred to by a few different names of which the most common is dynamic LS (DLS). As a result of the development of DLS the classical form of LS is now more generally referred to as static LS (SLS). An important parameter in scattering methods is the angle (relative to the incident beam) at which the scattered electromagnetic radiation is measured. In the case of SLS, for macromolecules that are larger than about 1/20 of the wavelength the incident light, the light scattered by the macromolecule will display an angular dependence due to inference effects from the scattered light that comes from different parts of the same macromolecule. The nature of this angular dependence can provide important information about the size and shape of the scattering macromolecules. However, given the visible nature of the light used in SLS and the fact that nearly all biopharmaceuticals (other than gene delivery products and other bio-nanoparticles) are significantly smaller than this 1/20 of the wavelength threshold, they will show no angular dependence in their scattered light. Light scattered from even small oligomers, such as dimers and trimers of biopharmaceuticals will not show angular dependence in their scattering. As a result, these macromolecules are referred to as isotropic scatters (or point scatters). The only information that can be obtained from these molecules by SLS, at relative

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low sample concentration, is their MW. However, if the wavelength of the incident light is reduced to that corresponding to X-rays, the impact of interference can be greatly enhanced and used to extract a great deal of information about the size, shape, and even dynamic of these macromolecules. In most cases, the range of angles that are employed in these studies covers only small angles. Under these conditions the method is commonly referred to as SAXS. The ability to use wider angles can also be employed where the method is commonly referred to as WAXS. In the case of DLS, it turns out that by using highly monochromatic light with appropriate phase alignment properties found in lasers, one can assess the actual fluctuations present in the scattered intensity of the light radiated by a macromolecule over a very wide time scale. Since this fluctuation is dependent on the diffusion induced movement of the macromolecule, using appropriate modern hardware and software an average translational diffusion coefficient, as well as a more detailed information concerning the distribution of translation diffusion coefficients that characterize these protein drugs can be assessed. Such diffusion coefficient data will yield useful information about the HOS of these drugs via information about the mass, size, and shape of these drug molecules and the polydispersity of the drug (i.e., in term of aggregation). Information on the size and shape of a biopharmaceutical by DLS extents down to much lower size levels than SLS. In addition, DLS is especially capable of detecting very low levels of very high MW material such as submicron particles (see Chapter 8 for further discussion).

3.5.8 Methods for particle analysis Particle analysis in the parenteral pharmaceutical area has been historically concerned with the presence of extraneous foreign particulate matter corresponding to visible and subvisible particles, generally unrelated to the API. In general, there are three classes of particles, visible, subvisible, and submicron. Visible particles are assessed via direct physical observation, and these are generally concerned with particles that are greater than about 100 mm [75]. Subvisible particles, we are typically particles between 1 and 100 mm and primarily use common light-obscuration techniques such as HIAC and microfluidic imaging (MFI) [75]. Submicron particles are those less than 1 mm and require techniques such as nanoparticle tracking analysis. It should be noted that the nature or source of these extraneous or foreign particles have been reported to arise from a range of material including fibers, glass, rubber, or metal particles or also silicone oil droplets, derived from packaging and storage materials [76e80]. In the biopharmaceutical area, the same extraneous particle analysis has been carried out for the same reasons used in the pharmaceutical area (as mentioned above). However, the biophysical properties of a protein biopharmaceutical give rise to the unique intrinsic problem of particle formation arising from the API itself due to their propensity to aggregate for various reasons [23]. One reason for this aggregation results from the interaction of protein drugs with foreign material [79,81]. Hence, the presence of particles has a much higher level of importance to biopharmaceuticals. Further compounding this additional source for particle formation from the API itself and the accelerating effect of foreign material that can

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enhance the particle formation from the API, protein-based biopharmaceuticals are also faced the unique property associated with the adverse issue of immunogenicity [82e85]. Given these additional specific problems surrounding protein-based drugs, the quaternary structure level of the HOS of these biopharmaceuticals has attracted a great deal of attention [80,86]. This attention has been further amplified by the realization that there was a significant gap in our analytical ability to detect particles <10 mm with particular concern with particles <1 mm. Complementing the issue surrounding protein particle detection has been the added concern surrounding the need to characterize the HOS of the individual monomeric drug molecules in these particles in terms of their secondary and tertiary structure. As a result, a multipronged approach of intense biophysical characterization using several tools discussed in the previous sections have been applied to detect, quantify, and characterize the particulate material in biopharmaceuticals [32,87e90]. At present, there are many different methods for the analysis of particles, which includes the following: DLS, nanoparticle tracking analysis, asymmetrical flow field flow fractionation, resonant mass measurements, electrical sensing zone method, light obscuration, flow microscopy, and visual inspection. Details associated with their basis for detection, practice, and applications of these techniques used for particle analysis for biopharmaceutical protein development will be addressed in Chapter 10.

3.5.9 Developing a better biophysical toolbox There are many different tools to investigate the biophysical properties of biopharmaceuticals. Instead of discussing each one, we will briefly focus on a few points concerning what is needed in developing better biophysical tools and methods, especially for the process development area of the biopharmaceutical industry. Hence, we would like to return to the beginning of this chapter, where we discussed the characteristics that give rise to a better and more informative method. One such attribute is the ability to detect signals from many small structural elements (i.e., at the amino acid or atomic level) with high spatial resolution that cover the entire biopharmaceutical or a large percentage of it (Fig. 3.2). Another important attribute is the ability of the method to disperse these signals (Fig. 3.1B), rather than provide a single summed output. If the ideal dispersion level of these signals could be achieved so there is no overlap, or if the overlaps were not significant, and these signals could be recorded with good precision and accuracy, then the method will be able to detect small changes in a biopharmaceutical’s HOS. Biophysical tools, such as H/DX-MS and NMR, are attracting considerable attention (see Chapters 12 and 13, respectively), because they display the attributes mentioned above. However, there are additional methodologies that we believe also contain some of these beneficial characteristics (i.e., high spatial sensitivity and resolution). They include antibody array methodology [91], proteolysis or even limited or kinetic proteolysis [73], and chemical reactivity [92]. In the former approach, arrays of specific antibodies are produced that recognize linear or conformational epitopes on a biopharmaceutical (e.g., monoclonal antibodies). The antibody arrays can measure and characterize the surface epitope distribution of a

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biopharmaceutical and establish the accessibility of the epitopes that are normally buried in the folded HOS of the biopharmaceutical to detect changes in their accessibility and therefore changes in the HOS of a biopharmaceutical. In the latter two cases, proteolytic digestion and or chemical modification, the methods are used to detect changes in a biopharmaceutical’s HOS by using reagents with fixed or limited exposure to monitor the accessibility of various target sites on the biopharmaceutical. Changes in the end-point, after a fixed period of exposure to a specific level of these reagents in terms of cleavage or chemical modification between biopharmaceutical samples could proxy for a change in the drug’s HOS (Fig. 3.4A). These methods could also operate in a kinetic mode (e.g., time-dependent exposure), as illustrated by Fig. 3.4B or C. It should also be noted that by quenching these reactions, further analysis via MS could provide more detailed information concerning the location of change(s) on the biopharmaceutical. Finally, in making a better biophysical toolbox, there is a need to develop approaches that enable the biopharmaceutical scientist to evaluate low-resolution data sets obtained from a number of biophysical tools collectively with the intent to extract more informative information. While the data from these tools independently may carry little value, there is growing evidence that collectively their data could be more powerful. Approaches for achieving this appear to be feasible using the appropriate analytical logic as exemplified and illustrated by the work of Middaugh et al. at the University of Kanas in their empirical phase diagrams [93e95].

3.6 Conclusion The biophysical characterization of protein biopharmaceuticals is principally focused on the monitoring and understanding of the HOS and biophysical properties of these complex biopolymers, which in collaboration with their chemical composition, empower these molecules with their unique biological or therapeutic activity (function). In the beginning of this chapter, we discussed how the ability of finding one biophysical tool capable of providing all the information about the spatial and temporal positions of all atoms in a biopharmaceutical to define their HOS is presently not practical and is only approached by very few techniques that are unfortunately not appropriate for implementing in the routine biopharmaceutical process development area. Hence, the path taken in biopharmaceutical development is to proxy indirect information from a number of different lower resolution, simpler to use, and low-cost biophysical tools that monitors the biophysical properties of biopharmaceuticals to assess their HOS. Although these biophysical tools provide lower resolution, collectively they can collaborate to develop a more fingerprint-like picture that can reveal more information about the HOS of these drugs and its consistency in a practical way. Nevertheless, all these biophysical tools have limitations, which can at times be difficult to assess and can vary from one biopharmaceutical to another. Hence, the use of orthogonal biophysical tools to probe the same basic biophysical properties can be very useful in ensuring our confidence in the data acquired.

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In this chapter it has been shown that the biophysical methods available to investigate the HOS of biopharmaceuticals span a wide spectrum of different methodologies. However, we are still left with significant gaps of knowledge when compared to the complexity of these molecules. Therefore, to improve our ability to better characterize (biophysically) biopharmaceuticals, the search for better tools must continue.

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[94] Alsenaidy MA, Jain NK, Kim JH, Middaugh CR, Volkin DB. Protein comparability assessments and potential applicability of high throughput biophysical methods and data visualization tools to compare physical stability profiles. Front Pharmacol 2014;5:39. [95] Joshi SB, Bhambhani A, Zeng Y, Middaugh CR. An empirical phase diagramehigh-throughput screening approach to the characterization and formulation of biopharmaceuticals. In: Jameel F, Hershenson S, editors. Formulation process dev strategies manuf biopharm. Hoboken (NJ): John Wiley & Sons, Inc.; 2010. p. 173e205.

Further reading [1] Jiskoot W, Crommelin DJA, editors. Methods for structural analysis of protein pharmaceutical. Arlington (VA): AAPS Press; 2005. [2] Narhi LO, editor. Biophysics for therapeutic protein development. New York (NY): Springer; 2013. [3] Jameel F, Hershenson S, editors. Formulation and process development strategies for manufacturing biopharmaceuticals. Hoboken (NJ): John Wiley and Sons; 2010.

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