Libo Wang

A defining requirement in biopharmaceutical development, formulation, and manufacturing is to elucidate and maintain the structure of the drug entity. That work relies on relevant protein characterization, a task complicated by how samples evolve through the drug development lifecycle. The requirement to change techniques to cope with progressively more concentrated, and complex, multicomponent formulations is a major issue when it comes to comparing datasets and safeguarding structural parity. In this article, we discuss a new infrared spectroscopy technique, microfluidic modulation spectroscopy (MMS), that delivers reproducible protein characterization over close to four orders of magnitude in protein concentration, from 0.1 to 200 mg/mL. This technique can transition with the sample from the earliest stages of development through to manufacture, and present experimental data demonstrating the concentration-independent data that can be generated.

	Elucidation of the structure-function relationships that define the clinical efficacy of therapeutic biologics such as monoclonal antibodies (mAbs) makes protein characterization critical to their development. Though the primary structure of proteins is relatively stable, higher order structure (HOS)-secondary, tertiary and quaternary-is labile, susceptible to change triggered by chemical or thermal stress. Therefore, delivery of a drug entity to the patient with its structure intact, unchanged by manufacture or storage, relies on detailed protein characterization across the biopharmaceutical lifecycle. This requirement creates the need for an analytical technique that can characterize structure reproducibly across a broad range of concentrations, and that remains viable and reliable in the presence of excipients and additives.

	Secondary structure develops from interactions between hydrogen bond donor and acceptor residues associated with repeating units in the primary peptide chains of the protein, and is typically characterized by Fourier transform infrared (FT-IR) spectroscopy and far ultra-violet circular dichroism (far-UV CD). The most common element of secondary structure is the α-helix, but β-sheet structures are also relatively prevalent, particularly within mAbs, which is one of the most widely used classes of therapeutic proteins. β-sheet structures consist of β-strands and stretched sections of peptide chain, linked via hydrogen bonds (1).

	With FT-IR, information relating to secondary structure is extracted from absorption spectra spanning the amide I band, resulting from the C=O stretch vibrations of peptide linkages along the protein backbone. These modes are highly sensitive to changes in secondary structure (2), with features of the amide I band correlated robustly with specific secondary structure elements; sensitivity to β-sheet structure is a particular benefit (3,4) of using the mid-IR. However, from a practical perspective, conventional FT-IR technology has some limitations, including the relatively high concentration required for measurement, optimally in the range of 10–150 mg/mL, which makes the technique less viable for the earlier stages of drug development, when sample concentration is typically low. In addition, FT-IR tends to exhibit background drift and poor repeatability, due to the complexities of water vapor subtraction and referencing.

	The technique of circular dichroism (CD) characterizes samples on the basis of the differential absorption of left-handed and right-handed circularly polarized light that occurs when a molecule contains one or more chiral chromophores. Such chromophores include the peptide bonds associated with secondary structure, which give rise to a CD signal in the far-UV region (180–240 nm) that can be analyzed to determine the relative quantities of major secondary structural elements (5). Far-UV CD is well-suited to more dilute, simple solutions, operating at a relative low concentration of 0.2 mg/mL, for example, when using a cell of 1-mm pathlength. However, commercial products are typically formulated at higher concentrations, and the presence of chiral molecules in more complex formulations is also problematic. Typically, a dialysis procedure must be performed to remove excipients in the formulation buffer that interfere with CD signals. Phosphate buffered saline (PBS), a buffer used routinely in biological research, is well-characterized for far-UV CD analysis, and can be used to dilute formulation components that interfere with CD measurements (5).

	Switching from far-UV CD to FT-IR as samples become more complex and concentrated, through formulation and into commercial manufacture, is one way to address the limitations of these techniques. However, this introduces the complicating factor of determining whether any conflicting observations are scientifically relevant, or simply an artifact of measurement or data analysis. An alternative strategy is to dilute samples to extend the application of far-UV CD. This is problematic from the perspective of data relevance, given that proteins are highly sensitive to their environment. The limitations of both of these strategies creates a need for innovative spectroscopy platforms for the direct measurement of native proteins.

	An important step forward in this respect is the development of microfluidic modulation spectroscopy (MMS), a technique that harnesses the advantages of IR spectroscopy for robust measurement across a very wide dynamic range, with minimal interference from formulation excipients and additives (see Figure 1). In the MMS system, the sample solution and a matching reference stream are rapidly modulated (1–5 Hz) across the laser path to produce differential absorbance spectra via a process of continuous autoreferencing. The result is nearly drift-free scans background compensated to the buffer of interest, and of superior quality to those produced via conventional FT-IR. Concentration limitations are also directly addressed through the use of a tunable mid-IR quantum cascade laser that generates an optical beam around 100 times brighter than that used in a typical FT-IR. Run in continuous wave mode to generate a very accurate, low noise beam, this laser, in combination with a fully optimized optical configuration, delivers highly repeatable, high sensitivity measurement over a concentration range from 0.1–200 mg/mL. The laser also enables the use of simple detectors with no requirement for nitrogen cooling.


	Figure 1: With MMS, rapid modulation with a matched reference stream produces highly sensitive differential IR scans, eliminating any potential interference from formulation excipients and additives; a tunable, cascade laser and optimized optical set-up enable measurement across a very wide concentration range.

	MMS effectively shapes IR spectroscopy into an efficient tool for protein characterization from early drug development through to manufacture. The following experimental study of a mAb in clinical development demonstrates the capabilities of MMS for the measurement of protein samples in the presence of different buffers and across a very broad concentration range.

	Dilution series were prepared from a mAb stock sample of concentration of 157 mg/mL made up in a formulation buffer. A first dilution series was prepared by mixing volumes of the stock solution with the formulation buffer to create a samples series covering a concentration range from 1 to 80 mg/mL (specifically 1, 5, 10, 20, 40, and 80 mg/mL). A second dilution series was prepared in a strictly analogous way, and covering the same concentration range, by mixing samples of the stock solution with a PBS buffer at pH 7.4. In addition, a series of samples of formulation buffer diluted in PBS buffer were prepared as matching buffers for the PBS dilution series. In MMS measurements, the sample is modulated with a relevant buffer, so there is a need for a series of PBS diluted formulation buffer samples matched to the PBS dilution series samples.

	All samples from both dilution series were analyzed by MMS (AQS

pro, RedShiftBio), generating differential absorbance spectra for each sample. All samples were tested at a modulation rate of 1 Hz. The instrument automatically determines the back pressure required for optimal fluid flow through the flow cell, to enhance data quality. For samples in the 1–40 mg/mL concentration range, the applied back-pressure was 5 psi and duplicate measurements were carried out for each sample. For the most concentrated 80 mg/mL samples, which were more viscous, higher back pressures were automatically determined (25 psi for the sample in the formulation buffer, and 10 psi for the sample in the PBS buffer). One measurement was carried out for both of these samples. The measurements made were subject to comprehensive data analysis using the instruments integral software package (AQS

	Analysis of the dataset for the first dilution series highlights key features of the data generated by MMS, and how it can be processed to reveal information about secondary structure, and to compare samples.

	Figure 2a shows the differential absorbance spectra produced for the first dilution series; the replicate measurements indicate the high repeatability and accuracy. A plot of the maximum diffAU value as a function of concentration (see Figure 2b) shows excellent linearity with an 

value of 0.9997, illustrating the potential of the technique for protein quantification. Samples of unknown concentration can be accurately quantified using this plot.


	Figure 2: In MMS, continuous modulation between the sample and reference streams produces (a) differential absorbance spectra, shown here for a dilution series; and (b) the plot of maximum diffAU against protein concentration showing strict linearity, highlighting the potential of the technique for quantification.

	With the buffer subtracted and concentration normalized, the absolute absorption spectra for each of the samples can be compared directly (see Figure 3). The high degree of overlap in these spectra indicate that the secondary structure of the protein is essentially unchanged by the dilution process across the full concentration range, which is not the case for all proteins.


	Figure 3: Buffer subtraction and concentration normalization produces absolute absorbance spectra for the mAb that can be used for detailed structural analysis.

	One way to accentuate structural differences between samples is to generate second-derivative spectra from absolute absorbance spectra, as a function of wavenumber. This data processing step produces the traces shown in Figure 4, which provide further evidence of the close similarity of the samples, particularly those in the 5 to 80 mg/mL range.


	Figure 4: One way to highlight difference, or conversely confirming similarity, between samples is to analyze the second derivative plots, as shown.

	Protein similarity can also be directly compared via area of overlap (AO) plots (data not shown) which quantify the extent to which the spectra for two samples are similar (see Table I). Using the average AO plot for the two 5 mg/mL samples as the reference for comparison, this analysis indicates that the 1 mg/mL samples have a similarity of 98.2 to 98.5%, while all the remaining samples, across the remainder of the concentration range, have a similarity in the range 99.6 to 99.9%.

	Finally, because absorption at specific wavenumbers is well-correlated with specific elements of secondary structure, the relative quantities of the different elements can be determined (see Figure 5). The secondary structure of the mAb is shown to consist predominantly of beta sheets (~60%) and beta turns (~30%), with a much smaller fraction of unordered structure and minimal levels of alpha-helix. Levels of each element are extremely consistent across each of the samples.


	Figure 5: Higher order structure (HOS) analysis determines the quantity of specific elements of secondary structure present in the mAb, including beta sheet, beta turn, unordered structure, and alpha helix.

	The data processing steps illustrated in the preceding stage were applied to elucidate the impact of diluting the mAb stock with PBS buffer. Figure 6 shows the protein concentration plot for the PBS dilution series alongside the absolute absorbance spectra.


	Figure 6: The protein concentration plot for the PBS dilution series showing (a) a very high degree of linearity; and (b) absolute absorbance spectra that are very closely matched, indicating a high degree of structural similarity in the samples.

	These data show that dilution in the PBS has a closely comparable impact to dilution in the formulation buffer. The plot of maximum diffAU against protein concentration, for example, shows similar, comparably high linearity, exhibiting an 

 value of 0.9998. The absolute absorbance spectra indicate that the structure of the protein is closely similar across the full concentration range, as confirmed by the HOS analysis (see Figure 7).

	These results are directly useful when it comes to comparing measurements with far-UV CD with MMS, or, indeed, FT-IR. The mAb stock solution was supplied at a concentration of 157 mg/mL, which is substantially outside the measurement range for far-UV CD, and there are limitations with respect to diluent choice, since formulation buffer cannot be used if it contains a significant level of chromophores. These data confirm that diluting the sample with PBS, an acceptable, well-characterized buffer for far-UV CD analysis, has no impact on the structure of the protein.


	Figure 7: The HOS of the mAb in the PBS dilution series is closely similar to the formulation dilution series with beta sheet and beta turn the predominant structural elements.

Analyzing Similarity across Both Dilution Series

	In a final analysis, the two datasets were merged to look at similarity with respect to both concentration and buffer. Figure 8 shows the second derivative spectra for all of the samples; Table II shows the associated HOS data.


	Figure 8: Combining second derivative spectra for all of the samples in both buffers confirms the exemplary similarity of secondary structure across both dilution series.

	These data illustrate that the secondary structure of the mAb is extremely consistent, regardless of concentration or buffer. When compared with the 5 mg/mL sample in the formulation buffer, the similarity of the 1 mg/mL samples in formulation buffer are in the range of 98.2 to 98.5%, while those of all the other samples, in both the formulation and PBS buffer, are between 99.0 and 99.9%, indicating that the structure of the mAb is highly comparable across all the samples. The ability of MMS to measure across the full concentration range, without interference from either buffer, makes it extremely valuable for elucidating differences between data sets and for effective buffer screening. Here, the results indicate that any discrepancies observed between far-UV CD and MMS data are not attributable to either concentration or buffer components.

	Instrumentation developed to characterize the secondary structure of proteins across a wide range of conditions, most especially over a broad concentration range, is particularly helpful to biopharmaceutical scientists. The development and manufacture of biopharmaceuticals calls for a detailed understanding and control of the drug entity, within the native formulation environment, and through manufacture and storage to the point of patient delivery. The use of different measurement techniques, especially under different protein conditions, significantly complicates the interpretation of and the analytical workflow. In particular, the comparison of datasets produced using alternative technologies can lead to discrepancies between results that cannot be attributed to the sample with any certainty.

	The ability of MMS to measure with high reproducibility and repeatability, across a very wide concentration range, and to generate data unaffected by formulation excipients and additives, is extremely valuable within this context. This capability significantly reduces the need to compare and transfer specifications from one technique or instrument to another, thereby simplifying and nullifying risk in the analytical workflow, while at the same time reducing the cost associated with the purchase and ongoing maintenance of multiple instruments. Such flexibility makes MMS superior to alternatives such as far-UV CD and FT-IR for the essential task of characterizing the secondary structure of protein, from the earliest stages of drug delivery through to commercial biopharmaceutical manufacture and release testing.

https://proteinstructures.com/Structure/Structure/secondary-sructure.html

, J.M. Chalmers and P.R. Griffiths, Eds. (John Wiley & Sons, Ltd., Chichester, United Kingdom, 2002), pp. 3399–3425.

Analytical Applications of FTIR to Molecular and Biological Systems, 

J.R. Durig, Ed. (D. Reidel, Boston, Massachusetts, 1980), pp. 241–255.

	(4) A. Dong, P. Huang, and W.S. Caughey, 

 are with RedShift BioAnalytics, Inc., in Burlington, Massachusetts. 

Ioannis A. Papayannopoulos and Shannon Renn-Bingham

 are with Celldex Therapeutics, in Fall River, Massachusetts. Direct correspondence to: 

Articles

A new infrared spectroscopy technique, microfluidic modulation spectroscopy (MMS), delivers reproducible protein characterization over close to four orders of magnitude in protein concentration (from 0.1 to 200 mg/mL). This technique characterizes samples from the earliest stages of development through to manufacture.

Native Measurement of a Biotherapeutic without Interference from Excipients Using Microfluidic Modulation Spectroscopy

Measurement and characterization of the secondary structure of proteins are critical in many research applications, especially the formulation and development of biotherapeutics. Traditional analytical tools are not optimized for the demanding requirements of these applications, which include high sensitivity, wide dynamic range, simplified workflow, and high repeatability. This article introduces a new infrared (IR) technique, microfluidic modulation spectroscopy (MMS), that is designed to address these needs, and presents data from measurements of commercially available proteins. The data demonstrate significant increases in sensitivity, dynamic range, and utility for the determination of protein similarity (fingerprinting), quantitation, protein secondary structure, and protein stability and aggregation through thermal and chemical denaturation methods. 

	Proteins are large molecules with complex three-dimensional (3D) structures that directly influence their efficacy and safety as biotherapeutics. The primary structure of proteins is the sequence of amino acids in the peptide backbone with secondary structures arising from interactions between hydrogen bond donor and acceptor residues associated with repeating peptide units. These interactions promote stabilized folding of the peptide chains principally to form α-helix or β-sheet–strand substructures, the geometry of which is defined by the intermolecular forces, bond angles, and planar configurations involved. However, the secondary and associated higher order structure of proteins is not fixed, but rather is dependent on the localized environment, changing in response to, for example, thermal or chemical stresses. This propensity for instability or conformational change that is often accompanied by 

, the process by which proteins start to bind together under different conditions and formulations, is a defining issue in the discovery, formulation, and manufacture of biotherapeutics and biosimilars.

Measuring Protein Structures in Drug Development, Formulation, and Manufacture

	Promising biological drug candidates, those that exhibit therapeutic activity and inherent stability, as assessed via simple screening techniques, become subject to increasing levels of structural elucidation as they progress through the pharmaceutical pipeline. Detailed definition of the structure of a drug molecule provides a basis for the identification of structure–function relationships, for understanding the mechanisms by which the drug is efficacious, but beyond this, structural characterization plays a critical role throughout the drug development life cycle. In particular, investigating structural changes is the key to understanding and controlling the factors and mechanisms associated with stability and aggregation.

	Defining an optimal formulation and manufacturing route relies on assessing the impact of variables such as pH, excipient choice, processing conditions including temperature and applied shear stress, and storage. Stress-induced structural changes may have significant consequences including a loss of efficacy, and in the worst case present a safety risk, so demonstrating comparability (that successive stages of formulation, manufacture, and storage do not materially impact the structure of the drug) up to the point of administration is essential.

	The demonstration of comparability relies on showing that the quality attributes of the drug remain "highly similar and that the existing knowledge is sufficiently predictive to ensure that any differences in quality attributes have no adverse impact upon safety or efficacy of the drug product" (1). The accurate measurement of secondary structure and changes in secondary structure underpin these efforts and are critical activities across the drug life cycle, including in the demonstration of biosimilarity. The use of biosimilars follow-on biotherapeutics with such closely comparable performance to an original drug that they can be used interchangeably, has the potential to drive down health care costs but is highly dependent on the rigorous demonstration of similarity in a wide range of attributes including protein content, activity, and stability. The demonstration of a comparable secondary structure via robust analysis is an efficient way to directly support claims of biosimilarity.

Using Vibrational Spectroscopy for Protein Characterization

	Vibrational spectroscopy is an established tool for protein characterization (2), with Fourier transform infrared (FT-IR) wavenumbers in the range 1700–1500 cm

 routinely used to probe the amide I (1700–1600 cm

) bands respectively. The amide I band is associated with the C=O stretch vibration of peptide linkages and quantifies the strength of the carbonyl bond along the protein backbone, which is highly sensitive to changes in secondary structure (3). Indeed, absorption features of the amide I band are well correlated with shifts in hydrogen bonding, dipole-dipole interactions, and geometric orientations in the α-helices, β-sheets, and turns, and with other less prevalent motifs of secondary structure such as random coils (4). The relative amounts of different elements of substructure can therefore be precisely determined by comparing measured amide I spectra with those gathered for proteins with well-characterized secondary structures (5) to provide valuable information for the assessment of, for example, chemical and thermal stability (6), aggregation (7), and biosimilarity (8,9). Notably, IR is particularly sensitive to the β-sheet structures that are prevalent in protein antibody drugs and is one of the very few analytical techniques that can be used to directly monitor aggregation processes because of its ability to measure the intermolecular β-sheet structures associated with aggregate formation (10).

	Though the potential of vibrational spectroscopy in protein analysis is widely recognized, current commercial presentations of the technology are not optimally matched with requirements for secondary structure measurement within biotherapeutic development, formulation, and manufacture. As a result, researchers routinely deploy a suite of diverse analytical techniques to study the outlined issues associated with protein structure, with each technique somewhat aligned with the constraints and requirements of different stages of the development cycle. Differential scanning calorimetry (DSC) is routinely used as an early screen for thermal stability, for example, while size-exclusion high performance liquid chromatography (HPLC) can be used to detect aggregation because of its capabilities for molecular size measurement. Neither of these techniques provides detailed structural characterization of the protein. FT-IR and Raman are both used for structural analysis but require sample concentrations of at least 10 mg/mL and 30 mg/mL, respectively (10), with FT-IR working optimally in the 10–150 mg/mL range. This concentration requirement can be a limitation for application in earlier stages of development when sample availability can be an issue. FT-IR also has other well-documented issues including a tendency to exhibit background drift, low sensitivity, and a requirement for close temperature control.

	Ultraviolet–circular dichroism (UV-CD), in contrast, is an established tool for structural analysis at more dilute concentrations, typically operating in the range 0.2–2.0 mg/mL (11), but it is unsuitable for direct measurement at high-formulation concentrations. Biotherapeutics are typically delivered intravenously or subcutaneously in liquid formulations, often at relatively high concentrations to minimize administration time within the limitation of having a stable formulation of practical viscosity. Sample dilution complicates measurement and introduces an additional source of analytical variability, and the extrapolation of measured results to higher concentrations is widely recognized as a potential source of error in biopharmaceutical characterization because of the sensitivity of protein structure to its surrounding environment. Furthermore, UV-CD is relatively insensitive to changes in β-sheet structure, especially intermolecular β-sheet structures, compromising its application in the study of aggregation mechanisms.

Introducing Microfluidic Modulation Spectroscopy: High Sensitivity IR Across a Wide Dynamic Range

	A new technique called microfluidic modulation spectroscopy (MMS) directly addresses the limitations of current technologies and provides an efficient tool for direct, label-free protein analysis. It uses a tunable mid-infrared quantum cascade laser to generate an optical beam that is 1000 times brighter than those used in conventional FT-IR, enabling the measurement of samples that are substantially more concentrated and the use of simpler detectors with no requirement for nitrogen cooling. The laser is run in continuous wave mode to generate a very high resolution (< 0.001 cm

 linewidth), low noise beam with minimal stray light that is focused through a microfluidic transmission cell with a relatively long (25 µm) optical pathlength onto a thermoelectrically cooled mercury cadmium tellurium (MCT) detector. This optical configuration delivers high sensitivity measurement over a concentration range of 0.1–200 mg/mL for structural characterization and down to 0.01 mg/mL for protein quantitation, giving MMS a far wider dynamic range than alternative protein characterization techniques.

	In an MMS instrument (see Figure 1), the sample (protein-in-buffer) solution and a matching buffer reference stream are introduced into the transmission cell under continuous flow and then rapidly modulated (1–10 Hz) across the laser beam path to produce nearly drift-free, background compensated, differential scans of the amide I band. The complete optical system is sealed and purged with dry air to minimize any interference from atmospheric water vapor, which absorbs across the 2000–1300 cm

 wavenumber range and can therefore compromise the use of IR spectroscopy for protein characterization. Advanced signal processing technology is the third key element of the instrument and converts the raw spectra into fractional contribution data for specific motifs of secondary structure, providing a structural fingerprint of the protein.


	Figure 1: In an MMS instrument the protein sample is rapidly modulated across the laser with a matching water-buffer stream to produce highly sensitive, differential IR scans of the amide I band that provide detailed structural information.

	MMS is continuously autoreferencing, eliminating the problems associated with background drift in FT-IR measurements through the use of rapid modulation and the associated generation of a differential measurement. It streamlines the utility of vibrational spectroscopy for biopharmaceutical analysis by combining high repeatability and sensitivity with the wide dynamic range needed to address measurement requirements from development through to quality control, and comparability assessment. The following examples show its application to routinely encountered proteins, demonstrate how performance compares with that of other techniques, and illustrate the value of MMS for protein quantification, structural conformation, the investigation of stability and aggregation, and similarity assessment.

	A series of experiments was carried out to demonstrate the dynamic range, repeatability, and sensitivity of MMS using a range of commercially available proteins. The specific details relating to the generation of each dataset are given below. All measurements were carried out using a prototype MMS platform (RedShift BioAnalytics Inc.) at a temperature of 21 °C with no cooling of the fluids or measurement cell.

Experiment I: Demonstrating Dynamic Range Through the Measurement of Bovine Serum Albumin Across a Range of Concentrations

	Bovine serum albumin (BSA) was obtained from Sigma-Aldrich and prepared in 0.016 M PBS (phosphate buffered saline, pH 7) buffer to generate a concentration series from 0.1 to 200 mg/mL. Measurements were performed under otherwise identical conditions using MMS. A modulation frequency of 1 Hz was applied during measurement and spectra were gathered from 1584 cm

Experiment II: Assessing Repeatability via Duplicate Measurements of Hen Egg White Lysozyme

	Multiple measurements of replicate hen egg white lysozyme (HEWL) samples (Sigma-Aldrich) were performed to assess the repeatability of secondary structure results. The samples were made up to a concentration of 10 mg/mL in water and measured at room temperature. The reference stream used during measurement was water and a modulation frequency of 5 Hz was applied. Again, spectra covered the amide I band.

Experiment III: Comparing the Sensitivity of MMS and FT-IR for the Detection of Structural Impurities by Spiking Immunoglobulin (IgG1) with BSA 

	To investigate the ability of MMS to detect structural impurities, BSA (a predominantly α-helical protein) was added to a solution of IgG1 (a predominantly β-sheet protein) in known concentrations and the resulting mixtures were then analyzed using the techniques of FT-IR and MMS. Data were gathered for a 100% IgG1 solution and for solutions of 2%, 4%, 6%, 8%, and 10% w/w BSA in IgG1 solutions. The buffer used for all solutions was 10 mM potassium phosphate, pH 7.0, and measurements were carried out at an overall concentration of 20 mg/mL. FT-IR measurements were made using an MCT detector and an attenuated total reflectance (ATR) cell (Bruker). MMS measurements were made using blank buffer as a reference fluid and a modulation frequency of 1 Hz. Data was gathered over a wavelength range of 1586–1715 cm

. With MMS all measurements were also repeated at a concentration of 1 mg/mL by further diluting the samples with the same buffer.

Robust Measurement Across a Wide Dynamic Range

	Data measured for BSA across a range of concentrations (Experiment I; see Figure 2) indicate that the secondary structure of the protein is dominated by an α-helix (alpha) substructure. This substructure absorbs at a wavenumber of 1656 cm

 and makes a fractional contribution of around 65% on average (65.29% mean, 4.23 standard deviation). Other major motifs include β-turns (turn) that absorb at a wavenumber of 1682 cm

 (extend). These can all be robustly identified from well-documented correlations between specific wavenumbers and secondary structure motifs (4). The consistency of these results over a concentration range that spans more than three orders of magnitude sets MMS apart from techniques such as FT-IR and UV-CD that measure over a much narrower concentration window.


	Figure 2: Measurements of BSA over the concentration range 0.1-200 mg/mL demonstrate the wide dynamic range of MMS, which is an important advantage for application in biotherapeutic development.

High Repeatability and Its Value in Biosimilarity Assessment

	Repeat protein sample measurements were performed to demonstrate the excellent repeatability made possible by MMS. HEWL material was obtained from Sigma-Aldrich and prepared in water at a concentration of 10 mg/mL. Four common structural motifs are identified: alpha-helix ("alpha"), beta sheet ("beta"), beta turn ("turn"), and unordered structures ("unord"). At 10 mg/mL, the standard deviation of the data is around 1% for the overall structure and equally low for each motif, less than 0.5 (0.14, 0.32, 0.24, and 0.06), highlighting the ability of the technique to repeatably quantify the fractional contribution of each secondary structure motif.


	Figure 3: Repeat measurements of HEWL made over the course of a month, at a concentration of 10 mg/mL demonstrate the very high repeatability of MMS, a feature that directly underpins its ability to sensitively detect structural change. (Mean and standard deviations for the fractional contribution of each motif are shown in the boxed text above each set of histograms.)

	High repeatability correlates directly with the ability of a technique to detect differences, and in the case of MMS, this enables the detection of slight changes in the protein secondary structure that may have a critical impact on biotherapeutic performance. The high repeatability and reproducibility of MMS means that it can detect such differences with greater sensitivity than alternative spectroscopic techniques, such as FT-IR, and this makes it a powerful tool for similarity assessment. Figure 4 shows the set of four BSA measurements (Experiment I) displayed in the form of overlaid spectra. A number of algorithms have been proposed for the comparison of spectra for the purposes of similarity assessment, including the correlation coefficient and the area of overlap methods (8,9). Using the overlap method similarity for these samples exceeds 97% in the middle concentration range, with a drop off in similarity at both the high and low ends of the range. For replicates at 1 mg/mL, MMS typically yields area of overlap similarity values of 98.5% or higher.


	Figure 4: Overlays of BSA data measured over several orders of magnitude in concentration, highlighting the value of MMS as a technique for similarity assessment throughout drug development.

	This similarity figure is significantly higher than values quoted for FT-IR in the literature, which produces a mean similarity of 86.37% ± 7.98% at a single concentration of 10 mg/mL for HEWL (9). These data suggested that with FT-IR, protein similarity values comparable to those observed with the MMS data for BSA could only be obtained at significantly higher concentrations. The practical implications of this comparison are that within established biotherapeutic workflows MMS will allow more sensitive similarity assessment across a wider range of concentrations than FT-IR, a capability that is valuable both for comparability assessments during formulation development and manufacture, for example, and for biosimilarity testing in support of a submission for a follow-on biologic. A technique that generates a lower similarity for samples that are identical or exhibits deviation over the concentration range of interest, has less ability to robustly differentiate samples with genuine structural differences, compromising its value. These data also confirm the ability of MMS to generate robust measurements at far higher concentrations than UV-CD, thereby avoiding the additional workflows and dilutions that may be needed to deploy UV-CD to assess the biosimilarity of formulations at representative concentrations.

	The measured BSA data also illustrate how MMS may be used as a protein quantification tool (Experiment I, Figure 5), more specifically the linearity of the 1656 cm

 peak as a function of concentration over three orders of magnitude of concentration, from 0.1 to 200 mg/mL. Spectroscopy is one of a wide range of assays routinely used for protein quantification, but most spectroscopic tools have a narrow dynamic range that stems from the limited linearity of many commercial spectrometers (12). This limited linearity, which can be attributed to the combined effects of stray light, instrument slit width (resolution), and poor detector linearity, forces researchers to adjust either sample concentration or cell pathlength to achieve accurate protein quantitation. These strategies tend to be time-consuming and can directly impact data integrity.


	Figure 5: Differential absorbance data measured at 1656 cm

 shows the linearity of the MMS measurements as a function of concentration highlighting the value of the technique for protein quantification.

	This linearity of the MMS data is attributable to the high resolution (<0.001 cm

) of the instrument in combination with its low stray light susceptibility and the fact that the technique utilizes a differential measurement, thereby eliminating issues such as background subtraction that can compromise the application of conventional spectrometers. The direct control of laser power further improves linearity by managing signal dynamic range and maintaining high detector linearity across the measurement range. UV–visible (vis) methods are currently a popular choice for protein quantification, but the absorption bands in the IR region are much narrower and less dependent on aromatic residues, making MMS less susceptible to interferences. Furthermore, because MMS is not dependent on a UV chromaphore the variation of extinction coefficient is much smaller, which can be an advantage in measuring unknown proteins. Combined with a minimum measurable concentration of less than 10 µg/mL (3 sigma, HEWL) and an upper limit of greater than 200 mg/mL, these benefits make MMS significantly advantageous relative to alternative assay methods for protein quantification.


	Figure 6: IgG1 has a secondary structure consisting predominantly of β-strands (left) whereas BSA is defined by an α-helix (right) motif. Spiking BSA into IgG1 therefore tests the sensitivity of a technique to changes in the most frequently encountered features of secondary structure.

Experiment III: Comparing the Sensitivities of MMS and FT-IR in a Protein Spike Experiment

	IgG1 has an almost exclusively β-strand secondary structure (86.5%) (13) while that of BSA is α-helix (94.3%) (14) (see Figure 6). Experiment III compares the relative ability of FT-IR and MMS to detect the changes that would be expected at the two wavenumbers associated with these motifs-1656 cm

 (β-strand)-as IgG1 is spiked with BSA, at a total protein concentration of 20 mg/mL. Plotting the change in the second derivative of the FT-IR absorbance spectra, as a function of wavenumber highlights the areas of greatest change, from sample to sample, which as expected are 1656 cm


	Figure 7: (a) FT-IR data for mixtures of BSA spiked into IgG1 indicate that the wavenumbers associated with maximum change are those associated with the α-helix and β-strand motifs, 1656 cm

, respectively. (b) The technique begins to lose sensitivity to changes in the strength of the β-sheet signal at concentrations of BSA below 8%.

	Comparable data derived from the MMS measurements at 20 mg/mL show a much more robust linear correlation between delta second derivative data and the %BSA present (Figure 8a) than is observed with FT-IR. Sensitivity is better than the minimum experimental spike amount of 2%. These results demonstrate the superior sensitivity of MMS relative to conventional FT-IR at 20 mg/mL. Data measured by MMS at 1 mg/mL (Figure 8b) indicates a detection limit of about 4% BSA. Taken together these results indicate that MMS produces far cleaner data than FT-IR, offering up to 20 times greater sensitivity. This is a major benefit in terms of the ability to detect differences in secondary structures and therefore directly impacts the value of MMS in all measurement applications.


	Figure 8: Plots of delta second derivative as a function of %BSA derived from MMS measurements (a) at 20 mg/mL and (b) at 1.0 mg/mL demonstrate the greater sensitivity of the technique relative to FT-IR.

	MMS is a valuable new technique for the characterization of proteins that harnesses the inherent advantages of IR spectroscopy in a configuration that is particularly well-suited to applications associated with the development, formulation, and manufacture of biotherapeutics. A key advantage is its wide dynamic range. MMS can measure over many decades of concentration, from 0.1 to 200 mg/mL (down to 0.01 mg/mL for protein quantification applications), in contrast to the single decade of concentration measurement associated with alternative techniques for measurement of the secondary structure of proteins, such as FT-IR and UV-CD. This wide dynamic range means it allows measurement at the concentration of interest from the early stages of biotherapeutic development through formulation into manufacture, with negligible requirements for sample dilution or concentration.

	Furthermore, MMS offers a streamlined workflow, with rapid and continuous autoreferencing that eliminates slow and inconsistent manual buffer subtraction. This characteristic results in exceptional sensitivity and repeatability, boosting its value as an analytical tool across the pharmaceutical pipeline. The recent introduction of MMS technology puts a powerful new tool in the hands of protein scientists that can be used for quantitation, the elucidation of stability and aggregation mechanisms, structural analysis, and biosimilarity assessment. Its use has the potential to streamline analyses for the commercialization of new products and bring valuable biotherapeutics to market more safely and efficiently.

	(1) International Conference on Harmonization, 

 "Comparability of Biotechnological/Biological Products Subject to Changes in the Manufacturing Process" Available at: 

http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q5E/Step4/Q5E_Guideline.pdf

, J.M. Chalmers and P.R. Griffiths, Eds. (John Wiley & Sons, Ltd., Chichester, UK, 2002), pp. 3399–3425.

Analytical Applications of FTIR to Molecular and Biological Systems

, J.R. Durig, Ed. (D. Reidel, Boston, Massachusetts, 1980), pp. 241–255.

	(5) A. Dong, P. Huang, and W.S. Caughey, 

	(7) B. Shivu, S. Seshadri, J. Li, K.A. Oberg, V.N. Uversky, A.L. Fink, 

	(8) B.S. Kendrick, A.C. Dong, S.D. Allison, M.C. Manning, and J.F. Carpenter, 

	(9) J. D'Antonio, B.M. Murphy, M.C. Manning, and W.A. Al-Azzam, 

 73–95 (2009). doi: 10.1016/ S0076-6879(09)63008-1.

	(13) E.O. Saphire, P.W. Parren, R. Pantophlet, M.B. Zwick, G.M. Morris, P.M. Rudd, R.A. Dwek, R.L. Stanfield, D.R. Burton, and I.A. Wilson, 

 is the Chief Technology Officer at RedShiftBio in Burlington, Massachusetts. 

 is the Senior Applications Specialist at RedShiftBio. 

 is the Vice President, Elion Labs, (Div. of KBI Biopharma) in Louisville, Colorado. Direct correspondence to: 

This article introduces a new infrared (IR) technique, microfluidic modulation spectroscopy (MMS), that is designed to address these needs, and presents data from measurements of commercially available proteins. The data demonstrate significant increases in sensitivity, dynamic range, and utility for the determination of protein similarity (fingerprinting), quantitation, protein secondary structure, and protein stability and aggregation through thermal and chemical denaturation methods.

Enhanced Protein Structural Characterization Using Microfluidic Modulation Spectroscopy

Measurement and characterization of the secondary structure of proteins are critical in many research applications, especially the formulation and development of biotherapeutics. Traditional analytical tools are not optimized for the demanding requirements of these applications, which include high sensitivity, wide dynamic range, simplified workflow, and high repeatability. This article introduces a new infrared (IR) technique, microfluidic modulation spectroscopy (MMS), that is designed to address these needs, and presents data from measurements of commercially available proteins. The data demonstrate significant increases in sensitivity, dynamic range, and utility for the determination of protein similarity (fingerprinting), quantitation, protein secondary structure, and protein stability and aggregation through thermal and chemical denaturation methods.

This article introduces a new IR technique, microfluidic modulation spectroscopy (MMS), that is designed to address the needs in biotherapeutics, and presents data from measurements of commercially available proteins.