Use of Pressure in Reversed-Phase Liquid Chromatography To Study

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Use of Pressure in Reversed-Phase Liquid Chromatography To Study Protein Conformational Changes by Differential Deuterium Exchange Alexey A. Makarov,* Wes A. Schafer, and Roy Helmy Department of Process and Analytical Chemistry, Merck Research Laboratories, 126 East Lincoln Ave., Rahway, New Jersey 07065, United States ABSTRACT: The market of protein therapeutics is exploding, and characterization methods for proteins are being further developed to understand and explore conformational structures with regards to function and activity. There are several spectroscopic techniques that allow for analyzing protein secondary structure in solution. However, a majority of these techniques need to use purified protein, concentrated enough in the solution to produce a relevant spectrum. In this study, we describe a novel approach which uses ultrahigh pressure liquid chromatography (UHPLC) coupled with massspectrometry (MS) to explore compressibility of the secondary structure of proteins under increasing pressure detected by hydrogen−deuterium exchange (HDX). Several model proteins were used for these studies. The studies were conducted with UHPLC in isocratic mode at constant flow rate and temperature. The pressure was modified by a backpressure regulator up to about 1200 bar. It was found that the increase of retention factors upon pressure increase, at constant flow rate and temperature, was based on reduction of the proteins’ molecular molar volume. The change in the proteins’ molecular molar volume was caused by changes in protein folding, as was revealed by differential deuterium exchange. The degree of protein folding under certain UHPLC conditions can be controlled by pressure, at constant temperature and flow rate. By modifying pressure during UHPLC separation, it was possible to achieve changes in protein folding, which were manifested as changes in the number of labile protons exchanged to deuterons, or vice versa. Moreover, it was demonstrated with bovine insulin that a small difference in the number of protons exchanged to deuterons (based on protein folding under pressure) could be observed between batches obtained from different sources. The use of HDX during UHPLC separation allowed one to examine protein folding by pressure at constant flow rate and temperature in a mixture of sample solution with minimal amounts of sample used for analysis.

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he market of protein therapeutics is exploding,1 and protein-based therapies may become one of the most effective clinical methods in the treatment of various diseases, ranging from cancer to metabolic disorders.2 Thus, it is no surprise that methods for the characterization of proteins are being further developed to understand and explore conformational structures of proteins in regards to function and activity. There are several spectroscopic techniques that enable the study of protein secondary structure in solution. One of the most commonly used techniques for this purpose is circular dichroism (CD). However, CD analysis requires the protein sample solution to be purified of interfering proteins and impurities, such as nucleotides or optically active buffers (e.g., glutamate), or preservatives/antioxidants, which might contribute to the spectrum. 3,4 NMR and mass-spectrometry techniques are often used to study purified protein samples. There are many examples of the use of pressure to study protein conformational changes, in some cases combined with the application of deuterium exchange to study a single protein in solution.5,6 In the 1970s, Brandts et al. performed systematic © XXXX American Chemical Society

studies using spectroscopy to study the protein unfolding of ribonuclease A with regards to pressure and temperature.7 Pressure is one the most important parameters in studying the various aspects of protein conformations.8−10 In recent years, there has been an increased usage of methods such as one- and two-dimensional NMR11,12 and MS5 to study the effects of pressure on the folding of proteins. Accordingly, the introduction of a technique that can be used for studying protein secondary structure in a mixture will be very helpful. Hydrogen−deuterium (H/D) exchange (HDX) is a wellestablished technique for chemical structure elucidation and mechanistic determinations.13 The replacement of labile or exchangeable protons in alcohols, carboxylic acids, amines, or amides by deuterium atoms is readily observed by mass spectrometry (MS) and nuclear magnetic spectroscopy (NMR),14 as well as by some other spectroscopic techniques. Received: November 20, 2014 Accepted: January 25, 2015

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DOI: 10.1021/ac5043494 Anal. Chem. XXXX, XXX, XXX−XXX

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EXPERIMENTAL SECTION Reagents and Chemicals. Ultrapure water was obtained from a Milli-Q Gradient A10 from Millipore (Bedford, MA, USA). Acetonitrile and trifluoroacetic acid (HPLC grade) were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Uracil and deuterium oxide were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Insulin from bovine pancreas (B1), insulin from bovine pancreas (meets USP specifications) (B2), insulin human (meets USP specifications) (H1), insulin human recombinant (H2), cytochrome c (from bovine heart), myoglobin (from horse heart), and bradykinin acetate were obtained from Sigma-Aldrich Inc. (St. Louis, MO). Instrumentation and Stationary Phases. An Agilent 1290 quaternary pump LC system (Agilent, Santa Clara, California), able to operate up to 1200 bar (17 400 psi) and equipped with an 160 Hz photodiode array detector, and MSD6130 Single Quadropole Mass-Spectrometer with standard ESI source were used. Only ESI positive mode was used in the experiments. The MSD-6130 mass accuracy was ±0.13 u within the calibrated mass range in scan mode, based on vendor specifications.26 The mass range for measurements was set to m/z 100−2000 which is well within the recommended mass range for this instrument (m/z 2−3000). The massspectrometer was calibrated using a standard calibration mixture obtained from Agilent before each experiment. Note: Agilent 1290 Quaternary pump is designed to mix mobile phases at atmospheric pressure (low pressure mixing). A pressure restrictor apparatus (Upchurch Scientific, P-880 adjustable back pressure regulator with a total volume of 9 μL) was set between the column and the detector on the UHPLC system. All the experiments were performed in isocratic mode. Agilent OpenLab ChemStation Edition for LC/MS C.01.05 with the deconvolution module was used for data acquisition and processing. Each spectrum was averaged based on spectra measurements across the total ion chromatogram peak of analyte. Nominal mass spectra deconvolution parameters included molecular weight assignment based on the curve fit with an agreement of 0.05%, noise cut off of 1000 counts, and minimum peaks in set 3; [H+] = 1.0079. A 2.1 × 150 mm, 1.7 μm BEH C-18 column with 130 Å pore size (Waters Corp. Milford, Massachusetts) was used in the experiments. Experimental Conditions at Constant Flow Rate. The chromatographic conditions consisted of an isocratic run (mobile phase mixed by an Agilent 1290 pump at atmospheric pressure), pumped at a flow rate of 0.4 mL/min and UV detection at 210 nm. At 210 nm, it was possible to detect all of the analytes in this study. Retention time reproducibility was found to be satisfactory for the purpose of the present study, with six consecutive injections at different pressures showing a relative standard deviation of no more than 0.4%. Nominal molecular weight determination reproducibility, based on deconvolution of multiple-charged species, was no more than 0.0036% RSD (n = 6). There was no attempt at quantification of analytes. In all experiments, the aqueous part of the mobile phase was 0.1 v/v% trifluoroacetic acid (TFA) in water or in deuterium oxide (pH ∼2), and the organic part of the mobile phase was 0.1 v/v% trifluoroacetic acid (TFA) in acetonitrile. The resultant mobile phases used for the experiments for different proteins were: 77:23, 68:32, 67:33, and 58:42, v/v aqueous part/organic part for bradykinin, cytochrome C, insulins, and myoglobin, respectively. For all experiments, the column temperature was kept constant at 45 °C. It is well

The presence of an exchangeable NH proton in every peptide bond (except proline) of a protein makes H/D exchange a powerful technique in understanding protein structure and folding. Unlike small molecules, proteins contain large hydrophobic interior regions with significant hydrogen bonding that do not readily undergo deuterium exchange, and this provides a means for evaluating their secondary and tertiary structure.15,16 There is at least one report of H/D exchange kinetics being proposed to control batch-to-batch reproducibility of insulin.17 It has also been reported that insulinrecombinant batches with identical molecular weights can be successfully differentiated by differences in conformational structure using kinetic HDX experiments.17 There are two main HDX workflows: continuous HDX and pulse HDX.15,16 In continuous HDX, the protein sample is exposed to the deuterium oxide in a physiologically relevant buffer (usually neutral) for some period of time, with aliquots of the sample quenched at different time intervals by low pH (usually pH 2.5) and low temperature (0 °C). The quenched sample is digested and analyzed for deuteron insertions.15,16 Pulse HDX workflow is similar except that one-time stressbased H/D exchange is employed.15,16 Both of these HDX approaches suffer from H/D-back-exchange during chromatographic separation and are not efficient in the case of labile protons located on side chains (protein surface).15,16 Kinetic rates of deuterium exchange for solvent-accessible amide protons occur readily within the chromatographic time frame and allow the use of deuterium oxide mobile phases in LC-MS as a very convenient means of performing H/D exchange.18 Deuterium exchange in the mobile phase for less accessible protons can be much slower (10 h or more) in nondenaturing conditions.17 Temperature19 and pressure20 have proven to be effective techniques in systematically and progressively probing these interior regions further using HD exchange.5 Pressure studies allow one to consider the thermodynamic effects of density and temperature separately. Complete denaturation of chymotrypsinogen was observed by monitoring the CO, C−N stretching, and N−H bending bands between 1600 and 1700 cm−1 at pressures up to 10 kbar. When the experiment was conducted in deuterium oxide, there was a clear increase in the amide II band at 1550 cm−1, associated with interior labile protons.21 NMR is able to clearly note the accelerated disappearance of amide protons in αlactalbumin and β-lactoglobulin treated in D2O at 2 kbar pressure over ambient exposure.22 However, it was reported that, for some proteins, pressure alone does not cause complete denaturation; this makes it necessary to combine pressure with temperature and/or denaturing agents.23,24 Pressure has appeared to be a unique tool for probing proteins close to the unfolded and the folded states.25 There are reports indicating that hydrostatic pressure allows one to study protein misfolding and protein aggregation.20 In this study, we report a novel approach that utilizes the ultrahigh pressure liquid chromatography (UHPLC) coupled with mass-spectrometry (MS) to explore the compressibility of the secondary structure of proteins under increasing pressure using H/D exchange. The proposed method can also be used for secondary structure integrity verification of proteins during the manufacturing process, such as purification, chemical modification, or solvent exchange. B

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proteins was compared in each experimental set using the same column, with temperature and flow rate kept constant. Isocratic methods were developed for each protein analyte to have reasonable retention, yet not use acetonitrile concentration, that would fully denature the protein. The minimal acetonitrile concentrations, at which each protein maintains partial α helix folding, were previously reported for the studied proteins using CD spectra measurements at 222 nm.33 A range of proteins differing by size and α-helical content in their native structure were used in this study (Table 1).

acknowledged that temperature may play an important role in chromatographic separation; however, since our goal was to investigate pressure effects alone, the temperature was kept constant (as was flow rate). A column temperature of 45 °C was used to enhance the rate of labile proton exchange without complete denaturation of the proteins. Each protein sample was injected at four different controlled column pressure settings (example for bradykinin (77/23 v/v 0.1 v/v% TFA in water/0.1 v/v% TFA in acetonitrile): 766, 882, 1000, and 1100 bar, each about ±3 bar), achieved with a pressure-restrictor apparatus. A separate experiment series was conducted at constant flow rate of 0.4 mL/min, 900 bar, and 75 °C column temperature. Each experimental trend was repeated at least three times on different dates using different mobile phases and sample preparation. Sample Preparation. A sample solution of uracil used for all experiments was prepared at about 0.1 mg/mL in deionized water. Sample solutions of proteins used for all experiments were prepared at about 0.2 mg/mL in 0.1 v/v% TFA in water or in 0.1 v/v% TFA in deuterium oxide. All analytes were completely dissolved before injections. Protein samples in deuterium oxide were incubated for 2 h at 45 °C. All solutions were stored at 10 °C until being injected and protected from light. The injection volumes for different analytes were 1−3 μL. The pH in the experiments was controlled by the pH of the mobile phase (pH ∼2).

Table 1. Compounds Used in the Study compound name

molecular weight, Dalton

number of amino acids

% α-helix in the native structure47−51,52

uracil bradykinin insulin B1/B2 insulin H1/H2 cytochrome C myoglobin

112 1060 5734 5808 12 228 16 950

NA 9 48 51 104 153

NA 0 58 58 38 77

Bradykinin, for example, does not have any α helix in the native structure and can be used as a control of deuterium exchange. Bradykinin structure consists of the 9 amino acid sequence: Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg with the number of labile protons correspondingly 6 + 0 + 0 + 1 + 1 + 2 + 0 + 1 + 6 = 17, making the total number of labile protons 17.34 Previous studies have shown an effect of pressure on the chromatographic separation of proteins and small molecules by RPLC, where changes in retention factor correlate with increasing molecular size for neutral analytes; pressure effects are more pronounced with larger size molecules.33,35−37 In the case of proteins, increased pressure can lead to shifts in the population of secondary structures such as alpha-helices, and we wondered if we could detect changes in protein folding under pressure by differences in the number of labile protons accessible for deuterium exchange. Differential Deuterium Exchange upon Pressure Increase. Two sets of experiments were performed to study proton/deuterium exchange in proteins under pressure: one set of experiments was performed using sample diluent for deuterium exchange when protein samples were dissolved in 0.1% TFA in deuterium oxide and incubated for 2 h at 45 °C (diluent HDX); in the other set of experiments, the aqueous portion of the mobile phase 0.1% TFA in deuterium oxide was used for proton/deuterium (H/D) exchange (mobile phase HDX). Figure 1 demonstrates the number of protons exchanged to deuterons in each set of experiments. The number of protons (ΔH) was calculated on the basis of the molecular mass difference between the protein after H/D exchange and the molecular mass of protein not exposed to deuterium oxide under the same chromatographic conditions. In the experiments with deuterium oxide in the sample diluent (Figure 1A), we observed a decrease in the number of protons exchanged to deuterons upon pressure increase. At the same time, bradykinin showed a constant zero exchange difference versus control upon pressure increase. This is consistent with bradykinin’s lack of secondary structure. Indeed, bradykinin has no secondary structure to obstruct its 17 labile protons from being exchanged. Bradykinin (as the control protein) demonstrates that the rate of H/D exchange itself was not a limiting factor in our experiments. The difference in the

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RESULTS AND DISCUSSION The goal of our study was to demonstrate the feasibility of studying protein secondary structure (or comparing protein folding) using pressure in a solution mixture by employing RP liquid chromatography combined with MS detection. Ideally, the method should be quick and straightforward and should not require special instrumentation to be supported by many analytical laboratories. The effect of pressure changes (up to about 1200 bar) on protein during UHPLC separation is likely to be synergetic, and in order to be detected and understood, it may require a careful experimental setup. It is well established that the heat generated from frictional heating in ultrahigh pressure chromatography can be an important factor influencing chromatographic performance,27−30 with radial heat dissipation having a negative effect on efficiency and longitudinal temperature gradient leading to variation in analyte retention. To mitigate the effect of nonuniform temperature gradients, all experiments in this study were performed at constant flow rate of 0.4 mL/min, where the effect of frictional heating would be constant. Compressibility of the mobile phase can also be a factor contributing to chromatographic error;31 however, mobile phase volume changes in these experiments were estimated to be no more than 2.6 × 10−5% (calculated for the worst-case scenario mixture of acetonitrile/water: 75/25 v/v). This variation can be considered negligible. The only variable parameter in the experiments was pressure, which was adjusted by the use of a postcolumn backpressure regulator, set to vary pressure from 0 to ∼414 bar; the overall column inlet pressure varied from ∼750 to ∼1170 bar. The overall inlet pressure (as recorded) was reported throughout this study. In order to assess the impact of pressure on retention factors, uracil elution was used throughout the entire study to serve as a void marker.32 The uracil elution did not change upon pressure increase, also demonstrating that solvent compression was negligible. The effect of pressure on different C

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Figure 1. Pressure effect on proton/deuterium exchange in proteins upon pressure increase at constant flow rate and temperature of 45 °C for different proteins. (A) In experiments with aqueous mobile phase (0.1%TFA in water) and (B) in experiments with deuterium oxide mobile phase (0.1% TFA in deuterium oxide). ΔH was calculated as the molecular weight difference between the deuterium exchange sample and the control sample (the control sample had no exposure to deuterium oxide). Note: each data point presented in the figure includes an experimental error bar (based on n = 3); however, some error bars are smaller than the size of the point marker.

cytochrome C. Note: the retention factors for proteins at column temperature of 75 °C were substantially smaller compared to 45 °C separation: 1.18 for bradykinin, 1.47 for cytochrome C, 2.97 for bovine insulin, and 0.57 for myoglobin. The goal of the experiments with two pairs of insulins, human and bovine obtained from different sources, was to demonstrate that it is feasible to distinguish between insulins based merely on folding behavior under pressure. Previously, it was demonstrated by an HDX kinetic study with recombinant insulins that it is possible, by using continuous H/D exchange, to observe differences in folding between pairs of recombinant insulins with identical molecular weights.17 Table 2 shows the results of the experiment using differential deuterium exchange in pairs of insulins from different sources: A, diluent HDX; B, mobile phase HDX. The number of protons exchanged (ΔH) was calculated based on molecular mass difference between the protein after H/D exchange and molecular mass of the protein not exposed to deuterium oxide at the same chromatographic conditions. The number of exchangeable protons in insulins upon pressure increase demonstrated the same trends as for other proteins shown in Figure 1. Insulin’s ΔH decreased upon pressure increase during diluent H/D exchange and ΔH was increased upon pressure increase during mobile phase H/D exchange. Pressure increase in both cases compressed insulin folding pockets to allow more protons to be exchanged to deuterons or vice versa, depending on the type of experiment. Small differences in the number of protons exchanged during diluent HDX versus mobile phase HDX indicated that labile protons may have a different location in the insulin molecule, and differential deuterium exchange allows this difference to be detected. Differential deuterium exchange under pressure did not reveal any difference in folding between human insulin recombinants. However, there was a small but reproducible difference between bovine insulins from different sources (Table 2). Note: The probe experiment for protein folding based on the number of protons exchanged during mobile phase HDX upon pressure increase may be obtained within half an hour, and the experiment does not require any special sample preparation.

number of protons (H/D exchanged) in this set of experiments depended on the number of deuterium atoms exchanged within folding pockets during the relatively long 2 h incubation at 45 °C but protected from exchange to protons during the short residence time of the UHPLC separation. During the UHPLC/ MS analysis, the exposed labile deuterium atoms were immediately exchanged back to protons, leaving only those protected in the folding pockets. Increasing the pressure compressed the folding pockets, exposing the labile deuterium atoms for exchange to protons. The number of protons exchanged to deuterium was decreased upon pressure increase, indicating that more folding pockets were compressed (denatured) by pressure. Complementary results were obtained in the second set of experiments with deuterated mobile phase used for H/D exchange (Figure 1B). The total number of protons exchanged in this experiment was much higher, since many of the labile protons were located on the protein surface or side chains and did not have any restrictions for H/D exchange. Again, bradykinin showed complete exchange of all 17 labile protons, and the number of protons exchanged was constant upon pressure increase, since bradykinin does not have any secondary structure. For other proteins, the number of exchanged protons increased upon pressure increase, indicating that more folding pockets were compressed (denatured) by pressure and made labile protons from folding pockets exchanged to deuterium atoms from the mobile phase. However, the pressure increase up to about 1200 bar in our experiments was not able to completely denature proteins, which indicated that pressure increases compressing proteins may lead not only to unfolding but also to possible folding (collapse) of some “pockets”. Complete denaturation for most of the proteins was obtained when the chromatographic separation was performed at a column temperature of 75 °C. Indeed, the difference in the number of protons exchanged (for proteins incubated at 45 °C for 2 h versus the control) at column temperature of 75 °C was zero for bradykinin (as the control) and zero for insulin and cytochrome C; myoglobin had a difference of only 8 protons. This experiment showed that at 75 °C there were no folding “pockets” left unexposed for exchange in insulin and D

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Molecular Molar Volume Change upon Pressure Increase. In several publications,33,35−40 it has been shown that k′ (retention factor) increases with an increase in pressure. While the increase for low molecular weight analytes is generally small, the increase for proteins has been found to be much larger.33,35,39,40 This effect was related to the changes in analyte molecular molar volume.33,35,36 As previously reported, insulin and chicken egg white lysozyme have demonstrated pressure-induced changes in retention (retention factor increased), which was attributed to a decrease in analyte molar volume by −100 to −130 mL/mol (using 26% acetonitrile in the mobile phase), as reported by McGuffin et al.41 and Liu et al.40 Molecular molar volume changes (ΔV) were in the range of −50 to −70 mL/mol for peptides in their report (Note: retention factor increased).40,41 As previously reported, the retention factor increase for proteins upon pressure increase may indicate changes in protein folding.33,35 Figure 2 illustrates retention factor changes upon pressure increase in experiments with diluent H/D exchange (A) and mobile phase H/D exchange (B). Indeed, the retention factor increase was significant for proteins with secondary structure (Table 1), namely, insulin, cytochrome c, and myoglobin (Figure 2). However, bradykinin demonstrated retention behavior under pressure similar to small analytes, for which retention factor increases upon pressure increase and is mostly driven by the size of the molecule and/or disruption of the analyte’ s solvation shell (as was reported and discussed in our previous publication35). Pressure impacts the molecular molar volume (including solvation shell) of an analyte at constant temperature, which leads to enhanced interaction with the stationary phase.33,36 Eq 141−44 shows how pressure-induced changes in analyte molecular molar volume (ΔV) correlate with the slope of the line of the natural logarithm of the retention factor, as a function of pressure, at constant temperature and constant flow rate.

Table 2. Differential Deuterium Exchange in Pairs of Insulins from Different Sourcesa A insulin-B1

insulin-B2

insulin-H1

insulin-H2

B

pressure (bar)

MW

ΔH

control sample 768 866 955 1090 control sample 768 866 955 1090 control sample 768 866 955 1090 control sample 768 866 955 1090

5732

0

5748 5747 5743 5741 5732

16 15 11 9 0

5747 5746 5742 5741 5806

15 14 10 9 0

5819 5817 5815 5813 5806

13 11 9 7 0

5819 5817 5815 5813

13 11 9 7

b

pressure (bar)

MWb

ΔH

control sample 825 925 1025 1125 control sample 825 925 1025 1125 control sample 825 925 1025 1125 control sample 825 925 1025 1125

5732

0

5802 5803 5803 5806 5732

70 71 71 74 0

5801 5802 5802 5805 5806

69 70 70 73 0

5880 5881 5883 5884 5806

74 75 77 78 0

5880 5881 5883 5884

74 75 77 78

a

A: in experiments with aqueous mobile phase (0.1%TFA in water) and with sample diluent 0.1% TFA in deuterium oxide; B: in experiments with deuterium oxide mobile phase (0.1% TFA in deuterium oxide) and with sample diluent 0.1%TFA in water. ΔH was calculated as the difference between deuterium exchange sample and control sample (the control sample had no exposure to deuterium oxide). %RSD for MW determination was 0.004% (n = 3). b Determined as nominal mass of the most abundant isotope.

Figure 2. Pressure effect at constant flow rate and temperature on retention factor in proteins. (A) In experiments with aqueous mobile phase (0.1% TFA in water) and (B) in experiments with deuterium oxide mobile phase (0.1% TFA in deuterium oxide). Note: each data point presented in the figure includes an experimental error bar (based on n = 3); however, some error bars are smaller than the size of the point marker. E

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⎛ β ⎞ ΔV P + ln⎜⎜ ⎟⎟ + ln(k ref ) RT ⎝ βref ⎠

same effect on the molecular molar volume with the deuterium oxide solvation shell. The amplitude of ΔV should depend on internal packing of proteins, and in this case, the pressure studies may have an important means of examining this fundamental protein property.5 The approach described may also be used as a quick way to confirm that the protein has remained unchanged, since, e.g., preparative chromatographic separation may alter protein properties.45,46 It may be especially important since the difference in conformational structure of protein-based drugs may imply a difference in the therapeutic and/or medicinal effect of the drug when administered. There is presently no commercially available technology to study proteins’ conformational changes (CD, ORD, NMR, etc.) by spectra combined with UHPLC, in order to directly evaluate protein conformational changes during UHPLC separation. The UV−vis (photodiode array) spectrum does not reflect protein conformational changes in many cases. In our study, we compared the UV (photodiode array) spectra of proteins and did not observe any apparent differences for different conformational structures of proteins at the studied conditions. However, the increasing number of new protein-based drugs requires one to develop fast, cheap, and sensitive methods to verify consistency between and/or within batches of biopharmaceuticals to ensure product quality, and the approach suggested in this study may also be used for this purpose. Although the biological activity is the authoritative assay for biopharmaceuticals, the ability to quickly detect changes in protein conformation in reaction mixtures from conjugation reactions would be of great use in the discovery and early process development space. Further studies may use HDX exchange to pinpoint the pressure value at which certain conformational structures undergo changes and may thus elucidate how changes in conformational structures may be related to changes in enzymatic activity.

(1)

In this equation, k′ is the analyte retention factor, ΔV is the change in the molecular molar volume of the analyte, P is pressure, T is temperature, R is the ideal gas constant, and β is the phase ratio. Here, kref and βref are the retention factor and the phase ratio, under reference conditions and at atmospheric pressure, respectively. An increase in pressure at constant temperature should lead to the reduction of analyte molecular molar volume (based on Ideal Gas Law principles), which will lead to an increase of analyte retention factor. Since the experimental conditions (besides pressure) were kept constant, the phase ratio is deemed to be constant (assuming negligible compressibility of the BEH-C18 stationary phase,36 under the studied conditions), as are kref and βref. The slopes of the lines of the natural logarithm of the retention factor, or ln(k′) vs pressure, were used to estimate molecular molar volume changes of the analyte under variable pressures. The changes in analyte molecular molar volume upon pressure increase are presented in Table 3, for different proteins. Note that the ΔV Table 3. Molar Volume Change upon Pressure Increase up to 1100 bara A

bradykinin insulin-B1 insulin-H1 cytochrome C myoglobin

B

−ΔV (cm / mol)

correlation (R2)

−ΔV (cm / mol)

correlation (R2)

18.5 58.2 55.5 97.8 108.4

0.91 0.99 0.98 1.00 1.00

15.9 44.9 39.7 66.1 82.0

0.99 0.99 0.99 1.00 1.00

3

3

a

A: in experiments with aqueous mobile phase (0.1%TFA in water); B: in experiments with deuterium oxide mobile phase (0.1% TFA in deuterium oxide). %RSD for ΔV (cm3/mol) determination was 7.59% (n = 3).

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CONCLUSIONS Pressure has a significant effect on a protein’s molecular molar volume under UHPLC separation conditions. The effect of pressure is especially pronounced for proteins that may remain partially folded under certain chromatographic conditions. It was demonstrated in this study that the increase of retention factors upon pressure increase at constant flow rate and temperature was based on a reduction of the protein’s molecular molar volume. The change in protein’s molecular molar volume was due to changes in protein folding, as was revealed by differential deuterium exchange. The degree of protein folding under certain UHPLC conditions could be controlled by pressure, at constant temperature and flow rate. By modifying pressure during UHPLC separations at constant flow rate and temperature, it was possible to achieve changes in protein folding, which manifested as changes in the number of labile protons exchanged to deuterons, or vice versa. This study evaluated the use of pressure during UHPLC separation to probe protein secondary structure. It was demonstrated that pressure combined with differential deuterium exchange can be a useful tool to compare protein folding. The small difference based on protein folding was observed between two bovine insulin batches obtained from different sources. Moreover, the time required to conduct the experiment to compare folding differences under different pressures using deuterium exchange was less than half an hour, and special sample preparation was not required. The use of

values were obtained at constant acetonitrile concentration in isocratic mode experiments, when only the pressure was different for each experiment and the system was under equilibrium at given experimental conditions. The results of molecular molar volume change upon pressure increase in proteins (Table 3) imply conformational changes by pressure, which are consistent with the results of H/D exchange under pressure. Indeed, the molecular molar volume changes for proteins that can have secondary structure were more significant than for bradykinin. The difference in ΔV in the experiments with diluent H/D exchange versus mobile phase H/D exchange could be explained by the difference in strength of hydrogen bonding in deuterium oxide versus water. Indeed, deuterium oxide has 11% higher density than water (Note: The deuterium oxide boiling point is 101.4 °C and the density is 1.107 g/mL from the Sigma-Aldrich safety data sheet). In this case, the difference in molecular molar volume in the two types of experiments could represent the difference in the protein solvation shell. For example, in the experiments with deuterium oxide in the mobile phase, deuterium oxide produces a solvation shell which more strongly interacts with protein analyte, and the same increase in pressure, which may have a significant effect on molecular molar volume by reducing the water solvation shell, would not be strong enough to have the F

DOI: 10.1021/ac5043494 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

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HDX during UHPLC separation allows for one to probe protein folding using pressure at constant flow and temperature in a mixture of sample (not purified) with a minimal amount of sample used for analysis.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (+1) 732-594-7735. Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/ac5043494 Anal. Chem. XXXX, XXX, XXX−XXX