Article pubs.acs.org/ac
Semiautomated pH Gradient Ion-Exchange Chromatography of Monoclonal Antibody Charge Variants Mohammad Talebi,*,† Robert A. Shellie,† Emily F. Hilder,† Nathan A. Lacher,‡ and Paul R. Haddad† †
Australian Centre for Research on Separation Science (ACROSS), School of Physical Sciences, University of Tasmania, Hobart, Tasmania 7005, Australia ‡ Analytical R&D, Pfizer BioTherapeutics Pharmaceutical Sciences, Chesterfield, Missouri 63017, United States ABSTRACT: A new approach using a chromatography system equipped with isocratic pumps and an electrolytic eluent generator (EG) is introduced, replacing external pH gradient delivery using conventional gradient systems, in which bottled buffers with preadjusted pH are mixed using a gradient pump. The EG is capable of generating high purity base or acid required for online preparation of the buffer at the point of use, utilizing deionized water as the only carrier stream. Typically, the buffer was generated from online titration of a reagent composed of low molecular weight amines. The reagent was delivered isocratically into a static mixing tee, where it was titrated to the required pH with electrolytically generated base or acid. The required pH gradient was thus conveniently generated by electrically controlling the concentration of titrant. Also, since the pH was adjusted at the point of use, this approach offered enhanced throughput in terms of eluent preparation time and labor, and with a more reproducible pH profile. The performance of the system was demonstrated by running pH gradients ranging from pH 8.2 to 10.9 on a polymer monolith cation-exchange column for high throughput profiling of charge heterogeneity of intact, basic therapeutic monoclonal antibodies. A high degree of flexibility in modulating the key parameters of the pH gradient, including the buffer concentration, the pH gradient slope and the operating pH range was demonstrated. This enabled fine-tuning of the separation conditions for each individual antibody in order to enhance the chromatographic resolution.
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and pH, and gradient specifications often need to be optimized for each individual antibody.5,6,8 Additionally, salt gradient IEC can often exhibit insufficient resolution and robustness for routine characterization of certain antibodies.9,10 Compared with IEC, capillary IEF may demonstrate superior resolution and shorter method development time.6 However, following focusing, the mobilization of the focused protein zones can lead to long analysis time, distortion of the pH gradient, and a loss of resolution because of nonuniform mobilization speed and diffusion effects.1 Lasdun et al. reported consistent separations between the two techniques but with poorer precision for capillary IEF.11 Developed by Sluyterman and co-workers in the late 1970s,12,13 chromatofocusing (CF) has emerged as a technique which combines the resolving power of IEF and the flexibility and simplicity of IEC. CF often employs a complex mixture of polyampholytes as eluents for generating a retained pH gradient inside a column containing a weak ion-exchanger. However, there are some limitations associated with polyampholytes, including the cost, batch-to-batch variability,
onoclonal antibodies (mAbs) represent an important class of biopharmaceuticals with a wide range of clinical indications. Like many other proteins, mAbs are also susceptible to chemical and enzymatic modifications that can occur during manufacture, formulation and storage. While chemical modifications, such as sialylation and deamidation, introduce additional negative charges to the molecules, leading to acidic species with slightly lower pI values, C-terminal processing of lysine residues introduces one or two additional positive charges to the molecules and generates basic species with slightly higher pI values.1−3 Some modifications, such as deamidation, can affect the biological activity of a protein,4 so detection and identification of molecular changes in therapeutic mAbs are very important in biopharmaceutical development. The large size of mAbs and the minor structural diversity between the variants make their separation very challenging. As a result, a range of orthogonal analytical and biochemical methods including ion-exchange chromatography (IEC) and isoelectric focusing (IEF), are typically employed for characterizing therapeutic mAbs and monitoring the batch-to-batch process consistency and product stability and purity.5,6 While IEC is considered the gold standard for chargesensitive analysis of mAbs,7 this approach is also known to require significant time for method development as several parameters, such as the column type, the eluent concentration © XXXX American Chemical Society
Received: June 28, 2014 Accepted: September 8, 2014
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electrical control in the EG was used for the titration of a weakly acidic reagent with predefined composition, thereby forming the pH gradient. Alternatively, methanesulfonic acid (MSA) generated in an EG was employed for the titration of a basic reagent to generate the pH gradient. A high-throughput separation of basic mAbs is demonstrated with this manner of pH gradient generation, coupled with the use of a cationexchange monolithic column. The flexibility of this approach for real-time control of both buffer concentration and pH for improving the resolution is also discussed.
binding to purified proteins, and difficulty in isolating buffer species from collected fractions.14−17 Frey et al. have been able to tackle some of these limitations by widening the scope of suitable columns and employing low molecular weight amines as buffering species.18−20 Very recently, they have also demonstrated the suitability of their approach for separating variants of acidic mAbs.21 Despite this progress, the inflexibility of CF in controlling the pH gradient slope is often an issue given that a linear pH gradient in this technique is only formed when the ratio of the buffering capacities in mobile and stationary phases varies linearly with pH.15−17,20 Instead of generating a pH gradient internally, the proportioning capability of a gradient pump can be utilized for external generation of pH changes by mixing two buffer solutions of different pH prior to the column. The utility of an external pH gradient in resolving variants of mAbs has been recently demonstrated by several workers.2,8,10,22−24 An external pH gradient allows for simpler method development and optimization as the slope and the pH gradient profile can be controlled by the pump proportioning with less dependence on the column chemistry and buffer composition. In agreement with previous studies,2,3,17 we have recently demonstrated that optimal resolution for each mAb can be achieved by fine-tuning of the buffer concentration and pH as well as the range and slope of the pH gradient.24 However, simultaneously changing both the pH and the ionic strength cannot be readily achieved in a conventional approach.25 Electrically controlled pH gradients have been introduced by Dasgupta and co-workers utilizing a commercial membranebased ion-exchange suppressor used in ion chromatography (IC).25 The suppressor device was fed with reagents containing different compositions of low molecular weight species and the pH gradient was controlled by the electric current programming of the device. A prototype suppressor was also designed by the same group to enable independent control of both pH and buffer concentration.26 Despite being successful in forming temporally linear and wide-range pH gradients (pH 3−12), these devices are limited to low-pressure conditions and their performance for HPLC applications has yet to be demonstrated. Automated electrolytic eluent generators (EGs) were introduced commercially in 1998.27 The idea behind this innovation was based on the fact that off-line preparation of IC eluents can be tedious, subject to operator error, and can introduce contaminants. Utilizing the electrolysis of water and charge-selective electromigration of counterions through ionexchange media, these new electrolytic devices have significantly changed the routine operation of IC separations. EGs offer several advantages, including the ability to produce highpurity acid or base eluents online using only deionized water as the carrier stream, and also having simple and robust construction and operation mechanisms. More importantly, the concentration of acid or base in the eluent is governed by the applied electrical current, so concentration gradients can be accomplished simply by programming the applied electrical current to the EG, instead of using gradient pumps and proportioning valves. Therefore, gradient separations can be performed using lower-cost isocratic pumps.28 Detailed descriptions of the operating principles of EGs have been discussed elsewhere.27,28 In this work, we demonstrate the feasibility of utilizing commercial EGs as electrolytic devices for the establishment of controlled pH gradients. Herein the KOH generated under
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EXPERIMENTAL SECTION Reagents and Chemicals. The buffering species, including N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), and diethanolamine (DEA) were obtained from Sigma−Aldrich (Sydney, NSW, Australia). Triethanolamine (TEA) was purchased from BDH (Poole, England). All chemicals were of analytical reagent grade. Water purified via a Milli-Q water purification system (Millipore, Bedford, MA) and filtered through a 0.2 μm nylon filter was used for reagent preparation and chromatography. Buffer design and calculation of buffer capacity and ionic strength were performed using Buffer Maker software (ChemBuddy, Marki, Poland). Calculated pH values for each buffer composition were also cross-validated with experimental measurements. Samples of three therapeutic IgG2 mAb formulations, which are referred to as mAb1, mAb2, and mAb3, were prepared by recombinant DNA technology at Pfizer Inc. mAb1, mAb2, and mAb3 have electrophoretic isoelectric points (pI) determined experimentally as 8.8, 8.5 and 9.0, respectively. Apparatus. All experiments were performed using a Dionex ICS-3000 IC system (Thermo Fisher Scientific, Lane Cove, Australia), equipped with a dual pump (DP) module, an eluent generator (EG) module, and an AS-1 autosampler fitted with a 1.5 mL vial tray. The EG module was equipped with both EGC II KOH and EGC III MSA cartridges for the generation of potassium hydroxide and methanesulfonic acid, respectively. UV detection was performed at 280 nm using a VWD-3400RS detector (Thermo Fisher Scientific). pH was measured online using a pH meter (Activon Model 210, Pennant Hills, NSW, Australia) fitted with an epoxy body combination pH electrode (part no. 121207, TPS, Springwood, QLD, Australia) after twopoint calibration with standard buffers. The pH electrode was fitted into a homemade pH flow-cell and the pH meter was interfaced to the Dionex Chromeleon software via a UI20 Universal Interface (Dionex). Separations were performed using a ProSwift SCX-1S (4.6 mm × 50 mm) monolithic column (Dionex). The injection volume was 10 μL and the column flow-rate was either 1.4 or 1.7 mL/min (depending on the generated buffer). Instrument control and data acquisition were performed using the Chromeleon software, version 6.80 SR8. Procedure. Figure 1 illustrates the plumbing scheme of the setup. An isocratic solution of reagent containing 5 mM each of CAPS and TAPS, or alternatively 10 mM of DEA and TEA, was isocratically pumped into a static mixing tee (part no. U-466, IDEX Health & Science, WA, USA) where it was titrated to form a buffer with the effluent (either KOH or MSA) generated in the EG module under electrical control. The pH range of the buffer solution was controlled by software-programming of the current applied to the EG module. The buffer was then B
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separation efficiency of mAbs, its control appears to be more important than the linearity of the pH profile.2,8,24,25 No attempts were therefore made in this study to maintain a constant buffer capacity. The first buffer system was composed of equimolar concentrations (5.0 mM) of CAPS (pKa 8.44) and TAPS (pKa 10.5). The reagent solution was pumped isocratically into the mixing tee at the flow rate of 0.7 mL/min, where it was titrated with an ascending linear concentration gradient of KOH, generated under electrical control in the EG at the same flow rate of 0.7 mL/min. This resulted in 2.5 mM concentration for each buffer species and a column backpressure of about 345 psi, well below the upper pressure limit (1000 psi) of the monolithic column used. Upon changing the concentration of KOH from 3.0 to 8.0 mM, a near-linear pH profile was established over 10 min, spanning the pH range 8.8−10.9 (see Figure 2). This pH range was found to be sufficiently wide for the purpose of this study, covering the pI range of studied mAbs.
Figure 1. Plumbing scheme of the setup. Flow-rates: CAPS-TAPS buffer system, F1 = F2 = 0.7 mL/min; DEA-TEA buffer system, F1 = 1 and F2 = 0.7 mL/min.
delivered into the column and the UV outlet was directed to the pH flow-cell. Typically, the column was equilibrated at the initial pH for 1.0 min before establishing the pH gradient over 10 min. Elution was then continued for the next 2.0 min with the high-pH buffer before stepping back to the initial pH and re-equilibrating the column for the next 5.0 min. The gradient cycle time was typically 20 min.
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RESULTS AND DISCUSSION Unlike CF, choosing an appropriate column for external pH gradient is not critical. However, we have shown that while monolithic columns can be conveniently utilized under very low ionic strength conditions, the use of packed columns with external pH gradients can be limited by the high back-pressure involved, evidently because of the generation of osmotic pressure inside the pores of the stationary phase particles.24 Similar observations have been reported recently by Zhang et al. that suggested modulation of the ionic strength by the addition of an appropriate amount of sodium chloride maintains the column back-pressure below the expected operating range.2 In comparison, the high permeability of monolithic materials can effectively compensate the effect of osmotic pressure at a very low ionic strength, as evidenced by constant back-pressure being observed during the application of external pH gradients.24 A strong cation-exchange monolithic column was therefore employed in this study, although at the utilized conditions a weak-cation exchange chemistry was also found to perform equally well in terms of the separation efficiency.24 CAPS−TAPS Buffer System. One approach for developing a buffer system for external pH gradient is to utilize appropriate concentrations of buffer species with equally spaced pKa values across the pH range of interest.29 The applicability of this simple approach in establishing sufficiently linear pH profiles has been demonstrated in several studies.2,8,15,23,30 Attempts have also been made to generate highly linear pH profiles by maintaining constant buffer capacity across the desired pH range through systematic variation (computer programming) of the concentration of buffering components.22,29 Although this approach often provides more linearity of pH profile across a wider pH range, the ionic strength of the buffer solution that correlates with the concentration of species and pH would remain uncontrolled unless balanced by the addition of an inert salt. Since the ionic strength has a great impact on the
Figure 2. pH gradient IEC of mAb samples. Reagent containing CAPS and TAPS (5 mM each) was titrated with an ascending gradient of KOH (dotted line), ranging from 3.0 to 8.0 mM in 10 min. Dashed line represents the pH profile. Mixing ratio of reagent to titrant = 1:1; total flow rate = 1.4 mL/min. Other conditions as per Experimental Section.
Figure 2 shows the separations achieved for the three studied mAbs, with insets showing more magnification of the separation zone. While the presence of minor acidic (having pI lower than the main isoform) and basic species is more apparent for mAb1 and mAb2, this was not the case for mAb3. As discussed previously, the chromatographic resolution in pH gradient IEC can be improved by reducing the pH gradient slope, due to its direct correlation to the peak width.24 Similar to CF, higher resolution can be achieved by shortening the pH gradient range. Figure 3 illustrates the change in resolution of mAb1 as a function of the pH gradient slope. Rather than using the conventional pump proportioning to control the gradient slope, the operating pH range was adjusted here simply by confining the concentration of generated KOH in the range of 3.0 to 5.0 mM over the same gradient time, corresponding to the pH range of 8.8 to 10.0 (Figure 3, lower panel). As can be seen, reduction of the gradient slope from 0.12 to 0.08 pH units/min led to expansion of the fine structure of the chromatogram with slight improvements in resolution, C
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Figure 4. pH gradient IEC of mAb1. Reagent containing DEA and TEA (10 mM each) was titrated with a descending gradient of MSA, ranging from 15 to 0.10 mM (dotted line). Dashed line represents the pH profile. Mixing ratio of reagent to titrant (1:0.7); total flow-rate 1.7 mL/min. Other descriptions as per Figure 2
TAPS buffer system, more resolution between the main isoform and both acidic and basic variants can be seen. In addition, some of the acidic isoforms previously recognizable in the magnified zone in Figure 2 seem to be merged together and appear as a single peak adjacent to the main isoform. Slight differences in the shape of pH profiles can also be noticed when Figure 4 and Figure 2 are compared. Unlike CAPS and TAPS, which are zwitterionic or negatively charged species in the studied pH range, DEA and TEA are partially positively charged and therefore can participate in electrostatic interactions with the cation-exchange stationary phase, leading to a slight deviation in the linearity of pH profiles.2,8 This effect would be strengthen by the fact that both the ionic strength and buffer capacity of the DEA−TEA buffer system decrease gradually by increasing the pH, whereas the opposite trend is the case for the CAPS−TAPS buffer system (see Figure 5). Despite these differences, however, comparison of the separation profiles in Figures 2 and 4 indicates the same elution pH for the main isoform of mAb1.
Figure 3. Effect of pH gradient slope on the separation of mAb1. The gradient slope was 0.08 pH units/min (upper panel) and 0.12 pH units/min (lower panel). Other descriptions as per Figure 2
although at the expense of some peak broadening (see Figure 3, upper panel). It is worth mentioning that since mixing of the buffer with the titrant occurs after the pumps, and because of the absence of a gradient mixer, the proposed setup features a high-pressure gradient system, which is known to offer lower dwell volume and faster system re-equilibration time than its low-pressure counterpart.31 It can be seen in Figure 3 that when applying a step change in the concentration of KOH, the observed onset of change in the pH response required only ∼45 s (1.3 mL), which corresponds to the combined void volumes of the chromatographic system, column and pH flow-cell. The residence volume of the pH flow-cell was estimated as ∼500 μL. Also no appreciable difference between the online measured pH and the pH of the collected aliquots was observed. After accounting for this time lag, the elution pH of the main isoform of mAb1 can be approximated as 9.2, which is slightly higher than its electrophoretic pI, 8.8. This is in agreement with our previous observations and those of others13,19,21,24 and can be related to the charge regulation effect, which implies that for large proteins such as mAbs the rate of change of the protein charge distribution with pH tends to be relatively high, particularly at low ionic strength conditions, which is apparently the case here, and determines the magnitude of the shift in pI.21,24 DEA−TEA Buffer System. The proposed approach also enables buffer formation by online titration of a basic reagent using the hydronium ion generated in an acid eluent generator. In this case, the composition of one of the buffer systems developed in our previous work24 was adopted and prepared here by online titration of a reagent consisting of equimolar concentrations (10 mM) of DEA (pKa 8.88) and TEA (pKa 7.76) with MSA. Again a nearly linear pH profile spanning from pH 8.2 to 10.2 was obtained over 10 min by driving a descending gradient of MSA concentration, ranging from 15 to 0.10 mM. Solutions of reagent and titrant were mixed at the ratio of 1:0.7 (total flow rate 1.7 mL/min), resulting in an operating concentration of ∼5.9 mM for each component. Figure 4 illustrates a typical separation obtained for mAb1 with overlaid traces of pH (dashed line) and MSA concentration (dotted line). In comparison to the CAPS−
Figure 5. Relationship between buffer capacity, ionic strength (IS), and pH for the pH gradients used.
Furthermore, the separation profile achieved with DEA− TEA was found to be almost identical to the profile obtained previously using imaged capillary electrophoresis.24 Considering the different separation mechanisms involved, such comparison can give insight into the separation quality and help for unambiguous assignment of protein variants. Nearbaseline resolution of the main isoform from other variants was then achieved by reducing the pH gradient slope to 0.1 pH units/min, although again with commensurate increase in the peak width (see Figure 6). D
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generated as before by titrating the reagent using a descending gradient of MSA ranging from 6.0 to 0.10 mM, which corresponds to the pH range of 8.20−10.0. By keeping the same mixing ratio as before, the output would contain 2.9 mM of DEA and TEA species. As an example, the separation achieved for mAb2 is illustrated in the lower panel of Figure 7. For comparison purposes, the upper panel shows the separation obtained without dilution of the buffer. Aside from having slightly different gradient slopes (0.20 vs 0.18 pH units/min), the pH profiles appear to be comparable. However, some differences can be seen in the separation profiles. While further resolution of acidic variants from the main isoform is evident with the diluted buffer, peaks also became wider and consequently the basic variants appeared as faint shoulders in the threshold of the main peak. This is in agreement with previous observations,8,24 suggesting that in pH-gradient elution at very low ionic strength, long-range electrostatic interactions are involved, which are readily suppressed by screening ions and can lead to decreased resolution.3 This emphasizes the importance of controlling both pH and buffer concentration in pH gradient IEC, which is easier to achieve by reprograming a current profile in the EG rather than by mechanically altering compositions or mixing ratios of multiple solutions. Repeatability. Repeatability of this approach was also studied by performing multiple injections of the mAb1 sample over two consecutive days and using the same batch of DEA− TEA reagent. Figure 8 illustrates an overlay of four expanded
Figure 6. Effect of pH gradient slope on the separation of mAb1. pH gradient slope was reduced from 0.2 (Figure 4) to 0.1 pH units/min by adjusting the MSA concentration in the range of 5−0.1 mM. Other descriptions as per Figure 4
While providing superior resolution for mAb1 variants, the DEA−TEA buffer system did not exhibit a markedly different separation profile for mAb2 variants than the CAPS−TAPS buffer (see Figure 7 upper panel), and in the case of mAb3 no
Figure 7. Effect of ionic strength on separation of mAb2. The concentration of each buffer component and pH gradient slope were 5.9 mM and 0.2 pH units/min (upper panel) and 2.9 mM and 0.18 pH units/min (lower panel). Other conditions as per Figure 4
Figure 8. Separation profiles obtained for mAb1 on two consecutive days using the same batch of reagent. Conditions as per Figure 4
elution from the column was observed. These observations can be explained by considering the role of the ionic strength of the eluent in the separation mechanism of pH-gradient IEC.2,24 In their attempts to develop a multiproduct pH gradient method for resolving charge variants of a wide range of mAbs, Zhang and co-workers have suggested that the pI of mAbs and the buffer concentration can differently affect the separation efficiency of charged variants and should be modulated individually to achieve optimal resolution.2 As evident from Figure 5, the ionic strength of the CAPS−TAPS buffer at pH 10.2, where elution of mAb3 occurred, was approximately 3.5 times higher than the DEA−TEA buffer system. The observed behavior of mAb3 is thus reasonable given higher pI values can imply stronger binding of the protein to the stationary phase requiring more ionic strength for elution than less basic proteins.2 As a proof of concept, the suitability of this approach for online control of the buffer concentration and pH was also briefly studied. To do so, the DEA−TEA reagent was diluted by 50% using pump proportioning before being delivered isocratically to the mixing tee. The buffer was subsequently
chromatograms obtained (two for each day). The percentage relative standard deviation for the retention time of the main peak was 0.60% and for individual peak areas of the acidic, main and basic variants values of
