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Jia Liu, PhD, is a process engineer in process sciences downstream at Bristol Myers Squibb Company.
Aaron Cheung is a chemical engineering student at the Massachusetts Institute of Technology.
John L. Hickey is associate director of process sciences downstream and project management at Bristol Myers Squibb.
Sanchayita Ghose, PhD, is the manager of the process sciences downstream group at Bristol Myers Squibb.
A stable alternative to Protein A chromatography.
A novel Protein A alternative stationary phase based on the variable heavy chain fragment of immune camelid antibody was evaluated and compared to commonly used commercial Protein A resins. The parameters evaluated were elution pH, equilibrium isotherm, dynamic binding capacity, and host cell protein clearance using a set of Chinese hamster ovary-derived monoclonal antibodies and Fc-fusion proteins. Linear retention experiments were used to compare the specificity of these resins for both non-IgG model proteins as well as antibodies and Fc-fusion proteins. The experimental results showed that the new camelid antibody resin behaved very similarly to Protein A resins in terms of retention of non-IgG model proteins and IgG-based molecules. Dynamic binding capacity was found to be comparable for Fc-fusion proteins and slightly lower for antibodies. Host cell protein clearance profiles were also similar under preparative conditions using complex biological feeds. Finally, the binding mechanism was explored by using different mobile-phase modifiers in linear pH gradient retention experiments.
Protein A affinity chromatography has been widely used for antibody purification in the biopharmaceutical industry because of its excellent selectivity and product recovery.1-5 In recent years, it has been recognized as the industry standard for capture and purification of antibodies and Fc-fusion proteins. The use of this highly selective and robust capture step allows for faster process development and has enabled the use of a platform approach for monoclonal antibody (MAb) purification.6,7 Despite all of its advantages, Protein A chromatography suffers from the limitations of high cost, ligand leaching, and caustic instability.8,9
In the last decade, several mixed mode and Protein A mimetic ligands have been developed as alternatives to Protein A chromatography. One is hydrophobic charge induction chromatography (HCIC), which uses heterocyclic ligands at high ligand densities that can get positively charged at low pH values.10 Similar to Protein A, adsorption on these resins can occur by hydrophobic interactions without high salt concentration, while elution can be controlled by lowering the pH to induce charge repulsion between the ionizable ligand and the bound protein.10,11 Protein A mimetic ligands were also developed based on the IgG binding domain of Protein A using techniques such as molecular modeling, protein engineering, phage display, and synthetic chemistry.12–15 Although initial studies from the resin manufacturers had shown some promise for the above-mentioned alternatives,16–22 recent and more comprehensive studies have shown that none of these resins possess the selectivity offered by Protein A chromatography.23,24
Recently, a novel technique was developed for the rapid identification of affinity ligands against a diverse set of targets using the variable heavy-chain (VHH) region of single-chain antibodies found in the Camelidae family.25 These molecules possess good specificity because of their enlarged hypervariable region and have very high physical and thermal stability because of their single-domain nature.25,26 Recombinantly expressed VHH fragments can be used as affinity ligands and have applications in laboratory-scale immunoaffinity and immunoperfusion chromatography.27 Their potential application at industrial scale became more promising after it was proven that these antibody fragments can be expressed efficiently in microorganisms such as the yeast Saccharomyces cerevisiae.28
This technology has been commercialized by the Bio Affinity Company (Naarden, The Netherlands) to generate ligands (called CaptureSelect ligands) that can be customized for any purification challenge. Unlike other proteinaceous ligands (such as Protein A), these ligands have the distinct advantage of being stable in strongly alkaline solutions.29–31 One such ligand was generated against the Fc-region of human IgGs. This was shown to bind to all human IgG subclasses and no cross-reactivity was found with bovine or mouse IgG.30 This ligand has the additional advantage of being specific for human IgGs only and unlike Protein A, it can bind IgG3s as well. In 2007, the ligand was immobilized on a highly cross-linked agarose-based backbone through a long, hydrophilic spacer arm and marketed through GE Healthcare (Uppsala, Sweden) as IgSelect affinity medium.32 Although this resin can potentially be an attractive and manufacturing-friendly alternative to Protein A chromatography, very limited data exists so far on the performance of this new resin.29
This article provides the first comprehensive evaluation of this new ligand for MAb purification. Using several industrial MAbs and Fc-fusion proteins, the performance of this resin was compared to two of the most commonly used commercial Protein A resins: MabSelect from GE Healthcare and ProSep-vA High Capacity from Millipore (Billerica, MA). These two Protein A resins are on two different backbones (agarose versus controlled pore glass) and were specifically chosen to represent a wide spectrum of Protein A resins. The parameters evaluated were binding affinity, dynamic binding capacity, selectivity, and binding thermodynamics. Finally, the binding mechanism of IgSelect resin also was explored by linear gradient retention experiments with different mobile-phase modifiers.
The three resins evaluated in this study (MabSelect, IgSelect, and Prosep-vA High Capacity) were purchased from their respective vendors. All resins were packed to 20 cm bed height in 1 cm I.D. columns made by GE Healthcare. The four test proteins—Fc-fusion proteins (A and B) and MAbs (C and D)—were expressed in Chinese hamster ovary (CHO) cells and produced at Bristol-Myers Squibb (Syracuse, NY). Model proteins such as horse cytochrome c and human serum albumin (HSA) samples were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals were purchased from Mallinckrodt Baker (Phillipsburg, NJ).
All chromatography experiments were carried out on an AKTAexplorer chromatographic system from GE Healthcare. High Performance Liquid Chromatography (HPLC) analysis was performed using Waters (Milford, MA) 2695 Separation Module and Waters 2996 Photodiode Array Detector. An Orbital Shaker 100 from ArmaLab (Bethesda, MD) was used for batch-adsorption experiments.
The elution pH of the various proteins was obtained by linear gradient experiments under analytical conditions using pulse injection of the samples. A gradient of pH was run from 6.5 to 2.5 over 10-column volumes in citrate buffer. The elution pH at peak maxima was calculated from the gradient and further verified from the effluent pH trace obtained from the online monitor pH/C-900 that is part of the AKTA system. In the binding mechanism exploration test, different mobile phase modifiers were added to citrate buffers at both pH values and an identical pH gradient was run for comparison with the original pH gradient.
Dynamic binding capacities were determined by performing breakthrough experiments at six minutes residence time. The column was equilibrated and regenerated using typical Protein A process conditions. Adsorption isotherms for the proteins on the various stationary phases were determined using batch experiments.
Selectivity of the three resins was compared under preparative condition using cell culture harvest material. Each resin was loaded to ~80% of its dynamic binding capacity for the respective protein. Typical Protein A equilibration, elution, and regeneration conditions were used for these experiments. Sample protein concentration was determined using an analytical Protein A assay. Host cell protein levels in the samples from the preparative experiments were determined using an in-house host cell protein ELISA assay.
A Langmuir Isotherm, as defined by the following equation, was used to compare the binding thermodynamics of the three resins in this study:
in which Q is the equilibrium concentration of the solute on the stationary phase (expressed in mg solute per mL column volume), C is the mobile phase solute concentration at equilibrium, Qmax is the maximum static binding capacity, and K is the affinity binding constant. The binding constant is thermodynamically representative of the protein's affinity to the resin.
Comparison of Binding Affinity and Elution pH
Both the ligands evaluated in this study (Protein A and the camelid antibody) use a similar operating scheme—the protein is bound at close to neutral pH and eluted with a lower pH. At lower pH values, the electrostatic repulsion between the ligand and the bound protein helps to overcome the attractive forces and hence cause elution.4,33 The pH of elution in a way can be used as a measure of the strength of interaction with the ligand and is a useful parameter to compare the affinity of various resins. Figure 1 plots the elution pH at peak maxima under linear gradient conditions using a wide variety of proteins. These proteins were carefully chosen to test a variety of possible protein–ligand interactions and included model proteins of varying hydrophobicity (Horse cytochrome c and HSA), MAbs (molecules C and D), as well as Fc-fusion proteins (molecules A and B). In an ideal case, a resin that is selective for IgG-based molecules should only bind those and not any other non-IgG–based model protein.
Figure 1. Elution pH of model proteins and IgGs on MabSelect, IgSelect, and ProSep-vA resins
As shown in Figure 1, the model proteins cytochrome c and HSA had an elution pH of around 6.5 (binding condition as described in the experimental section) on all three resins clearly indicating that they were not retained under these conditions. This is in contrast to results shown for other Protein A alternatives such as HCIC and mimetic ligands that bound model proteins as well as Ig-based molecules.24 The non-binding of the model protein to the IgSelect resin (similar to what is seen for Protein A resins) is indicative of its selectivity. Moreover, the elution pHs for all the IgG-based molecules (antibodies and Fc-fusion proteins) were somewhat comparable across the three resins and fell in the pH range of 3.5–3.9. Thus, it can be said that the new camelid antibody ligand had very similar affinity to IgG molecules as a conventional Protein A ligand under the conditions tested.
Comparison of Dynamic Binding Capacity
Dynamic binding capacity (DBC) is one of the most important performance parameters for chromatography resins. This is particularly true for Protein-A–based resins, which are very expensive, and are used as a capture step in antibody purification. DBC was calculated for all four antibodies and Fc-fusion proteins using the methodology described in the experimental section. Figure 2 plots the 1% breakthrough capacity on all three resins.
Figure 2. Dynamic biniding capacity (DBC, 1%) results of the four IgG molecules on MabSelect, IgSelect, and ProSep-vA resins. The tests were carried out at pH 7.0 and six minutes residence time.
From Figure 2, it can be seen that the two Fc-fusion proteins (molecules A and B) had very similar capacities on all three resins. Furthermore, the Fc-fusion proteins had a lower binding capacity than the two antibodies (C and D), particularly on MabSelect. This has been noted in the literature and has been explained by the differences in the steric hindrance exerted by Fc-fusion proteins versus MAbs.34 On the other hand, a difference in DBC was seen amongst the three resins for the two antibodies, molecules C and D. Both antibodies showed the highest capacity on MabSelect. The capacity of the new resin IgSelect was less than MabSelect but comparable (or even slightly higher for D) to ProSep-vA. Controlled pore glass resins such as ProSep-vA have been shown to have lower binding capacities but improved mass transport properties.35,36 The performance of the new resin was in-between the two Protein A resins with respect to DBC. The dynamic binding capacity on all these resins were compared at 6 minutes residence time. These resins might have different responses with varying residence time based on the transport properties of their backbone. However, the response of MabSelect and IgSelect would be very similar because they are based on a similar cross-linked agarose backbone.
It has been conjectured in the literature that the monomeric VHH fragments might have a lower binding capacity because they do not have the capability to bind multiple IgGs like Protein A, which has multiple binding domains.29 This was found to be true by our experimental results with antibodies, particularly on MabSelect and IgSelect because their backbone and ligand densities are comparable. Interestingly, the single-binding domain did not seem to have any negative effect on Fc-fusion proteins because they have a larger steric hindrance and thereby cannot optimally access the multiple binding domains of the Protein A ligand.
Comparison of Adsorption Isotherm
Figure 3 shows the adsorption isotherms of Fc-fusion protein A at pH 7.0 for all the three resins. Table 1 lists the thermodynamic parameters (Qmax and K). It shows that amongst the three resins, MabSelect had the highest Qmax value, which was consistent with DBC results. On the other hand, Qmax for ProSep-vA was slightly lower than IgSelect even though their relative trend for dynamic binding capacity was opposite. This can be explained by improved flow properties for the glass-bead–based ProSep-vA resin, which causes its DBC to be closer to its equilibrium capacity. Furthermore, K values for all three resins were very similar, indicating that the binding strength for Protein A and this new ligand was very similar.
Figure 3. Comparison of adsorption isotherms on MabSelect, IgSelect, and ProSep-vA resins at pH 7.0
Comparison of Selectivity
As mentioned before, it has been shown in the literature that none of the small-molecule synthetic ligands developed as Protein A alternatives were able to provide the lock-and-key induced fit as Protein A.24,29 The non-specific binding of host cell proteins to these ligands have made their selectivity much lower than that of Protein A resins. On the other hand, the VHH fragment has the capability to recognize unique conformational epitope and hence has the promise of showing greater selectivity.
Table 1. Langmuir Isotherm data at pH 7.0
To compare the selectivity of the new resin to Protein A, host cell protein clearance was compared for the four antibodies and Fc-fusion proteins using complex cell-culture fluid. The details of these preparative experiments are outlined in the experimental section. Protein recoveries for all of these experiments were comparable and >90%. The elution pools from these experiments were collected and analyzed for host cell protein (Chinese hamster ovary protein, CHOP) levels. Figure 4 plots the CHOP log reduction values (LRVs) from these experiments. The higher the LRV, the more selective is the resin. Figure 4 shows that IgSelect demonstrated comparable host cell protein clearance to the Protein A resins. In fact, for molecules A, B, and D, the LRV values for IgSelect were slightly lower or comparable to MabSelect but higher than the other Protein A resin ProSep-vA. ProSep resins are known to give slightly lower CHOP clearance than the agarose-based Protein A resins because of non-specific interactions of CHOP with their silica backbone.37 On the whole, the selectivity shown by this new camelid antibody resin appears to be very promising.
Figure 4. Log reduction value (LRV) of Chinese hamster ovary host cell proteins (CHOP) under preparative conditions on MabSelect, IgSelect, and ProSep-vA resins
Exploration of Binding Mechanism
The binding mechanism of IgGs on Protein A ligand has been studied in detail by x-ray crystallography and it has been shown that the interactions consist of hydrophobic interactions along with some hydrogen bonding and two salt bridges.15,38 To explore the differences in the binding mechanisms of Protein A ligand and the IgSelect ligand, linear gradient experiments were conducted with different modifiers in the mobile phase. Molecule A and D (one Fc-fusion protein and one antibody) were used as the test proteins. Only one of the representative Protein A resins (MabSelect) was used for comparison.
As summarized in Table 2, the results for both molecules with no additive in mobile-phase buffer showed similar elution pH on both MabSelect and IgSelect resins, which proved the reproducibility of the data previously included in Figure 1. Change in the elution pH on addition of mobile-phase modifiers was used as an indicator of the change in the strength of binding. An increase in the elution pH would suggest a decrease in binding strength and vice versa. Ethylene glycol (20%) was used in the mobile phase to test the role of hydrophobic interactions in IgG binding on IgSelect resin because this mobile-phase modifier can reduce the effect of hydrophobic interactions. Compared to the control (i.e., with no additives), both molecules A and D had higher elution pHs on MabSelect and IgSelect resins. This indicated that 20% ethylene glycol weakened bindings on both the ligands to a similar extent.
Table 2. Elution pH values with different mobile phase modifiers
A suppressant of surface charge interactions (300 mM NaCl) was added to the mobile phase to investigate the role of electrostatic interaction in IgG binding on the IgSelect resin. Compared to control, both proteins had lower elution pHs on IgSelect resin on adding salt to the mobile phase. In fact, molecule D did not elute from IgSelect even at pH 2.5 under this condition. This indicated that the binding between IgGs and the new ligand was significantly increased after the addition of a medium concentration of salt. In comparison, MabSelect actually showed slightly higher elution pH for both molecules under the same conditions. These results showed that a medium concentration of NaCl affected IgG binding on the two resins in different ways and it required further investigation to determine if the stronger binding on IgSelect was because of decreased electrostatic interactions or increased hydrophobic interactions or a combination of both.
NaCl used in the previous set of experiments could have contributed to increased hydrophobic interactions while decreasing electrostatic interactions. To explore this effect further, a stronger kosmotropic salt (100 mM sodium citrate) was tested as the third mobile-phase modifier to explore the effect of increased hydrophobic interactions with minimum surface charge shielding. Compared to the data with no additives, 100 mM sodium citrate caused a significant binding increase on IgSelect resin while the effect on MabSelect resin was very small. This proved that salt played a much bigger role in the binding on IgSelect resin than MabSelect resin. When the mobile phase contains medium concentration of Kosmotropic salt or even NaCl, the binding of IgG molecules with CaptureSelect ligand can be significantly increased. In comparison, the Protein A ligand did not seem to be significantly affected by the tested salt concentration.
Gaining a fundamental understanding of the interaction mechanism on this new ligand requires additional sophisticated analytical techniques (such as x-ray crystallography and docking calculations) and was beyond the scope of this study. The results shown here demonstrate a clear difference in the way Protein A and the new ligand interact with IgGs.
As the newest alternative to Protein A chromatography, the camelid antibody ligand provided very good selectivity for IgG-based molecules. This resin showed similar binding affinity and somewhat comparable dynamic binding capacity to the commonly used commercial Protein A resins. For antibodies, it did have a lower capacity than the leading agarose-based Protein A resin; however, this shortcoming might be compensated by the vendor by further optimizing the ligand density and appropriate resin pricing. This resin does offer the distinct advantage of being base stable. Different from previously reported mixed mode and Protein A mimetic ligands, this ligand showed as good host cell protein clearance as the Protein A resins used for comparison. Although the new resin has been primarily marketed for IgG3 purification, the results presented in this study demonstrate that this can potentially be used very effectively for industrial MAb purification. Parameters that remain to be evaluated comprehensively before its widespread acceptance and use are column lifetime and viral clearance. Finally, the binding mechanism exploration studies indicated a difference in ligand–IgG interactions between the two ligands. For IgSelect, medium to high concentration of kosmotropic salt should be avoided in the background buffer to maximize antibody recovery during purification.
The authors would like to acknowledge the process biochemistry and analytical groups at Bristol-Myers Squibb for their analytical assay support.
Jia Liu, PhD, is a process engineer in process sciences downstream, John L. Hickey is associate director of process sciences downstream and project management, and Sanchayita Ghose, PhD, is the manager of the process sciences downstream group, all at Bristol Myers Squibb Company, Syracuse, NY, 315.431.7930, firstname.lastname@example.orgAaron Cheung is a chemical engineering student at the Massachusetts Institute of Technology.
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