A comparison of Protein A chromatographic stationary phases: Performance characteristics for monoclonal antibody purification
Zhuo Liu Sigma S. Mostafa ∗ Abhinav A. Shukla
KBI Biopharma, Process Development and Manufacturing, Research Triangle Park, NC, USA
Abstract Protein A chromatography remains the dominant capture step used during the downstream purification of monoclonal antibodies (mAbs). With the recent expiry of the Repligen patent on recombinant Protein A, a variety of new Protein A resins have been introduced in the market. Given productivity limitations during downstream processing that have come into sharper focus with the recent increase in cell culture titers for mAbs, the selection of an appropriate Protein A resin has direct implications on the overall process economics of mAb production. The performance of seven different Protein A chromatographic resins was compared with respect to static
binding capacity and dynamic binding capacity as a function of flow rate. This data was translated into a comparison of productivity (g mAb purified per unit resin volume per unit time) for the seven stationary phases. In addition, elution pH and host cell protein impurity levels after product capture on each of these resins were determined. The current article provides an effective methodology and dataset for the selection of the optimal Protein A chromatographic C 2014 International Union of Biochemistry and Molecular Biology, resin. Inc. Volume 00, Number 0, Pages 1–11, 2014
Keywords: monoclonal antibodies, productivity, Protein A chromatography
1. Introduction Monoclonal antibodies (mAbs) have grown significantly into the predominant class of biopharmaceuticals undergoing clinical development today [1–3]. A large number of mAbs have been successfully commercialized (>30 till date) starting in the mid 1990s and have proven to be a highly effective and versatile therapeutic modality. From a process perspective, titers of mAbs obtained from fed-batch cell culture are now exceeding 5 g/L, and downstream process platforms have enabled increasing standardization of the manufacturing processes used for this class of products [4–6]. From a downstream process perspective, despite strong interest in nonchromatographic separation technologies [7, 8] and in alternative chromatographic
Abbreviations: mAbs, monoclonal antibodies; HCP, host cell protein; CV, column volumes. ∗ Address for correspondence: Abhinav A. Shukla, PhD, KBI Biopharma, Process Development and Manufacturing, 2 Triangle Drive, Research Triangle Park, NC 27709, USA. Tel.: +919-479-9898; e-mail: [email protected]. Received 23 October 2013; accepted 7 May 2014 DOI: 10.1002/bab.1243
Published online in Wiley Online Library (wileyonlinelibrary.com)
capture steps [9], Protein A chromatography is currently firmly established as the capture step with the most prevalent use because of its generic nature and the high degree of purification it affords. On the other hand, the Protein A resin has widely been recognized as the single most costly raw material during mAb purification. This is particularly true during early clinical production because the resin is not used to the full extent of its lifetime (typically up to 100 cycles or more are possible). However, even during commercial production, a high proportion of the raw material cost is directly linked to the Protein A operation. The benefits in terms of reliability, ease of development, and robustness still tend to weigh the argument in favor of employing Protein A chromatography. However, because several different Protein A resins are on the market, it is of definitive interest to biopharmaceutical companies to employ the most optimal Protein A resin. Since the expiry of the Repligen patent on the manufacture of recombinant Protein A ligand in 2009, several new vendors have entered the market with their Protein A resins. It is important to be able to compare these resins with one another on an even footing to be able to make the right process choices. Preparative Protein A resins differ in terms of the Protein A ligand that is employed (base-stable vs. standard-recombinant Protein A) and in terms of the base matrix for the stationary
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Properties of seven different Protein A resins
TABLE 1 Resin
Manufacturer
Matrix
rProtein A Sepharose GE Healthcare Fast Flow
4% cross-linked agarose
MabSelect SuRe
GE Healthcare
MabSelect SuRe LX
Ligand rProtein A (E. coli)
Mean particle Operational flow size (μm) rate (cm/h)
pH range
90
30–300
2–11
Highly cross-linked Alkali-stabilized agarose protein A (E. coli)
85
100–500
2–13
GE Healthcare
Highly cross-linked Alkali-stabilized agarose protein A (E. coli)
85
100–500
2–13
CaptivA PriMAB
Repligen
4% cross-linked agarose
Recombinant Staphylococcal protein A (E. coli)
90
30–300
2–11
POROS MabCapture A
Life Technology Cross-linked poly(styrenedivinylbenzene)
rProtein A
45
700
2–10
Amsphere Protein A JWT203
JSR Life Sciences
Polymethacrylate
rProtein A (E. coli)
49
100–600
3–12
Prosep Ultra Plus
Millipore
Porous glass
rProtein A (E. coli)
60
600
1.5–8.5
phase. These properties are summarized for seven leading resins on the market in Table 1. As can be seen from the table, the resins from GE Healthcare are on their proprietary agarose backbone. The rProtein A Sepharose FF resin is on a more compressible, 4% cross-linked agarose backbone, whereas the MabSelect resins are on a highly cross-linked agarose bead that is less compressible and hence permits taller bed heights and faster flow rates. The CaptivA resin from Repligen is a recent market entrant and is also on a 4% cross-linked agarose bead. In contrast, the other three resins on the list move away from agarose as the base matrix. The POROS MabCapture A resin uses the POROS polystyrene divinyl benzene base bead, which is quite hydrophobic in nature but very stable in terms of resistance to pressure and flow. The Amsphere Protein A resin from JSR Life Sciences is another new entrant in the Protein A resin market. This is on a polymethacrylate base bead. The Prosep Ultra Plus is on an incompressible controlled pore glass bead, which makes it highly resistant to compression and enables operation at high flow rates. Several resins in Table 1 now employ base-stable Protein A ligands. These include the MabSelect SuRe, MabSelect SuRe LX, and the Amsphere Protein A resins. Several aspects of each of these resins are important for use in process applications. High dynamic binding capacities are important to minimize resin volumes needed for the process. On the other hand, the ability to handle a high load flow rate is important to reduce cycle times in operation. The ratio of dynamic binding capacity to operation time is important, as this governs the productivity of the process [10]. A dual-flow-rate strategy with rapid flow rates initially followed by slower flow
2
was demonstrated to increase throughput on Protein A resins by enabling more complete binding to the stationary phase [11]. It is also important to recognize that these priorities can shift as drug development progresses. In early clinical development, Protein A resin cost considerations might drive one toward selecting a resin that has a high dynamic binding capacity and a low resin cost per unit volume. On the other hand, during commercial operation, during which time it is assumed that the resin is used to the full extent of its lifetime, one might be driven toward selection of a resin with a higher productivity to reduce cycle times in manufacturing. The regulatory burden of making a process change in resin during clinical development also needs to be considered in case a switch in resin is considered. Additional considerations are also key in selecting the right Protein A resin. It has been shown that host cell protein (HCP) levels after Protein A chromatography do vary as a result of the hydrophobicity of the base matrix for the resin [12]. This can be a basis for resin selection, as this dictates the difficulty of developing a suitable polishing step for the downstream process as well as the risk of any HCPs persisting through the process. A small amount of Protein A ligand also leaches with each use into the product stream [13]. With improvements in stability of the ligand linkage with the resin, this has become much less of an issue than it used to be with the first generation of commercial Protein A resins. Another important factor to take into consideration is the alkaline stability of the Protein A ligand. Base-stable Protein A was first introduced on the market in 2005 [14]. The key advantage of this resin was its ability to tolerate high concentrations of sodium hydroxide during brief exposure periods used for clean-in-place and column
Protein A Chromatographic Stationary Phases
regeneration. This has huge implications for bioburden control for process intermediates during downstream processing. Even for the base-stable ligands, it has been shown that ligand denaturation is the key attribute that limits Protein A resin lifetime [15]. The current article compares seven different Protein A chromatographic resins that are on the market today. The goal is to provide a practical roadmap for comparison of Protein A resins that can be readily employed in a biopharmaceutical process development setting to help the selection of the most appropriate resin for a particular application. In the present article, these resins are compared with respect to their static and dynamic binding capacities and elution pH. The dynamic binding capacity data at different flow rates is then used to calculate productivity for mAb purification for these resins. Residual HCP levels after Protein A chromatography are also compared in addition to their process performance. This provides a useful dataset to help guide selection of Protein A chromatography resins for mAb downstream processing.
2. Materials and Methods 2.1. Materials Recombinant Protein A Sepharose Fast Flow, MabSelect SuRe, and MabSelect SuRe LX resins were from GE Healthcare (Piscataway, NJ, USA). CaptivA PriMAB, POROS MabCapture, Amsphere Protein A, and Prosep Ultra Plus Protein A resins were obtained from Repligen Bioprocessing, Life Technologies (Grand Island, NY, USA), JSR Life Sciences (Tsukuba, Ibaraki, Japan), and Millipore Corporation (Bedford, MA, USA), respectively. All media were packed in Vantage-L columns (internal diameter 1.1 cm) obtained from Millipore Corporation. POROS Protein A column (4.6 × 50 mm) used for analytical quantification was purchased from Life Technologies. TSKgel G3000SWxl column (7.8 × 300 mm) used for size-exclusion chromatography analysis was obtained from Tosoh Biosciences (Montgomeryville, PA, USA). Citric acid, sodium citrate, glacial acetic acid, sodium acetate, sodium chloride, sodium hydroxide, sodium phosphate monobasic, sodium phosphate dibasic, phosphate-buffered saline (PBS) 10× power concentrate, TRIS base, hydrochloric acid, and benzyl alcohol were purchased from Fisher Scientific (Tampa, FL, USA). The monoclonal antibodies mAb 1 and mAb 2 used in this study were expressed in Chinese hamster ovary (CHO) cells and produced at KBI Biopharma (Durham, NC, USA). Both mAbs are IgG1 s with molecular weights ranging from 145 to 155 kDa and basic isoelectric points between 8.0 and 8.5.
2.2. Equipment All chromatographic experiments were carried out using an AKTA Avant chromatographic system from GE Healthcare (Uppsala, Sweden) with built-in ultraviolet (UV), conductivity, and pH detectors. The UV absorbance of protein samples was measured using Biophotometer Plus UV spectrophotometer
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from Eppendorf International (Hamburg, Germany). Highperformance liquid chromatography analysis was carried out using Agilent 1100 series system from Agilent Technologies (Santa Clara, CA, USA). Adsorption isotherm measurements were carried out in the batch mode by using an end-over-end rotator from Glas-Col (Terre Haute, IN, USA). For the HCP enzyme-linked immunosorbent assay a microtiter plate shaker (Thermo Scientific) and a Molecular Devices (Sunnyvale, CA) SpectraMax 96-well plate reader were used.
2.3. Methods 2.3.1. Adsorption isotherm measurement Adsorption isotherms for mAb 1 were measured in a set of batch experiments at room temperature. For each Protein A resin, a 25% slurry of the resin was made in PBS, pH 7.4, containing 137 mM sodium chloride, 2.7 mM potassium chloride, and 11.9 mM phosphates. Purified mAb 1 at a concentration of 11.11 g/L in the same buffer was used for these experiments. Varying volumes of the protein solution (0.25–2.0 mL) were mixed with a fixed volume of the Protein A resin slurry (1 mL). Additional PBS was added to keep the final volume same for each data point. The system was rotated on an end-over-end rotator for 24 H to achieve equilibrium. After the equilibrium, the unbound protein concentrations in the supernatant were determined by UV absorbance at 280 nm. The amount of the bound protein was then obtained from a mass balance and was used to calculate the amount of protein adsorbed per unit packed bed volume of resin.
2.3.2. Dynamic binding capacity measurements For all frontal-analysis experiments, each resin was packed into a 1.1 cm internal diameter × 10 cm bed height column. The chromatographic column was equilibrated with 5 column volumes (CVs) of equilibration buffer (25 mM citrate buffer, pH 6.0). The mAb 1 clarified cell culture fluid with titer of 1.84 g/L corresponding to target load of 70 g antibody/L resin was loaded onto each column. Then, the chromatographic column was perfused with 5 CVs of wash buffer (25 mM citrate buffer, pH 6.0), 5 CVs of elution buffer (25 mM acetate buffer, pH 3.5), 3 CVs of acid strip buffer (100 mM citric acid, pH 2.0), 3 CVs of regeneration buffer (50 mM NaOH, 1 M NaCl), and 3 CVs of storage buffer (25 mM citrate buffer, 2% benzyl alcohol, pH 5.0). No regeneration step was used for Prosep Ultra Plus resin because of poor resin base bead stability under alkaline conditions. Column effluent fractions (2 CVs) were collected during the loading step, and the antibody concentration in each fraction was determined using analytical Protein A analysis on a POROS Protein A column (4.6 × 50 mm) from Life Technologies. Antibody concentration at the column outlet as a fraction of the inlet feed concentration was plotted against column loading to obtain the breaking-through curve. The breakthrough volume was multiplied by the inlet feed concentration and normalized with respect to the packed bed volume to express the x-axis of the breakthrough curves in the same units as the dynamic binding capacity. Dependent on the operational flow
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Biotechnology and Applied Biochemistry rate for each resin, four out of five different linear flow rates (100, 200, 300, 400, 600 cm/H) were used for the loading, which corresponded to residence times of 6, 3, 2, 1.5, and 1 Min, respectively. The linear flow rate used for the nonloading step was 300 cm/H.
2.3.3. Analytical Protein A analysis Analytical Protein A analysis was carried out to determine antibody concentration in breakthrough fractions. Each sample was loaded onto POROS Protein A column, using loading buffer (50 mM phosphate buffer, 100 mM NaCl, pH 7.0), and to be eluted by elution buffer (100 mM phosphate buffer, 100 mM NaCl, pH 2.5).
2.3.4. Size-exclusion chromatography Analytical size-exclusion chromatography was conducted under isocratic conditions with a TSKgel G3000SWxl column (7.8 × 300 mm) from Tosoh Biosciences, with PBS, pH 7.4, buffer and a flow rate of 0.5 mL/Min. The column effluent was monitored at 280 nm by using a photodiode array detector.
2.3.5. Elution pH determination The elution pH of mAb 1 and mAb 2 was obtained from linear gradient elution experiments, using pulse injections of the samples (0.5 mL injection of protein samples at 5 mg/mL protein concentration). A gradient of pH was run from pH 6.0 to 2.5 over 10 CVs in 25 mM 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 built-in pH detector from GE AKTA Avant system.
2.3.6. Purification of mAbs from clarified cell culture fluids For column purification performance experiments, previous chromatographic columns for generation of breakthrough curves were used. The column was equilibrated with 5 CVs of equilibration buffer (25 mM citrate buffer, pH 6.0). The mAb 1 and mAb 2 clarified cell culture fluids with titers of 1.84 and 2.76 g/L corresponding to the target load of 20 g antibody/L resin were loaded onto each column. Then the chromatographic column was perfused with 3 CVs of wash buffer (25 mM citrate buffer, pH 6.0), 5 CVs of elution buffer (25 mM acetate buffer, pH 3.5), 3 CVs of acid strip buffer (100 mM citric acid, pH 2.0), 3 CVs of regeneration buffer (50 mM NaOH, 1 M NaCl), and 3 CVs of storage buffer (25 mM citrate buffer, 2% benzyl alcohol, pH 5.0). Only a low-pH strip was used for Prosep Ultra Plus resin because of poor resin stability under alkaline conditions. The elution pool pH was immediately adjusted to 5.5 by 1 M Tris buffer, pH 8.0.
2.3.7. Sample analysis for yields, purities, and HCP levels The yields for purified mAbs were calculated according to the protein concentrations determined by UV absorbance at 280 nm with extinction coefficients for mAb 1 and mAb 2. The purified mAb purities were determined by analytical size-exclusion
4
chromatography, using TSKgel G3000SWxl column. The CHO HCP third-generation kit for CHO HCP detection purchased from Cygnus Technologies (Southport, NC, USA) was used to determine HCP levels in each eluate sample. The assay was performed according to the manufacturer’s protocol. Samples were serially diluted twofold in the assay diluent so that the absorbance reading fell within the range of the standard curve (1–100 ng/mL). Data were normalized against the baseline control run for analysis.
3.4. Theory The isotherm data for each resin were fitted to a Langmuir isotherm model to obtain the value of the static binding capacity (Qmax ): Q = Q max C / (K d + C ) ,
(1)
where Q, C, Kd , and Qmax are the concentration of the bound protein (mg protein/mL resin), the concentration of the unbound protein (mg protein/mL solution), the dissociation constant (mg/mL), and the maximum binding capacity (mg protein/ mL resin), respectively. Productivity is the amount of protein purified per unit of packed resin per unit time, which can be expressed as following equation [16]: Rpv =
Lc
1 1 C 0 U1
+
1 Q d U1
,
(2)
where Rpv , Lc , C0 , Ul , N, Qd , and Ue are volumetric production rate (g protein/L resin/H), bed height (cm), feed load titer (g/L), linear flow rate for load (cm/H), number of CVs for nonload steps (unitless), dynamic binding capacity (g protein/L resin), and linear flow rate for nonload steps (cm/H). This equation assumes that the column is loaded either at the maximum binding capacity or a fixed percentage of that across all the resins that are compared in terms of their productivity. The Qd can be expressed as a function of residence time (τ ) by the following empirical model [17]: Qd = Q∞ d τ/ (τd + τ ) ,
(3)
where Qd is the 10% breakthrough dynamic binding capacity at long residence times and τ d is the residence time constant. The dynamic binding capacities versus residence time data were fitted into this equation to obtain Qd and τ d . Therefore, volumetric production rate can be expressed as a function of column length (Lc ) and load linear flow rate (Ul ). The productivity is calculated by assuming that a total of 22 CVs are needed for nonload steps including equilibration, wash, elution, acid strip, regeneration, and storage, which are run at maximum operational flow rate for each resin.
4. Results and Discussion The current article compares leading Protein A chromatography stationary phases in terms of binding capacity, productivity, and product purity levels. The intent is to provide a practical
Protein A Chromatographic Stationary Phases
TABLE 2
Static binding capacity (Qmax ) and dissociation constant (Kd ) for seven different Protein A resins (from Langmuir isotherm linear form fit)
Resin
Qmax (mg/mL) Kd (10−8 M)
R2
rProtein A Fast Flow
64.4
9.73
99.9
MabSelect SuRe
65.9
8.00
99.9
MabSelect SuRe LX
87.8
24.3
98.8
CaptivA PriMAB
62.5
14.7
99.8
POROS MabCapture A
48.0
28.3
99.8
Amsphere Protein A JWT203
62.8
5.40
99.9
Prosep Ultra Plus
70.6
18.0
99.7
database that facilitates the selection of resins for this important step and also provides an easily implementable methodology for their comparison.
4.1. Static and dynamic binding capacities
FIG. 1
Static binding capacity and Langmuir isotherm fits (left panel) and linearized Langmuir fits (right panel) of isotherm for mAb 1 on seven different Protein A resins.
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Static binding capacities were measured on all seven stationary phases by using purified mAb protein. Figure 1 shows the adsorption isotherms measured on the seven chromatographic stationary phases. As expected, the adsorption isotherms show a steep increase in binding capacity even at very low mobilephase protein concentrations. This is expected given the strong affinity this resin has for mAbs. Instead of a direct fit of the isotherm data to Eq. (1), it was found useful to fit linearized data (C/Q vs. C) as shown in Fig. 1 to obtain the Langmuir isotherm parameters as indicated in Table 2. As can be seen in Table 2, static binding capacities at saturation do vary between the various Protein A stationary phases. The MabSelect SuRe LX was a standout with about 90 mg/mL binding capacity because of a concerted effort to optimize the bead structure and Protein A ligand density to optimize mAb binding capacity. The POROS MabCapture A resin was somewhat on the low end of the binding capacity at 48 mg/mL. Other stationary phases gave close to a 65 mg/mL static binding capacity for this molecule. Table 2 also provides the calculated values for the Langmuir isotherm parameters Kd and Qmax for each of these resins. Whereas Qmax correlates directly with the observed maximum static binding capacities, Kd should correlate with the strength of the binding between the mAb and the resin. The higher the Kd value, the less steep the adsorption isotherm, especially at low mobile-phase protein concentrations. In this regard, the MabSelect SuRe LX had the highest Kd , whereas the Amsphere Protein A had the lowest Kd . Although the MabSelect SuRe and the SuRe LX resins have the same Protein A ligand, their Kd values are significantly different. The Kd value may have less to do with the strength of interaction of the mAb with a single Protein A ligand than with an average interaction strength for multiple ligands on the resin surface. It has been shown in the literature [18] that each Protein A ligand binds multiple mAb
5
Biotechnology and Applied Biochemistry molecules and that steric effects dominate the binding capacity. These steric effects can occur on a single Protein A ligand as well as between adjacent Protein A ligands. On the MabSelect SuRe LX resin, because a lot more Protein A ligand has been placed per unit area of the stationary phase, it is conceivable that inter-Protein A ligand steric effects may begin to dominate and reduce the avidity of binding. Dynamic binding capacities were measured at four different linear flow rates on each of the seven Protein A chromatographic resins, and the breakthrough curves at four different linear flow rates are shown in Fig. 2. As can be seen from the figure, certain resins show a greater dependency of flow rate on binding capacity than others. For example, binding to the POROS MabCapture A and the Prosep A Ultra Plus resins are relatively independent of flow rate, whereas that on MabSelect SuRe LX shows the greatest dependency on flow rate. This could again relate to accessibility of the Protein A ligands for binding. Both the POROS and Prosep A resins have a more rigid bead with larger pores that allow for greater flow through them, thus making the ligands more readily available for binding. The same properties of course decrease the available surface area per unit bead volume, leading to a decrease in static binding capacity. Similar trends have been reported in the literature with respect to dynamic binding capacity versus flow rate for MabSelect versus Prosep A resins [19]. Clearly, there is a tradeoff between binding capacity and flow rate for some of the resins with the highest static binding capacities. Thus a comparison of productivity for these resins is warranted to determine if the high capacities on some of the resins can be effectively exploited during process operation. To enable this comparison, the dynamic binding capacity data in Fig. 2 were fit to Eq. (3), and the coefficients Qd and τ d were calculated as listed in Table 3. Analogous to the Langmuir isotherm fitting shown earlier, fitting the plots of τ /Qd versus τ was found to be useful to obtain the coefficients given in Table 3. The fits for the dynamic binding capacity versus residence time data are shown in Fig. 3. As can be seen from the figure, Eq. (3) was successful in fitting the data for all the resins. It must be noted that Eq. (3) is an empirical equation in contrast with the Langmuir isotherm; hence there is no correlation to be expected between Qmax values and Qd values presented in Tables 2 and 3, respectively.
4.2. Productivity As discussed earlier, the amount of product produced per unit time per unit resin volume is an important consideration during commercial manufacturing. Increased time of operation can be a significant concern when the focus is on minimizing suite time to enable a bioreactor batch to be processed in the shortest time possible. Depending on the facility design, this could also be an important consideration earlier in clinical development, particularly if the plant is designed to have multiple bioreactor trains feeding into a single downstream-processing train. The high titers now being routinely achieved in cell culture also increase the emphasis on higher productivity in the downstream
6
process, which is still reliant on batch chromatographic unit operations with a limitation on the maximum size of columns that can be employed. Equation (2) relates the productivity on a given Protein A resin to the bed height and linear flow velocity through the use of Eq. (3), which was fit to the dynamic binding capacity versus residence time data in Fig. 3. This enables productivity to be plotted as a function of linear flow velocity and column bed height for these resins. Figure 4 shows a series of plots for each chromatographic resin at three different load product concentrations from 1 to 5 g/L. As can be seen from the figures, productivity often shows an optimum with respect to linear flow velocity. As linear flow velocity increases, productivity is expected to increase because the operation takes less time. This is offset by the decrease in dynamic binding capacity. These two opposing effects result in an optimum in the productivity for the Protein A process step. In general, productivity decreases with an increase in column bed height while linear flow velocity is held constant, which is plotted on the z-axis in Fig. 4. This is to be expected, as a taller bed height will result in a longer duration for operation and thus decrease productivity. The productivity surface goes through an optimum at a specific column length and linear flow velocity combination. As can be seen in Fig. 4, this is typically at the lower end of bed height and at an optimal linear flow velocity, as explained earlier. As can be seen from the figure, different resins will exhibit this optimum at different linear flow velocities. A resin that has its dynamic binding capacity relatively invariant with respect to flow rate such as the POROS MabCapture A resin will show an increase of productivity at a higher linear flow rate, whereas one whose dynamic binding capacity varies more significantly with flow rate (e.g., MabSelect SuRe LX) will show an optimum at a lower linear flow velocity. Productivity also varies with the concentration of product mAb in the harvest load material. In general, a higher productivity would be achieved with a higher load protein concentration because the load cycle will no longer take as much time. In fact, at a sufficiently high protein concentration in the load material, the load portion of the cycle will not have much significance, and productivity would be dictated by the nonload parts such as equilibration, wash, and elution. For the purpose of the analysis conducted here, the flow rate during the nonload portion of the cycle was assumed to be at the maximum operational flow rate for each resin as listed in Table 1. In actual practice, this will be limited by the compressibility of the resin. A less compressible resin should have productivity advantages in this situation. This is illustrated by the fact that there is a wider optimal region for high-productivity operation for less compressible resins such as the POROS MabCapture A than for resins that are more compressible such as the rProtein A Sepharose FF. In terms of how high the productivity levels are when comparing resins, two sets of resins show themselves to be the highest. One set has resins with high dynamic binding capacity such as the MabSelect SuRe LX. The other set has resins with a lower dynamic binding capacity but possessing dynamic
Protein A Chromatographic Stationary Phases
FIG. 2
Breakthrough curves for mAb 1 with seven different Protein A resins.
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Biotechnology and Applied Biochemistry
TABLE 3
Dynamic binding capacity at long residence time (Qd ) and residence time constant (τ d ) for seven different Protein A resins (from Langmuir isotherm linear form fit)
Qd (mg/mL)
τ d (H)
R2
rProtein A Fast Flow
62.8
0.03694
99.1
MabSelect SuRe
69.6
0.03438
99.2
MabSelect SuRe LX
101.1
0.05221
98.4
CaptivA PriMAB
66.0
0.04733
97.8
POROS MabCapture A
33.0
0.00586
100
Amsphere Protein A JWT203
56.6
0.01866
100
Prosep Ultra Plus
46.6
0.01026
100
Resin
binding capacity that is less dependent on flow rate; this includes resins such as the Prosep A Ultra Plus, the Amsphere Protein A and CaptivA PriMAb. One caveat to this entire analysis is the nondependency assumed for the nonload flow rates. If the nonload flow rate was also assumed to be directly related to residence time, the shape of the surfaces in Fig. 4 would show a constant productivity region at a particular residence time (in other words, a transverse “ridge” for particular bed height and linear flow velocity combinations; data not shown). The overall comparison of productivity between resins still holds in that situation, with the exception that the shortest column length does not turn out to be the one with the highest productivity. The analysis shown above provides a means of comparing productivity between Protein A resins and can be adapted to the specific situation encountered during process operation. The analysis clearly shows that high binding capacity is not the only desirable attribute for Protein A resins, instead a high dynamic binding capacity that does not vary as much with residence time is the goal.
4.3. Elution pH Elution pH is another important attribute for Protein A chromatographic resins, albeit one that would seem to depend more on the ligand employed than on the backbone bead. A lower elution pH is often associated with potential degradation of product quality, including an increase in high molecular aggregate levels. Most resins still use the recombinant Protein A ligand from Repligen (rProtein A FF, CaptivA PriMAb, POROS MabCapture A, and Prosep A Ultra Plus) and would be expected to have a similar elution pH. On the other hand the MabSelect SuRe, MabSelect SuRe LX, and Amsphere Protein A all use an engineered base-stable Protein A ligand. It has been shown in the literature [14] that for VH3 subfamily antibodies, the elution pH is dictated by interactions with the fragment antigen-binding region rather than with the fragment crystallizable region. For
8
FIG. 3
Empirical fits of dynamic binding capacity (Qd ) versus residence time (τ ) using Eq. (3) (left panel) or linearized form (right panel) for mAb 1 on seven different Protein A resins.
Protein A Chromatographic Stationary Phases
FIG. 4
Productivity for seven different resins as a function of linear flow velocity (U) and column length (L) assuming different feed titer as 1 g/L (left panel), 2.5 g/L (center panel), and 5 g/L (right panel).
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TABLE 4
Purification performance for mAb 1 and mAb 2 with seven different Protein A resins
Resin
HCP (ppm)
2.29
97.3
99.2
449
mAb 2
2.38
96.1
98.4
309
mAb 1
2.05
95.7
99.1
606
mAb 2
2.10
96.5
98.2
398
mAb 1
2.09
99.7
98.8
591
mAb 2
1.93
99.5
98.4
310
mAb 1
2.38
95.3
99.0
563
mAb 2
2.44
96.2
98.3
271
mAb 1
1.70
97.9
99.5
886
mAb 2
1.58
99.4
98.3
476
Amsphere Protein mAb 1 A JWT203
4.99
98.1
98.7
7938
mAb 2
3.50
95.4
98.1
3791
Prosep Ultra Plus mAb 1
2.31
99.8
98.7
2338
mAb 2
2.43
99.9
98.1
828
MabSelect SuRe LX
CaptivA PriMAB
Elution pH for mAb 1 and mAb 2 with seven different Protein A resins.
Purity (%)
rProtein A mAb 1 Sepharose Fast Flow
MabSelect SuRe
FIG. 5
Elution CV Yield Molecule number (%)
POROS MabCapture A
there are some important trends in elution pH between resins. In general, the Amsphere Protein A resin had the lowest elution pH, whereas the POROS MabCapture A had the highest elution pH. Overall, elution pH varied between 3.5 and 3.9 for the two mAbs. It can be speculated that interactions with the resin backbone as well as accessibility of the ligands on the resin surface could play a role in determining elution pH for a given resin. Elution pH could be a selection factor for an aggregationprone molecule for which even the relatively small difference in elution pH between 3.5 and 3.9 could be significant. FIG. 6
HCP levels after Protein A capture for mAb 1 and mAb 2 with seven different Protein A resins.
this subclass of mAbs, the engineered base-stable Protein A ligand that is based on a single domain of the Protein A ligand shows a more uniform elution pH across antibodies because they all interact through their fragment crystallizable regions. The two mAbs selected for this study were not from the VH3 subclass and hence do not show significant differences in elution pH between resins with the engineered single-domain versus recombinant multidomain ligands (Fig. 5). Nevertheless,
10
4.4. Process performance and HCP impurity levels To compare their performance in an actual downstream process, the two mAbs were captured on the seven Protein A resins. Table 4 shows a comparison of some of the key outcomes from this purification experiment. To enable an unbiased comparison of purification performance, all seven resins were loaded at 20 g/L capacity such that the column loading did not significantly influence elution pool purity. As can be seen from Table 4, both the mAbs eluted from all the Protein A resins are with high step yields. The number of CVs required for product elution was also fairly similar with variation between 2 and 2.5 CVs observed. An exception was
Protein A Chromatographic Stationary Phases
the Amsphere Protein A resin that had a much wider elution peak ranging up to 5 CVs. This could possibly be the result of nonspecific product interactions with the resin backbone and correlates with the need for lower elution pH for this resin as well. No significant differences were observed in product purity by analytical size-exclusion chromatography for the elution pools. HCP levels are an important performance criterion that drives the development of the polishing steps for mAbs [4]. It has been previously shown that HCPs can associate with mAbs and copurify through Protein A chromatography [12]. An additional mechanism was also observed in which HCPs could associate with Protein A resins that present a more hydrophobic resin backbone and result in enhanced HCP levels in the elution pools. As shown in Fig. 6, HCP levels were found to be relatively consistent for most of the Protein A resins and varied between 300 and 600 ppm. A notable exception to this was seen on the resins with the most hydrophobic base bead. Whereas the POROS MabCapture A resin only showed a slight enhancement in HCP levels, this effect was more dramatic on the Amsphere and the Prosep A resins. It must be noted that the purity levels obtained in Table 4 did not include any specific HCP reduction washes as have been shown to be useful in the literature [12]. Nevertheless, an enhanced HCP level after the capture step is often viewed with concern during purification process development because obtaining quantitative clearance over the polishing steps is not always guaranteed.
5. Conclusions The results presented in the present article represent a comprehensive comparison of Protein A resins currently on the market with respect to static and dynamic binding capacities, productivity, elution pH, and HCP reduction capabilities. This offers insight into the mass transfer and surface properties of the various resins and forms a useful database for the resin developer as well as the process development scientist. The article also presents a methodology for comparing various Protein A resins with each other via a calculated process pro-
Biotechnology and Applied Biochemistry
ductivity (amount of product purified per unit resin volume per unit time). As discussed in the article, no single property dominates the resin selection process. Additional factors such as resin cost, reliability of supply, and supporting documentation in the form of drug master files from the resin supplier also play into the selection of Protein A resins. The ultimate selection of a Protein A chromatographic resin takes into account some of this complexity in addition to many of the performance parameters such as productivity and clearance of impurities.
6. References [1] Beck, A., Wurch, T., Bailly, C., and Corvaia, N. (2010) Nat. Rev. Immunol. 10, 345–352. [2] Piggee, C. (2008) Anal. Chem. 80, 2305–2310. [3] Reichert, J. (2008) Curr. Pharm. Biotechnol. 9, 423–430. [4] Shukla, A., and Thommes, J. (2010) Trends Biotechnol. 28, 253–261. [5] Shukla, A., Hubbard, B., Tressel, T., Guhan, S., and Low, D. (2007) J. Chromatogr. B 848, 28–39. [6] Kelley, B. (2009) mAbs 1, 443–452. [7] Gagnon, P. (2012) J. Chromatogr. A 1221, 57–70. [8] Thommes, J., and Etzel, M. (2007) Biotechnol. Progr. 23, 42–45. [9] Ghose, S., Hubbard, B., and Cramer, S. (2006) J. Chromatogr. A 1122, 144–152. [10] Tugcu, N., Roush, D., and Goklen, K. (2008) Biotechnol. Bioeng. 99, 599–613. [11] Ghose, S., Nagrath, D., Hubbard, B., Brooks, C., and Cramer, S. (2004) Biotechnol. Progr. 20, 830–840. [12] Shukla, A., and Hinckley, P. (2008) Biotechnol. Progr. 24, 1115–1121. [13] Carter-Franklin, J., Victa, C., McDonald, P., and Fahrner, R. (2007) J. Chromatogr. A 1163, 105–111. [14] Ghose, S., Allen, M., Hubbard, B., Brooks, C., and Cramer, S. (2005) Biotechnol. Bioeng. 92, 665–673. [15] Jiang, C., Liu, J., Rubacha, M., and Shukla, A. (2009) J. Chromatogr. A 1216, 5849–5855. [16] Fahrner, R., Whitney, D., Vanderlaan, M., and Blank, G. (1999) Biotechnol. Appl. Bioc. 30, 121–128. [17] McCue, J., Kemp, G., Low, D., and Quinones-Garcia, I. (2003) J. Chromatogr. A 989, 139–153. [18] Ghose, S., Hubbard, B., and Cramer, S. (2007) Biotechnol. Bioeng. 96, 768–779. [19] Hahn, R., Schlegel, R., and Jungbauer, A. (2003) J. Chromatogr. B 790, 35–51.
11
Zhuo Liu Sigma S. Mostafa ∗ Abhinav A. Shukla
KBI Biopharma, Process Development and Manufacturing, Research Triangle Park, NC, USA
Abstract Protein A chromatography remains the dominant capture step used during the downstream purification of monoclonal antibodies (mAbs). With the recent expiry of the Repligen patent on recombinant Protein A, a variety of new Protein A resins have been introduced in the market. Given productivity limitations during downstream processing that have come into sharper focus with the recent increase in cell culture titers for mAbs, the selection of an appropriate Protein A resin has direct implications on the overall process economics of mAb production. The performance of seven different Protein A chromatographic resins was compared with respect to static
binding capacity and dynamic binding capacity as a function of flow rate. This data was translated into a comparison of productivity (g mAb purified per unit resin volume per unit time) for the seven stationary phases. In addition, elution pH and host cell protein impurity levels after product capture on each of these resins were determined. The current article provides an effective methodology and dataset for the selection of the optimal Protein A chromatographic C 2014 International Union of Biochemistry and Molecular Biology, resin. Inc. Volume 00, Number 0, Pages 1–11, 2014
Keywords: monoclonal antibodies, productivity, Protein A chromatography
1. Introduction Monoclonal antibodies (mAbs) have grown significantly into the predominant class of biopharmaceuticals undergoing clinical development today [1–3]. A large number of mAbs have been successfully commercialized (>30 till date) starting in the mid 1990s and have proven to be a highly effective and versatile therapeutic modality. From a process perspective, titers of mAbs obtained from fed-batch cell culture are now exceeding 5 g/L, and downstream process platforms have enabled increasing standardization of the manufacturing processes used for this class of products [4–6]. From a downstream process perspective, despite strong interest in nonchromatographic separation technologies [7, 8] and in alternative chromatographic
Abbreviations: mAbs, monoclonal antibodies; HCP, host cell protein; CV, column volumes. ∗ Address for correspondence: Abhinav A. Shukla, PhD, KBI Biopharma, Process Development and Manufacturing, 2 Triangle Drive, Research Triangle Park, NC 27709, USA. Tel.: +919-479-9898; e-mail: [email protected]. Received 23 October 2013; accepted 7 May 2014 DOI: 10.1002/bab.1243
Published online in Wiley Online Library (wileyonlinelibrary.com)
capture steps [9], Protein A chromatography is currently firmly established as the capture step with the most prevalent use because of its generic nature and the high degree of purification it affords. On the other hand, the Protein A resin has widely been recognized as the single most costly raw material during mAb purification. This is particularly true during early clinical production because the resin is not used to the full extent of its lifetime (typically up to 100 cycles or more are possible). However, even during commercial production, a high proportion of the raw material cost is directly linked to the Protein A operation. The benefits in terms of reliability, ease of development, and robustness still tend to weigh the argument in favor of employing Protein A chromatography. However, because several different Protein A resins are on the market, it is of definitive interest to biopharmaceutical companies to employ the most optimal Protein A resin. Since the expiry of the Repligen patent on the manufacture of recombinant Protein A ligand in 2009, several new vendors have entered the market with their Protein A resins. It is important to be able to compare these resins with one another on an even footing to be able to make the right process choices. Preparative Protein A resins differ in terms of the Protein A ligand that is employed (base-stable vs. standard-recombinant Protein A) and in terms of the base matrix for the stationary
1
Biotechnology and Applied Biochemistry
Properties of seven different Protein A resins
TABLE 1 Resin
Manufacturer
Matrix
rProtein A Sepharose GE Healthcare Fast Flow
4% cross-linked agarose
MabSelect SuRe
GE Healthcare
MabSelect SuRe LX
Ligand rProtein A (E. coli)
Mean particle Operational flow size (μm) rate (cm/h)
pH range
90
30–300
2–11
Highly cross-linked Alkali-stabilized agarose protein A (E. coli)
85
100–500
2–13
GE Healthcare
Highly cross-linked Alkali-stabilized agarose protein A (E. coli)
85
100–500
2–13
CaptivA PriMAB
Repligen
4% cross-linked agarose
Recombinant Staphylococcal protein A (E. coli)
90
30–300
2–11
POROS MabCapture A
Life Technology Cross-linked poly(styrenedivinylbenzene)
rProtein A
45
700
2–10
Amsphere Protein A JWT203
JSR Life Sciences
Polymethacrylate
rProtein A (E. coli)
49
100–600
3–12
Prosep Ultra Plus
Millipore
Porous glass
rProtein A (E. coli)
60
600
1.5–8.5
phase. These properties are summarized for seven leading resins on the market in Table 1. As can be seen from the table, the resins from GE Healthcare are on their proprietary agarose backbone. The rProtein A Sepharose FF resin is on a more compressible, 4% cross-linked agarose backbone, whereas the MabSelect resins are on a highly cross-linked agarose bead that is less compressible and hence permits taller bed heights and faster flow rates. The CaptivA resin from Repligen is a recent market entrant and is also on a 4% cross-linked agarose bead. In contrast, the other three resins on the list move away from agarose as the base matrix. The POROS MabCapture A resin uses the POROS polystyrene divinyl benzene base bead, which is quite hydrophobic in nature but very stable in terms of resistance to pressure and flow. The Amsphere Protein A resin from JSR Life Sciences is another new entrant in the Protein A resin market. This is on a polymethacrylate base bead. The Prosep Ultra Plus is on an incompressible controlled pore glass bead, which makes it highly resistant to compression and enables operation at high flow rates. Several resins in Table 1 now employ base-stable Protein A ligands. These include the MabSelect SuRe, MabSelect SuRe LX, and the Amsphere Protein A resins. Several aspects of each of these resins are important for use in process applications. High dynamic binding capacities are important to minimize resin volumes needed for the process. On the other hand, the ability to handle a high load flow rate is important to reduce cycle times in operation. The ratio of dynamic binding capacity to operation time is important, as this governs the productivity of the process [10]. A dual-flow-rate strategy with rapid flow rates initially followed by slower flow
2
was demonstrated to increase throughput on Protein A resins by enabling more complete binding to the stationary phase [11]. It is also important to recognize that these priorities can shift as drug development progresses. In early clinical development, Protein A resin cost considerations might drive one toward selecting a resin that has a high dynamic binding capacity and a low resin cost per unit volume. On the other hand, during commercial operation, during which time it is assumed that the resin is used to the full extent of its lifetime, one might be driven toward selection of a resin with a higher productivity to reduce cycle times in manufacturing. The regulatory burden of making a process change in resin during clinical development also needs to be considered in case a switch in resin is considered. Additional considerations are also key in selecting the right Protein A resin. It has been shown that host cell protein (HCP) levels after Protein A chromatography do vary as a result of the hydrophobicity of the base matrix for the resin [12]. This can be a basis for resin selection, as this dictates the difficulty of developing a suitable polishing step for the downstream process as well as the risk of any HCPs persisting through the process. A small amount of Protein A ligand also leaches with each use into the product stream [13]. With improvements in stability of the ligand linkage with the resin, this has become much less of an issue than it used to be with the first generation of commercial Protein A resins. Another important factor to take into consideration is the alkaline stability of the Protein A ligand. Base-stable Protein A was first introduced on the market in 2005 [14]. The key advantage of this resin was its ability to tolerate high concentrations of sodium hydroxide during brief exposure periods used for clean-in-place and column
Protein A Chromatographic Stationary Phases
regeneration. This has huge implications for bioburden control for process intermediates during downstream processing. Even for the base-stable ligands, it has been shown that ligand denaturation is the key attribute that limits Protein A resin lifetime [15]. The current article compares seven different Protein A chromatographic resins that are on the market today. The goal is to provide a practical roadmap for comparison of Protein A resins that can be readily employed in a biopharmaceutical process development setting to help the selection of the most appropriate resin for a particular application. In the present article, these resins are compared with respect to their static and dynamic binding capacities and elution pH. The dynamic binding capacity data at different flow rates is then used to calculate productivity for mAb purification for these resins. Residual HCP levels after Protein A chromatography are also compared in addition to their process performance. This provides a useful dataset to help guide selection of Protein A chromatography resins for mAb downstream processing.
2. Materials and Methods 2.1. Materials Recombinant Protein A Sepharose Fast Flow, MabSelect SuRe, and MabSelect SuRe LX resins were from GE Healthcare (Piscataway, NJ, USA). CaptivA PriMAB, POROS MabCapture, Amsphere Protein A, and Prosep Ultra Plus Protein A resins were obtained from Repligen Bioprocessing, Life Technologies (Grand Island, NY, USA), JSR Life Sciences (Tsukuba, Ibaraki, Japan), and Millipore Corporation (Bedford, MA, USA), respectively. All media were packed in Vantage-L columns (internal diameter 1.1 cm) obtained from Millipore Corporation. POROS Protein A column (4.6 × 50 mm) used for analytical quantification was purchased from Life Technologies. TSKgel G3000SWxl column (7.8 × 300 mm) used for size-exclusion chromatography analysis was obtained from Tosoh Biosciences (Montgomeryville, PA, USA). Citric acid, sodium citrate, glacial acetic acid, sodium acetate, sodium chloride, sodium hydroxide, sodium phosphate monobasic, sodium phosphate dibasic, phosphate-buffered saline (PBS) 10× power concentrate, TRIS base, hydrochloric acid, and benzyl alcohol were purchased from Fisher Scientific (Tampa, FL, USA). The monoclonal antibodies mAb 1 and mAb 2 used in this study were expressed in Chinese hamster ovary (CHO) cells and produced at KBI Biopharma (Durham, NC, USA). Both mAbs are IgG1 s with molecular weights ranging from 145 to 155 kDa and basic isoelectric points between 8.0 and 8.5.
2.2. Equipment All chromatographic experiments were carried out using an AKTA Avant chromatographic system from GE Healthcare (Uppsala, Sweden) with built-in ultraviolet (UV), conductivity, and pH detectors. The UV absorbance of protein samples was measured using Biophotometer Plus UV spectrophotometer
Biotechnology and Applied Biochemistry
from Eppendorf International (Hamburg, Germany). Highperformance liquid chromatography analysis was carried out using Agilent 1100 series system from Agilent Technologies (Santa Clara, CA, USA). Adsorption isotherm measurements were carried out in the batch mode by using an end-over-end rotator from Glas-Col (Terre Haute, IN, USA). For the HCP enzyme-linked immunosorbent assay a microtiter plate shaker (Thermo Scientific) and a Molecular Devices (Sunnyvale, CA) SpectraMax 96-well plate reader were used.
2.3. Methods 2.3.1. Adsorption isotherm measurement Adsorption isotherms for mAb 1 were measured in a set of batch experiments at room temperature. For each Protein A resin, a 25% slurry of the resin was made in PBS, pH 7.4, containing 137 mM sodium chloride, 2.7 mM potassium chloride, and 11.9 mM phosphates. Purified mAb 1 at a concentration of 11.11 g/L in the same buffer was used for these experiments. Varying volumes of the protein solution (0.25–2.0 mL) were mixed with a fixed volume of the Protein A resin slurry (1 mL). Additional PBS was added to keep the final volume same for each data point. The system was rotated on an end-over-end rotator for 24 H to achieve equilibrium. After the equilibrium, the unbound protein concentrations in the supernatant were determined by UV absorbance at 280 nm. The amount of the bound protein was then obtained from a mass balance and was used to calculate the amount of protein adsorbed per unit packed bed volume of resin.
2.3.2. Dynamic binding capacity measurements For all frontal-analysis experiments, each resin was packed into a 1.1 cm internal diameter × 10 cm bed height column. The chromatographic column was equilibrated with 5 column volumes (CVs) of equilibration buffer (25 mM citrate buffer, pH 6.0). The mAb 1 clarified cell culture fluid with titer of 1.84 g/L corresponding to target load of 70 g antibody/L resin was loaded onto each column. Then, the chromatographic column was perfused with 5 CVs of wash buffer (25 mM citrate buffer, pH 6.0), 5 CVs of elution buffer (25 mM acetate buffer, pH 3.5), 3 CVs of acid strip buffer (100 mM citric acid, pH 2.0), 3 CVs of regeneration buffer (50 mM NaOH, 1 M NaCl), and 3 CVs of storage buffer (25 mM citrate buffer, 2% benzyl alcohol, pH 5.0). No regeneration step was used for Prosep Ultra Plus resin because of poor resin base bead stability under alkaline conditions. Column effluent fractions (2 CVs) were collected during the loading step, and the antibody concentration in each fraction was determined using analytical Protein A analysis on a POROS Protein A column (4.6 × 50 mm) from Life Technologies. Antibody concentration at the column outlet as a fraction of the inlet feed concentration was plotted against column loading to obtain the breaking-through curve. The breakthrough volume was multiplied by the inlet feed concentration and normalized with respect to the packed bed volume to express the x-axis of the breakthrough curves in the same units as the dynamic binding capacity. Dependent on the operational flow
3
Biotechnology and Applied Biochemistry rate for each resin, four out of five different linear flow rates (100, 200, 300, 400, 600 cm/H) were used for the loading, which corresponded to residence times of 6, 3, 2, 1.5, and 1 Min, respectively. The linear flow rate used for the nonloading step was 300 cm/H.
2.3.3. Analytical Protein A analysis Analytical Protein A analysis was carried out to determine antibody concentration in breakthrough fractions. Each sample was loaded onto POROS Protein A column, using loading buffer (50 mM phosphate buffer, 100 mM NaCl, pH 7.0), and to be eluted by elution buffer (100 mM phosphate buffer, 100 mM NaCl, pH 2.5).
2.3.4. Size-exclusion chromatography Analytical size-exclusion chromatography was conducted under isocratic conditions with a TSKgel G3000SWxl column (7.8 × 300 mm) from Tosoh Biosciences, with PBS, pH 7.4, buffer and a flow rate of 0.5 mL/Min. The column effluent was monitored at 280 nm by using a photodiode array detector.
2.3.5. Elution pH determination The elution pH of mAb 1 and mAb 2 was obtained from linear gradient elution experiments, using pulse injections of the samples (0.5 mL injection of protein samples at 5 mg/mL protein concentration). A gradient of pH was run from pH 6.0 to 2.5 over 10 CVs in 25 mM 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 built-in pH detector from GE AKTA Avant system.
2.3.6. Purification of mAbs from clarified cell culture fluids For column purification performance experiments, previous chromatographic columns for generation of breakthrough curves were used. The column was equilibrated with 5 CVs of equilibration buffer (25 mM citrate buffer, pH 6.0). The mAb 1 and mAb 2 clarified cell culture fluids with titers of 1.84 and 2.76 g/L corresponding to the target load of 20 g antibody/L resin were loaded onto each column. Then the chromatographic column was perfused with 3 CVs of wash buffer (25 mM citrate buffer, pH 6.0), 5 CVs of elution buffer (25 mM acetate buffer, pH 3.5), 3 CVs of acid strip buffer (100 mM citric acid, pH 2.0), 3 CVs of regeneration buffer (50 mM NaOH, 1 M NaCl), and 3 CVs of storage buffer (25 mM citrate buffer, 2% benzyl alcohol, pH 5.0). Only a low-pH strip was used for Prosep Ultra Plus resin because of poor resin stability under alkaline conditions. The elution pool pH was immediately adjusted to 5.5 by 1 M Tris buffer, pH 8.0.
2.3.7. Sample analysis for yields, purities, and HCP levels The yields for purified mAbs were calculated according to the protein concentrations determined by UV absorbance at 280 nm with extinction coefficients for mAb 1 and mAb 2. The purified mAb purities were determined by analytical size-exclusion
4
chromatography, using TSKgel G3000SWxl column. The CHO HCP third-generation kit for CHO HCP detection purchased from Cygnus Technologies (Southport, NC, USA) was used to determine HCP levels in each eluate sample. The assay was performed according to the manufacturer’s protocol. Samples were serially diluted twofold in the assay diluent so that the absorbance reading fell within the range of the standard curve (1–100 ng/mL). Data were normalized against the baseline control run for analysis.
3.4. Theory The isotherm data for each resin were fitted to a Langmuir isotherm model to obtain the value of the static binding capacity (Qmax ): Q = Q max C / (K d + C ) ,
(1)
where Q, C, Kd , and Qmax are the concentration of the bound protein (mg protein/mL resin), the concentration of the unbound protein (mg protein/mL solution), the dissociation constant (mg/mL), and the maximum binding capacity (mg protein/ mL resin), respectively. Productivity is the amount of protein purified per unit of packed resin per unit time, which can be expressed as following equation [16]: Rpv =
Lc
1 1 C 0 U1
+
1 Q d U1
,
(2)
where Rpv , Lc , C0 , Ul , N, Qd , and Ue are volumetric production rate (g protein/L resin/H), bed height (cm), feed load titer (g/L), linear flow rate for load (cm/H), number of CVs for nonload steps (unitless), dynamic binding capacity (g protein/L resin), and linear flow rate for nonload steps (cm/H). This equation assumes that the column is loaded either at the maximum binding capacity or a fixed percentage of that across all the resins that are compared in terms of their productivity. The Qd can be expressed as a function of residence time (τ ) by the following empirical model [17]: Qd = Q∞ d τ/ (τd + τ ) ,
(3)
where Qd is the 10% breakthrough dynamic binding capacity at long residence times and τ d is the residence time constant. The dynamic binding capacities versus residence time data were fitted into this equation to obtain Qd and τ d . Therefore, volumetric production rate can be expressed as a function of column length (Lc ) and load linear flow rate (Ul ). The productivity is calculated by assuming that a total of 22 CVs are needed for nonload steps including equilibration, wash, elution, acid strip, regeneration, and storage, which are run at maximum operational flow rate for each resin.
4. Results and Discussion The current article compares leading Protein A chromatography stationary phases in terms of binding capacity, productivity, and product purity levels. The intent is to provide a practical
Protein A Chromatographic Stationary Phases
TABLE 2
Static binding capacity (Qmax ) and dissociation constant (Kd ) for seven different Protein A resins (from Langmuir isotherm linear form fit)
Resin
Qmax (mg/mL) Kd (10−8 M)
R2
rProtein A Fast Flow
64.4
9.73
99.9
MabSelect SuRe
65.9
8.00
99.9
MabSelect SuRe LX
87.8
24.3
98.8
CaptivA PriMAB
62.5
14.7
99.8
POROS MabCapture A
48.0
28.3
99.8
Amsphere Protein A JWT203
62.8
5.40
99.9
Prosep Ultra Plus
70.6
18.0
99.7
database that facilitates the selection of resins for this important step and also provides an easily implementable methodology for their comparison.
4.1. Static and dynamic binding capacities
FIG. 1
Static binding capacity and Langmuir isotherm fits (left panel) and linearized Langmuir fits (right panel) of isotherm for mAb 1 on seven different Protein A resins.
Biotechnology and Applied Biochemistry
Static binding capacities were measured on all seven stationary phases by using purified mAb protein. Figure 1 shows the adsorption isotherms measured on the seven chromatographic stationary phases. As expected, the adsorption isotherms show a steep increase in binding capacity even at very low mobilephase protein concentrations. This is expected given the strong affinity this resin has for mAbs. Instead of a direct fit of the isotherm data to Eq. (1), it was found useful to fit linearized data (C/Q vs. C) as shown in Fig. 1 to obtain the Langmuir isotherm parameters as indicated in Table 2. As can be seen in Table 2, static binding capacities at saturation do vary between the various Protein A stationary phases. The MabSelect SuRe LX was a standout with about 90 mg/mL binding capacity because of a concerted effort to optimize the bead structure and Protein A ligand density to optimize mAb binding capacity. The POROS MabCapture A resin was somewhat on the low end of the binding capacity at 48 mg/mL. Other stationary phases gave close to a 65 mg/mL static binding capacity for this molecule. Table 2 also provides the calculated values for the Langmuir isotherm parameters Kd and Qmax for each of these resins. Whereas Qmax correlates directly with the observed maximum static binding capacities, Kd should correlate with the strength of the binding between the mAb and the resin. The higher the Kd value, the less steep the adsorption isotherm, especially at low mobile-phase protein concentrations. In this regard, the MabSelect SuRe LX had the highest Kd , whereas the Amsphere Protein A had the lowest Kd . Although the MabSelect SuRe and the SuRe LX resins have the same Protein A ligand, their Kd values are significantly different. The Kd value may have less to do with the strength of interaction of the mAb with a single Protein A ligand than with an average interaction strength for multiple ligands on the resin surface. It has been shown in the literature [18] that each Protein A ligand binds multiple mAb
5
Biotechnology and Applied Biochemistry molecules and that steric effects dominate the binding capacity. These steric effects can occur on a single Protein A ligand as well as between adjacent Protein A ligands. On the MabSelect SuRe LX resin, because a lot more Protein A ligand has been placed per unit area of the stationary phase, it is conceivable that inter-Protein A ligand steric effects may begin to dominate and reduce the avidity of binding. Dynamic binding capacities were measured at four different linear flow rates on each of the seven Protein A chromatographic resins, and the breakthrough curves at four different linear flow rates are shown in Fig. 2. As can be seen from the figure, certain resins show a greater dependency of flow rate on binding capacity than others. For example, binding to the POROS MabCapture A and the Prosep A Ultra Plus resins are relatively independent of flow rate, whereas that on MabSelect SuRe LX shows the greatest dependency on flow rate. This could again relate to accessibility of the Protein A ligands for binding. Both the POROS and Prosep A resins have a more rigid bead with larger pores that allow for greater flow through them, thus making the ligands more readily available for binding. The same properties of course decrease the available surface area per unit bead volume, leading to a decrease in static binding capacity. Similar trends have been reported in the literature with respect to dynamic binding capacity versus flow rate for MabSelect versus Prosep A resins [19]. Clearly, there is a tradeoff between binding capacity and flow rate for some of the resins with the highest static binding capacities. Thus a comparison of productivity for these resins is warranted to determine if the high capacities on some of the resins can be effectively exploited during process operation. To enable this comparison, the dynamic binding capacity data in Fig. 2 were fit to Eq. (3), and the coefficients Qd and τ d were calculated as listed in Table 3. Analogous to the Langmuir isotherm fitting shown earlier, fitting the plots of τ /Qd versus τ was found to be useful to obtain the coefficients given in Table 3. The fits for the dynamic binding capacity versus residence time data are shown in Fig. 3. As can be seen from the figure, Eq. (3) was successful in fitting the data for all the resins. It must be noted that Eq. (3) is an empirical equation in contrast with the Langmuir isotherm; hence there is no correlation to be expected between Qmax values and Qd values presented in Tables 2 and 3, respectively.
4.2. Productivity As discussed earlier, the amount of product produced per unit time per unit resin volume is an important consideration during commercial manufacturing. Increased time of operation can be a significant concern when the focus is on minimizing suite time to enable a bioreactor batch to be processed in the shortest time possible. Depending on the facility design, this could also be an important consideration earlier in clinical development, particularly if the plant is designed to have multiple bioreactor trains feeding into a single downstream-processing train. The high titers now being routinely achieved in cell culture also increase the emphasis on higher productivity in the downstream
6
process, which is still reliant on batch chromatographic unit operations with a limitation on the maximum size of columns that can be employed. Equation (2) relates the productivity on a given Protein A resin to the bed height and linear flow velocity through the use of Eq. (3), which was fit to the dynamic binding capacity versus residence time data in Fig. 3. This enables productivity to be plotted as a function of linear flow velocity and column bed height for these resins. Figure 4 shows a series of plots for each chromatographic resin at three different load product concentrations from 1 to 5 g/L. As can be seen from the figures, productivity often shows an optimum with respect to linear flow velocity. As linear flow velocity increases, productivity is expected to increase because the operation takes less time. This is offset by the decrease in dynamic binding capacity. These two opposing effects result in an optimum in the productivity for the Protein A process step. In general, productivity decreases with an increase in column bed height while linear flow velocity is held constant, which is plotted on the z-axis in Fig. 4. This is to be expected, as a taller bed height will result in a longer duration for operation and thus decrease productivity. The productivity surface goes through an optimum at a specific column length and linear flow velocity combination. As can be seen in Fig. 4, this is typically at the lower end of bed height and at an optimal linear flow velocity, as explained earlier. As can be seen from the figure, different resins will exhibit this optimum at different linear flow velocities. A resin that has its dynamic binding capacity relatively invariant with respect to flow rate such as the POROS MabCapture A resin will show an increase of productivity at a higher linear flow rate, whereas one whose dynamic binding capacity varies more significantly with flow rate (e.g., MabSelect SuRe LX) will show an optimum at a lower linear flow velocity. Productivity also varies with the concentration of product mAb in the harvest load material. In general, a higher productivity would be achieved with a higher load protein concentration because the load cycle will no longer take as much time. In fact, at a sufficiently high protein concentration in the load material, the load portion of the cycle will not have much significance, and productivity would be dictated by the nonload parts such as equilibration, wash, and elution. For the purpose of the analysis conducted here, the flow rate during the nonload portion of the cycle was assumed to be at the maximum operational flow rate for each resin as listed in Table 1. In actual practice, this will be limited by the compressibility of the resin. A less compressible resin should have productivity advantages in this situation. This is illustrated by the fact that there is a wider optimal region for high-productivity operation for less compressible resins such as the POROS MabCapture A than for resins that are more compressible such as the rProtein A Sepharose FF. In terms of how high the productivity levels are when comparing resins, two sets of resins show themselves to be the highest. One set has resins with high dynamic binding capacity such as the MabSelect SuRe LX. The other set has resins with a lower dynamic binding capacity but possessing dynamic
Protein A Chromatographic Stationary Phases
FIG. 2
Breakthrough curves for mAb 1 with seven different Protein A resins.
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TABLE 3
Dynamic binding capacity at long residence time (Qd ) and residence time constant (τ d ) for seven different Protein A resins (from Langmuir isotherm linear form fit)
Qd (mg/mL)
τ d (H)
R2
rProtein A Fast Flow
62.8
0.03694
99.1
MabSelect SuRe
69.6
0.03438
99.2
MabSelect SuRe LX
101.1
0.05221
98.4
CaptivA PriMAB
66.0
0.04733
97.8
POROS MabCapture A
33.0
0.00586
100
Amsphere Protein A JWT203
56.6
0.01866
100
Prosep Ultra Plus
46.6
0.01026
100
Resin
binding capacity that is less dependent on flow rate; this includes resins such as the Prosep A Ultra Plus, the Amsphere Protein A and CaptivA PriMAb. One caveat to this entire analysis is the nondependency assumed for the nonload flow rates. If the nonload flow rate was also assumed to be directly related to residence time, the shape of the surfaces in Fig. 4 would show a constant productivity region at a particular residence time (in other words, a transverse “ridge” for particular bed height and linear flow velocity combinations; data not shown). The overall comparison of productivity between resins still holds in that situation, with the exception that the shortest column length does not turn out to be the one with the highest productivity. The analysis shown above provides a means of comparing productivity between Protein A resins and can be adapted to the specific situation encountered during process operation. The analysis clearly shows that high binding capacity is not the only desirable attribute for Protein A resins, instead a high dynamic binding capacity that does not vary as much with residence time is the goal.
4.3. Elution pH Elution pH is another important attribute for Protein A chromatographic resins, albeit one that would seem to depend more on the ligand employed than on the backbone bead. A lower elution pH is often associated with potential degradation of product quality, including an increase in high molecular aggregate levels. Most resins still use the recombinant Protein A ligand from Repligen (rProtein A FF, CaptivA PriMAb, POROS MabCapture A, and Prosep A Ultra Plus) and would be expected to have a similar elution pH. On the other hand the MabSelect SuRe, MabSelect SuRe LX, and Amsphere Protein A all use an engineered base-stable Protein A ligand. It has been shown in the literature [14] that for VH3 subfamily antibodies, the elution pH is dictated by interactions with the fragment antigen-binding region rather than with the fragment crystallizable region. For
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FIG. 3
Empirical fits of dynamic binding capacity (Qd ) versus residence time (τ ) using Eq. (3) (left panel) or linearized form (right panel) for mAb 1 on seven different Protein A resins.
Protein A Chromatographic Stationary Phases
FIG. 4
Productivity for seven different resins as a function of linear flow velocity (U) and column length (L) assuming different feed titer as 1 g/L (left panel), 2.5 g/L (center panel), and 5 g/L (right panel).
Biotechnology and Applied Biochemistry
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Biotechnology and Applied Biochemistry
TABLE 4
Purification performance for mAb 1 and mAb 2 with seven different Protein A resins
Resin
HCP (ppm)
2.29
97.3
99.2
449
mAb 2
2.38
96.1
98.4
309
mAb 1
2.05
95.7
99.1
606
mAb 2
2.10
96.5
98.2
398
mAb 1
2.09
99.7
98.8
591
mAb 2
1.93
99.5
98.4
310
mAb 1
2.38
95.3
99.0
563
mAb 2
2.44
96.2
98.3
271
mAb 1
1.70
97.9
99.5
886
mAb 2
1.58
99.4
98.3
476
Amsphere Protein mAb 1 A JWT203
4.99
98.1
98.7
7938
mAb 2
3.50
95.4
98.1
3791
Prosep Ultra Plus mAb 1
2.31
99.8
98.7
2338
mAb 2
2.43
99.9
98.1
828
MabSelect SuRe LX
CaptivA PriMAB
Elution pH for mAb 1 and mAb 2 with seven different Protein A resins.
Purity (%)
rProtein A mAb 1 Sepharose Fast Flow
MabSelect SuRe
FIG. 5
Elution CV Yield Molecule number (%)
POROS MabCapture A
there are some important trends in elution pH between resins. In general, the Amsphere Protein A resin had the lowest elution pH, whereas the POROS MabCapture A had the highest elution pH. Overall, elution pH varied between 3.5 and 3.9 for the two mAbs. It can be speculated that interactions with the resin backbone as well as accessibility of the ligands on the resin surface could play a role in determining elution pH for a given resin. Elution pH could be a selection factor for an aggregationprone molecule for which even the relatively small difference in elution pH between 3.5 and 3.9 could be significant. FIG. 6
HCP levels after Protein A capture for mAb 1 and mAb 2 with seven different Protein A resins.
this subclass of mAbs, the engineered base-stable Protein A ligand that is based on a single domain of the Protein A ligand shows a more uniform elution pH across antibodies because they all interact through their fragment crystallizable regions. The two mAbs selected for this study were not from the VH3 subclass and hence do not show significant differences in elution pH between resins with the engineered single-domain versus recombinant multidomain ligands (Fig. 5). Nevertheless,
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4.4. Process performance and HCP impurity levels To compare their performance in an actual downstream process, the two mAbs were captured on the seven Protein A resins. Table 4 shows a comparison of some of the key outcomes from this purification experiment. To enable an unbiased comparison of purification performance, all seven resins were loaded at 20 g/L capacity such that the column loading did not significantly influence elution pool purity. As can be seen from Table 4, both the mAbs eluted from all the Protein A resins are with high step yields. The number of CVs required for product elution was also fairly similar with variation between 2 and 2.5 CVs observed. An exception was
Protein A Chromatographic Stationary Phases
the Amsphere Protein A resin that had a much wider elution peak ranging up to 5 CVs. This could possibly be the result of nonspecific product interactions with the resin backbone and correlates with the need for lower elution pH for this resin as well. No significant differences were observed in product purity by analytical size-exclusion chromatography for the elution pools. HCP levels are an important performance criterion that drives the development of the polishing steps for mAbs [4]. It has been previously shown that HCPs can associate with mAbs and copurify through Protein A chromatography [12]. An additional mechanism was also observed in which HCPs could associate with Protein A resins that present a more hydrophobic resin backbone and result in enhanced HCP levels in the elution pools. As shown in Fig. 6, HCP levels were found to be relatively consistent for most of the Protein A resins and varied between 300 and 600 ppm. A notable exception to this was seen on the resins with the most hydrophobic base bead. Whereas the POROS MabCapture A resin only showed a slight enhancement in HCP levels, this effect was more dramatic on the Amsphere and the Prosep A resins. It must be noted that the purity levels obtained in Table 4 did not include any specific HCP reduction washes as have been shown to be useful in the literature [12]. Nevertheless, an enhanced HCP level after the capture step is often viewed with concern during purification process development because obtaining quantitative clearance over the polishing steps is not always guaranteed.
5. Conclusions The results presented in the present article represent a comprehensive comparison of Protein A resins currently on the market with respect to static and dynamic binding capacities, productivity, elution pH, and HCP reduction capabilities. This offers insight into the mass transfer and surface properties of the various resins and forms a useful database for the resin developer as well as the process development scientist. The article also presents a methodology for comparing various Protein A resins with each other via a calculated process pro-
Biotechnology and Applied Biochemistry
ductivity (amount of product purified per unit resin volume per unit time). As discussed in the article, no single property dominates the resin selection process. Additional factors such as resin cost, reliability of supply, and supporting documentation in the form of drug master files from the resin supplier also play into the selection of Protein A resins. The ultimate selection of a Protein A chromatographic resin takes into account some of this complexity in addition to many of the performance parameters such as productivity and clearance of impurities.
6. References [1] Beck, A., Wurch, T., Bailly, C., and Corvaia, N. (2010) Nat. Rev. Immunol. 10, 345–352. [2] Piggee, C. (2008) Anal. Chem. 80, 2305–2310. [3] Reichert, J. (2008) Curr. Pharm. Biotechnol. 9, 423–430. [4] Shukla, A., and Thommes, J. (2010) Trends Biotechnol. 28, 253–261. [5] Shukla, A., Hubbard, B., Tressel, T., Guhan, S., and Low, D. (2007) J. Chromatogr. B 848, 28–39. [6] Kelley, B. (2009) mAbs 1, 443–452. [7] Gagnon, P. (2012) J. Chromatogr. A 1221, 57–70. [8] Thommes, J., and Etzel, M. (2007) Biotechnol. Progr. 23, 42–45. [9] Ghose, S., Hubbard, B., and Cramer, S. (2006) J. Chromatogr. A 1122, 144–152. [10] Tugcu, N., Roush, D., and Goklen, K. (2008) Biotechnol. Bioeng. 99, 599–613. [11] Ghose, S., Nagrath, D., Hubbard, B., Brooks, C., and Cramer, S. (2004) Biotechnol. Progr. 20, 830–840. [12] Shukla, A., and Hinckley, P. (2008) Biotechnol. Progr. 24, 1115–1121. [13] Carter-Franklin, J., Victa, C., McDonald, P., and Fahrner, R. (2007) J. Chromatogr. A 1163, 105–111. [14] Ghose, S., Allen, M., Hubbard, B., Brooks, C., and Cramer, S. (2005) Biotechnol. Bioeng. 92, 665–673. [15] Jiang, C., Liu, J., Rubacha, M., and Shukla, A. (2009) J. Chromatogr. A 1216, 5849–5855. [16] Fahrner, R., Whitney, D., Vanderlaan, M., and Blank, G. (1999) Biotechnol. Appl. Bioc. 30, 121–128. [17] McCue, J., Kemp, G., Low, D., and Quinones-Garcia, I. (2003) J. Chromatogr. A 989, 139–153. [18] Ghose, S., Hubbard, B., and Cramer, S. (2007) Biotechnol. Bioeng. 96, 768–779. [19] Hahn, R., Schlegel, R., and Jungbauer, A. (2003) J. Chromatogr. B 790, 35–51.
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