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Purification of monoclonal antibodies by hydrophobic
interaction chromatography under no-salt conditions
Sanchayita Gho a, Yinying T ao a, Lynn Conley a & Douglas Cecchini b
a Department of Process Biochemistry, Biogen Idec; Rearch Triangle Park, NC USA
b Cambridge Center Bio7-6; Cambridge, MA USA
Published online: 26 Jun 2013.
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mAbs 5:5, 795–800; September/October 2013; © 2013 Landes Bioscience
RepORt
RepORt
*Correspondence to: Sanchayita Gho; Email: Sanchayita. Submitted: 05/21/13; Revid: 06/25/13; Accepted: 06/25/13/10.4161/mabs.25552
Introduction
H ydrophobic interaction chromatography (H IC) occupies a unique niche as a polishing step in many monoclonal antibody (mAb) purification process.1,2 This mode of chromatography is particularly uful for aggregate removal, and it provides good clearance of other process-related impurities such as host cell pro-tein (HCP), leached Protein A and endogenous virus.3-6 HIC is bad on interactions between hydrophobic (aliphatic or aro-matic) ligands on the stationary pha with hydrophobic patches on the surface of proteins.7 Interactions of proteins on HIC are usually promoted by kosmotropic salts, e.g., ammonium sulfate, sodium citrate, potassium phosphate.8 Kosmotropic salts interact with water molecules to reduce solvation of protein molecules in solution and expo their hydrophobic patches to promote bind-ing.9 Elution is usually facilitated by decreasing salt concentration or by u of organic mobile pha modifiers.
Despite its orthogonal lectivity, the u of HIC in any purifi-cation process prents two primary challenges. In general, bind-ing capacity has been traditionally limited on H IC, especially in compari
son to ion exchange chromatography (IEX).10,11 Resin vendors have lately tried to optimize the pore size and ligand den-sity in an effort to maximize capacity;12 however, 10% break-through capacities of > 40 mg/mL of resin have not yet been reported.13 To circumvent this issue, HIC is sometimes ud in the
Hydrophobic interaction chromatography (HIC) is commonly ud as a polishing step in monoclonal antibody purification process. HIC offers an orthogonal lectivity to ion exchange chromatography and can be an effective step for aggregate clearance and host cell protein reduction. HIC, however, suffers from the limitation of u of high concentrations of kosmotropic salts to achieve the desired paration. the salts often po a disposal concern in manufacturing facilities and at times can cau precipitation of the product. Here, we report an unconventional way of operating HIC in the flowthrough (Ft) mode with no kosmotropic salt in the mobile pha. A very hydrophobic resin is lected as the stationary pha and the pH of the mobile pha is modulated to achieve the required lectivity. Under the pH conditions tested (pH 6.0 and below), antibodies typically become positively charged, which has an effect on its polarity and overall surface hydrophobicity. Optimum pH conditions were chon under which the antibody product of interest flowed through while impurities such as aggregates and host cell proteins bound to the column. this
strategy was tested with a panel of antibodies with varying pI and surface hydrophobicity. performance was comparable to that obrved using conventional HIC conditions with high salt.
Purification of monoclonal antibodies by hydrophobic interaction chromatography
under no-salt conditions
Sanchayita Gho,1,* Yinying tao,1 Lynn Conley 1 and Douglas Cecchini 2
1
Department of process Biochemistry, Biogen Idec; Rearch triangle park, NC USA; 2Cambridge Center Bio7-6; Cambridge, MA USA
Keywords: HIC, flowthrough, monoclonal antibodies, no salt, aggregates
flowthrough mode in which the product of interest flows while the more hydrophobic impurities remain bound to the column. This strategy has been particularly popular as a polishing step in antibody process since aggregates are usually more highly retained on HIC.14 Second, the u of high concentrations of salts is highly undesirable in any manufacturing process becau it can cau
corrosion of stainless steel tanks. Due to municipal waste water concerns, it is very expensive to dispo of ammonium sul-fate, the most commonly ud kosmotropic salt.15 In addition, the prence of salt in the load material, elution pool or the FT pool from the H IC step also complicates sample manipulation and requires significant dilution, or an ultrafiltration/diafiltra-tion unit operation, between processing steps.13
Efforts to operate H IC under reduced or no-salt conditions have been reported. Arakawa and rearchers 16,17 tried to u argi-nine to promote binding and facilitate elution in HIC systems. Recently, Gagnon 18 reported the u of glycine in HIC systems to keep conductivities low. Kato et al.19 ud HIC at low salt con-centration for capture of mAbs using a critical hydrophobicity approach, but with limited success.
Here, we report a novel u of HIC in the flowthrough mode with no kosmotropic salt in the mobile pha. Instead of the addi-tion of salt, the pH of the mobile pha was modulated to alter the surface charge of the protein, and thereby influence lectiv-ity. The effect of pH on retention in HIC is usually unpredictable
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conditions. A lesr hydrophobic resin would require even higher salt concentration to provide the same lectivity. To compare the hydrophobicity of various resins on an even basis, linear reten-tion of lysozyme in a decreasing salt (ammonium sulfate) gradi-ent was determined on commonly ud commercial HIC resins. More hydrophobic ligands, e.g., phenyl, butyl, hexyl, octyl, were lected for this experiment, and less hydrophobic ligands such as ether and PPG were excluded. The resins chon for screen-ing were Phenyl Sepharo FF HS (control resin), Capto Phenyl H S, Butyl Sepharo 4FF and Octyl Sepharo 4FF from GE Healthcare, and Phenyl Toyopearl, Butyl Toyopearl and Hexyl Toyopearl from Tosoh.
The linear retention data on all of the resins is shown in Figure 1. Phenyl Sepharo FF HS was actually more hydropho-bic than most other resins. The only resin that was more hydro-phobic than the control resin was Hexyl Toyopearl, and hence this resin was lected for further optimization. Hexyl Toyopearl also offers the advantage of a rigid polymeric backbone and allows faster flow rate a
nd ea of packing at larger scale. Interestingly, Hexyl Toyopearl has traditionally not been lected for bind and elute applications due to overly strong antibody-resin interactions leading to low product recovery.13
Process optimization. To determine the pH of the mobile pha needed for the FT step, pH gradients were run initially under analytical conditions with all four antibodies on the Hexyl Toyopearl resin. A pH range of 6.0–3.5 was chon for the gradi-ent becau most of the antibodies ud in the study were not very stable beyond this range. The pH at which each mAb eluted in the gradient is shown in Figure 2 and the exact values are listed
and thus pH is not frequently studied as a parameter during HIC optimization. In practice, however, it can influence protein reten-tion by titrating charged patches clo to the hydrophobic patches on the protein surface.20 For our examination of the effects of pH adjustment, we lected a very hydrophobic resin to promote maximum interaction with the stationary pha under no-salt conditions.
中国人民大学研招网Results
Four mAbs (mAbs A-D) with varying pIs (~6.5–8.7) and surface hydrophobicity were ud in this stud
y. The antibodies had a HIC FT step in their manufacturing process that primarily rved to reduce aggregates and HCPs. Ammonium sulfate was ud as the kosmotropic salt to achieve the desired lectivity; the concentra-tion lected in the process was dependent on the hydrophobicity of the molecule and the paration desired. The ammonium sul-fate concentration needed for each molecule and the dilution that was required to prepare the load sample for its respective H IC (Phenyl Sepharo Fast Flow [FF] High Substitution [HS]) FT step are shown in Table 1. The aim of this study was to devi an alternative HIC FT step using no-salt conditions that would be comparable in process performance to the existing HIC FT step, which rved as the control.
Resin lection. The first step in the optimization process was to lect a resin that was more hydrophobic than the Phenyl Sepharo FF HS resin ud in the existing process. In the FT mode, only a more hydrophobic resin than the control resin has the potential of achieving the same paration under reduced salt Table 1. Ammonium sulfate concentrations ud in the control HIC (phenyl Sepharo) Ft process and corresponding dilutions with concentrated salt solution required to achieve the required ammonium sulfate concentration Molecule
Ammonium sulfate concentration needed in the existing HIC process
苏州园林导游词
% Dilution needed to achieve the needed salt concentration
A 200 mM 14
B 650 mM 33
C 220 mM
26
D
欧诺拉公主
Control HIC process did not exist
Linear retention of lysozyme on 7 commercially available HIC resins in a decreasing ammonium sulfate gradient.D o w n l o a d e d b y [1.202.15.126] a t 00:30 07 J a n u a r y 2015
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was chon for this study. Since the HIC step was designed to be ud as the 2nd polishing step, eluate from the 1st polishing step was ud as load for this study. All experiments were performed at 100 mg/ml resin loading. Table 4 summarizes the yield and product quality data and shows the consistent performance across all three resin lots.
Discussion
The results shown here demonstrate a new way of utilizing the lective power of a HIC step without using high salt solutions. Operating an H IC step in the abnce of kosmotropic salts in
in Table 2. MAbs B and D were practically unretained and hence eluted at pH 6.0, the starting point of the gradient (Fig. 2).
The pH values listed in Table 2 was ud as the starting point for further optimization of the preparative flowthrough conditions. The amount of protein loaded during the prepara-tive experiment
s was kept the same as the control process for an unbiad comparison. H igher pH s caud the antibody mono-mer to bind more strongly, resulting in lower step yields, while lower pHs caud the high molecular weight (HMW) species to flow through along with the monomer. The goal was to find the optimum pH that gave the best compromi between recovery and HMW clearance. The mobile pha pH was optimized for each molecule to give comparable performance as its respective control step in terms of step yield and impurity (H MW and HCP) clearance (detailed optimization data not shown). Figure 3 shows a reprentative chromatogram for mAb B from the no-salt H IC flowthrough step. The final conditions developed for the new H IC FT step for each antibody are listed in Table 3. A comparison of the data in Tables 2 and 3, indicates that the final optimum pH conditions were fairly clo to tho obtained from the analytical pH gradient experiments. H ence, this can be ud as quick method development tool for this process step. It is also interesting to note that mAbs B and D had the same optimum pH (pH 6.0) despite having pIs at the two ends of the range (8.7 vs. 6.5). This was probably due to the fact that the two mAbs were significantly different in their surface hydrophobicity as determined by linear retention on the control HIC resin (Fig. 4). mAb B is less hydrophobic than mAb D (Fig. 4), which likely counteracted the effect of higher pI. Thus, it can be said that the optimum pH needed by each molecule was influenced by both its pI and surface hydrophobicity. As shown in Table 3, the process data (step recovery and impurity clearance) from th
e two HIC steps (no-salt and high salt control process) indicates that perfor-mance comparable to the control was en in all cas.
Further optimization studies were conducted with mAb B to evaluate the effect of column loading on step performance. Figure 5 plots step yield and H MW level of the FT pool as a function of column loading on the Hexyl resin. Only HMW was monitored becau it was the critical impurity that needed to be removed by this step. Protein A eluate with a higher HMW % was ud for this study to test the worst-ca scenario; hence, the HMW levels here are slightly higher than that reported in Table 3. As en in Figure 5, both yield and HMW levels incread as a function of column loading. This is typical for any flow-through step where the optimum column loading is lected bad on best compromi between yield and desired HMW level. The rate of increa in this ca was found to be similar to what had been en with the historic high salt HIC step. An average loading of ~100 g/L was chon for this process to consistently meet target HMW level of < 1%.
After finalizing the mobile pha conditions and column loading, a resin lot-to-lot variability study was also completed to ensure process robustness at manufacturing scale (Table 4). This was considered important becau resin hydrophobicity was a major contributor to the lectivity of this step. Three lots of Hexyl resin spanning the manufacturer’s specification range
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Table 2. elution pH at peak maxima in a decreasing pH gradient on Hexyl toyopearl data
Molecule
pH at peak maxima
A 5.5
B 6.0
C 5.6D
6.0
*elution pH of 6.0 implies the antibody was un-retained in the gradient.
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Toyopearl 650M, Butyl Toyopearl 650M, and Hexyl Toyopearl 650C were obtained from Tosoh Bioscience. TSK gel G3000 SWXL column (7.8 mm × 300 mm) ud for SEC analysis was purchad from Tosoh Bioscience. All chemicals and salts were purchad from JT Baker.
Equipment. All chromatographic experiments were per-formed on AKTA Explorer chromatographic systems from GE Healthcare. H PLC analysis was performed in a Waters H PLC e2695 Separation Module. Absorbance of protein samples was
the mobile pha can have substantial implications for large scale protein purification process. For example, the method elimi-nates the need for the addition of relatively high concentrations of ammonium sulfate or other kosmotropic salts to the mobile pha prior to the HIC step and avoids the associated dilution of the feed stream. In our ca, this enabled the scale up of a highly productive (high titer) mAb production process in an existing facility by overcoming tank volume limitations. Minimizing pool volumes also had an economic impact as it helped to significantly reduce the size of the costly viral filter that followed the HIC step. Furthermore, removing ammonium sulfate from the manufac-turing process helped reduce disposal costs and was considered more compatible with environmental considerations. While the proof-of-concept described here was demonstrated with mAbs and Hexyl Toyopearl resin and is particularly uful for high titer antibody process, in theory the concept can be extended to any other protein and resin of similar hydrophobicity.
Materials and Methods
Materials. All mAbs ud in this study were produced internally at Biogen Idec in a CHO cell line. MAbs A-D were IgG1s with isoelectric points of ~7.2, 8.7, 7.4, and 6.5, respectively. Model protein lysozyme was purchad from Sigma. Agaro-bad resins such as Phenyl Sepharo H S, Capto Phenyl H S, Butyl Sepharo 4FF and Octyl Sepharo 4FF were obtained from GE Healthcare. Met
hacrylate-bad HIC resins such as Phenyl
Table 3. process performance comparison between high-salt and no-salt HIC Ft step for each antibody mAb
Loading g/L
HIC FT condition
Mobile pha composition
Mobile pha cond
ms/cm
Step Yield %
Product Quality in FT pool HMW %
HCP level ppm
Load – eluate from the first polishing step
0.810A
理在情先
35
Control 200 mM AmSO 4 in 50 mM sodium acetate pH 5.2
39
850.33< 3No salt
10 mM sodium citrate
pH 5.5
2.686
0.21 3.8Load – eluate from the first polishing step
0.725B
65
Control 650 mM AmSO 4 in 20 mM sodium acetate pH 5.695
780.10 4.8No salt
5 mM sodium citrate, pH 6.0 1.388
0.13 4.7Load – eluate from capture step
2.5100C*
70
Control 220 mM AmSO4 in 50 mM sodium acetate pH 5.5
38
860.3138No salt
10 mM sodium citrate
pH 5.5
2.688
0.3423Load – eluate from the first polishing step多彩校园
2.210D
55
Control**----No salt
10 mM sodium citrate
pH 6.0
2.6
90
0.37
< 1.4
*HIC ud as the 2nd polishing step for mAb A, B, D and as the 1st polishing step for mAb C; **Control HIC process did not exist for mAb D, only the new low salt HIC step was developed. Abbreviations: AmSO 4, ammonium sulfate; Ft, flowthrough; HCp, host cell protein; HMW, high molecular weight;
cond, conductivity.
Figure 4. elution salt concentration of mAb B and D on a decreasing ammonium sulfate gradient using phenyl toyopearl resin (Lower elu-tion salt concentration implies greater hydrophobicity).
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