Optimization of cief to Resolve Challenging MAb Samples of Clinical Immunodiagnostic Relevance Sushma Rampal, Sr. Application Scientist, Beckman Coulter Life Sciences, Brea, CA, USA Marcia Santos, Staff Applications Scientist, Beckman Coulter Life Sciences, Brea, CA, USA Ryan Bonn, Senior Scientist, Abbott Diagnostics Division, Abbott Park, IL, USA Introduction Capillary Isoelectric Focusing (cief) analysis is a highly reproducible electrophoretic technique used for the high resolution separation of proteins based on their isoelectric point. This technique is widely used in the biopharmaceutical industry for heterogeneity determination of recombinant monoclonal antibody (rmab) charge isoforms. Beckman Coulter has previously published cief application bulletins describing methods in which stepwise optimization of sample preparation resulted in reproducible separation of charge isoforms 1,2,3. This separation method, described in detail elsewhere 4, has been validated for use on Beckman Coulter s PA 800 Series capillary electrophoresis (CE) systems configured with a UV detector for analysis at 280 nm. Beckman Coulter's 32 Karat control software further supports integration and analysis necessary for accurate pi determination. While an optimized method combines Beckman Coulter's latest CE technology, the PA 800 plus Pharmaceutical Analysis System, and chemical reagents to achieve highest reproducibility, there exist cases in which inadequate resolution results in co-migrating isoform peaks and possible deterioration of a neutral coated capillary. Previously, we found that this phenomenon can be caused by the tendency of the rmab to precipitate on the capillary surface leading to a loss of resolution and reduced capillary life span 1. A systematic approach in which optimization of the sample preparation and addition of capillary rinsing and conditioning procedures were employed with the goal of taining resolution and improving capillary run life. The work reported here illustrates implementation of simple method modifications to successfully resolve these challenging separations 5. We found that replacement of 4.3 M urea, originally described as a capillary cleaning solution to remove precipitation and aggregation, with Sample Loading Solution (SLS) (Beckman Coulter (BEC) PN 608082) aids in taining reproducibility and prolonging capillary life. The resulting procedure is recommended for enhanced performance of both the Neutral Coated Capillary (BEC PN 477441) and N-CHO Capillary (BEC PN 477601). With this new method, capillary life of at least 100 separations was achieved using the N-CHO capillary. Blood Banking Capillary Electrophoresis Centrifugation Flow Cytometry Genomics Lab Automation Lab Tools Particle Characterization Experimental Procedure The reagents described below were all purchased from Sigma-Aldrich and used without any further purification. The corresponding catalog numbers are referenced as necessary. The anolyte was a 200 mm phosphoric acid solution prepared by diluting 685 µl of 85% phosphoric acid (catalog no. 345,245) in 50 ml of double deionized DDI water. The catholyte was a 300 mm sodium hydroxide (NaOH) solution prepared by diluting 15 ml of 1M NaOH solution (catalog no. 72082) in DDI water to a final volume of 50 ml. The chemical mobilizer solution consisted of a 350 mm solution of acetic acid prepared by diluting 1.0 ml of glacial acetic acid (catalog no. 537,020) in DDI water to a final volume of 50 ml. The cathodic stabilizer solution was a 500 mm arginine solution prepared by dissolving 0.87 g of arginine (catalog no. A5006) into DDI water to a final volume of 10 ml. Due to the alkaline nature of the cathodic stabilizer solution, it is important to keep this solution in a sealed flask to avoid absorption of CO 2 from air. The anodic stabilizer solution was a 200 mm solution prepared by dissolving 0.26 g of iminodiacetic acid (catalog no. 220,000) into DDI water to a final volume of 10 ml. All solutions described above were filtered through a 0.2 μm syringe filter and can be kept at room temperature for up to 30 days following preparation. The urea-cief gel solution used in sample preparation consisted of 1.8 g of urea (catalog no. U0631) dissolved in 7.0 ml of cief gel (BEC PN 477497) in a polypropylene conical tube. It is necessary to vortex vigorously until all the solid material has been dissolved. Dissolution of urea may take up to 15 minutes. The solution must be filtered through a 5 µm pore-size low protein bind syringe filter. The urea-cief gel solution must be stored at 2-8 o C and can be used for up to 30 days. IB-18071A
Sample buffer exchange was performed using 20 mm Tris ph 8, prepared by dilution of 4 ml of a 50 mm Tris ph 8 solution (BEC PN 477427) in 6 ml of DDI water to a final volume of 10 ml. This solution must be prepared fresh and should be discarded after use. The ampholyte solution (Pharmalyte 3-10) was purchased from GE-Healthcare Bio-Sciences AB, (catalog no. 17-0456-01). Samples Two proteins are being reported in this study. MAK-33 is a lyophilized monoclonal immunoglobulin G (IgG) antibody purchased from Roche (PN 11200941), and was reconstituted in 20 mm Tris ph 8.0 to a concentration of 5 mg/ml. MAb 1 is a proprietary clinical diagnostic protein property of Abbott Diagnostic Division. Sample Buffer Exchange A sample stock solution was prepared by combining 300 µl of each MAb with 150 µl of 20 mm Tris ph 8.0 in an Amicon Ultra Filter 30 kd cutoff (Millipore, catalog no. UFC900308) and subjected to centrifugation at 12,000 g for 10 min. Additional 20 mm Tris at ph 8.0 (300 µl) was added to the filtered protein solution and this buffer replacement procedure was repeated for two more cycles. The reing concentrated samples were then diluted in additional exchange buffer to yield a final 5 mg/ml stock concentration. Sample Preparation The cief sample mix was prepared by mixing 200 µl of urea-cief gel, 12.0 µl of Pharmalyte 3-10, 20.0 µl of cathodic stabilizer, 2.0 µl of anodic stabilizer, 2.0 µl of pi markers 10, 9.5, 5.5 and 4.1 from the pi Marker Kit (Beckman Coulter PN A58481). The ampholyte mixture is a very dense solution and tends to sit at the bottom of the vial, therefore vigorous vortexing of the ampholytes with the 3M urea gel for 1 minute was necessary followed by centrifugation for approximately 15 seconds. Finally, 20 µl of buffer exchanged sample was added to 200 ul of the resulting mixture and mixed by pipetting up and down several times. Two hundred microliters of the sample mix was transferred to a micro vial (BEC PN 144709) and placed in a universal sample vial (BEC PN A62251). Table 1 indicates the volume of reagents used in each buffer vial, and figure 1 shows the position of the buffer vials in each inlet and outlet buffer tray. Table 1: Volume in Buffer Vials Reagents Vol (ml) # of vials for 25 Runs Water-Dip 1.5 2 Anolyte 1.5 1 Catholyte 1.5 1 Sample Loading Solution 1.5 1 Chemical Mobilizer 1.5 1 Water-Rinse 1.5 2 Water-Waste 0.8 3
Figure 1: Buffer vial positions in inlet and outlet buffer trays Modified instrument method setup used in this study is shown in Fig. 2, 3 and 4 below. Figure 2: Initial conditions for Modified cief Separation Method. Figure 3: UV detector initial conditions for Modified cief Separation Method. Figure 4: Time program of the Modified cief Separation Method. The capillary conditioning and shutdown methods consist of a 5 min/50 psi rinse with Sample Loading Solution followed by 50 psi/5 min rinse with DDI water. In addition, the shutdown method includes a lamp off step followed by a wait step in which the instrument places the ends of the capillary in DDI water for storage.
Results and Discussion: In cief, suppression of the electroosmotic flow (EOF) within the capillary is critical to prevent molecules from sticking to the capillary surface and to tain separation reproducibility 6. Suppression of EOF can be achieved by using coated capillaries which provide a neutral surface in order to better control analyte migration. In isolated cases, even though a neutral surface coating has been applied, proteins can still adhere to the capillary wall resulting in loss of peak resolution and shape, poor reproducibility of separation profile, migration time, and pi, and finally a capillary that has been rendered useless following only a few separations (Figure 5). A B Figure 5: A) Typical full cief separation including focusing and mobilization of a MAb1 which includes internal pi peptide markers 10.0, 9.5, and 4.1. This was run #1 on a newly conditioned neutral capillary employing original methods outlined elsewhere 4. The fingerprint profile of MAb 1 contains a pi range of 6.7-6.3 of six distinct isoform peaks. (B) Select runs from the first 12 consecutive runs on the capillary from (A) of MAb 1. Each electropherogram is offset by 1.7 minutes and 0.013 AU for clarity. Each injection is derived from the same sample vial. Electropherograms 12, 9, and 7 at the top of the figure indicate poor capillary performance. From R. Bonn et al 5. Replacement of the 4.3 M urea solution with the Sample Loading Solution (SLS) alleviated the poor profile reproducibility when using either N-CHO or Neutral coated capillaries (Figures 6 and 7).
RUN #12 RUN #1 Figure 6: Reproducibility of MAb 1 using Sample Loading Solution in a N-CHO Capillary. Stacking of 12 runs obtained on a N-CHO coated capillary with the Improved cief Separation Method which incorporates rinsing with Sample Loading Solution in between runs. RUN #12 RUN #1 Figure 7: Reproducibility of a MAb 1 using Sample Loading Solution in a Neutral Capillary. Stacking of 12 runs obtained on a Neutral coated capillary with the Improved cief Separation Method which incorporates rinsing with Sample Loading Solution in between runs.
CV for detection time, pi, and area percent for the replicate runs 49 through 60 respectively using a N-CHO capillary is reported in Table 2. By replacing the cleaning solution with Sample Loading Solution, the capillary is stable and performs reproducibly for at least 60 runs. Table 2: Detection time reproducibility for MAb1 using a single N-CHO capillary. Data for separations 49-60 shown. Detection time reproducibility (reps 49-60) for MAb1 Basic Basic Acidic Acidic1 Acidic2 Basic Predicted pi of Mab 1 isoforms Basic Acidic Acidic1 Acidic2 Basic Area % composition of Mab A 1 isoforms rep 49 26.90 27.09 27.26 27.45 27.59 6.64 6.57 6.51 6.43 6.38 11.96 25.00 28.06 20.60 14.39 rep 50 26.88 27.07 27.23 27.43 27.58 6.64 6.57 6.50 6.43 6.37 12.16 24.22 27.78 21.42 14.41 rep 51 26.88 27.06 27.23 27.43 27.57 6.64 6.57 6.51 6.43 6.37 12.04 24.46 28.61 20.39 14.50 rep 52 26.85 27.04 27.21 27.40 27.55 6.64 6.57 6.50 6.43 6.37 12.31 24.86 28.03 20.78 14.01 rep 53 26.84 27.03 27.20 27.40 27.54 6.66 6.58 6.52 6.44 6.39 12.02 24.59 28.26 21.02 14.11 rep 54 26.84 27.03 27.20 27.39 27.54 6.63 6.56 6.49 6.42 6.36 11.63 24.39 28.64 21.10 14.23 rep 55 26.84 27.03 27.20 27.39 27.53 6.63 6.55 6.49 6.41 6.36 11.73 24.74 28.86 21.03 13.64 rep 56 26.82 27.01 27.18 27.38 27.52 6.63 6.55 6.49 6.41 6.35 11.87 24.80 28.89 20.47 13.97 rep 57 26.83 27.01 27.18 27.38 27.52 6.63 6.56 6.49 6.42 6.36 12.26 24.35 28.77 20.81 13.81 rep 58 26.81 27.00 27.17 27.37 27.51 6.63 6.55 6.49 6.41 6.36 11.88 24.77 28.35 20.92 14.08 rep 59 26.81 26.99 27.17 27.36 27.50 6.62 6.55 6.49 6.41 6.36 12.07 24.19 28.89 20.75 14.10 rep 60 26.79 26.98 27.16 27.36 27.49 6.63 6.56 6.49 6.42 6.37 11.83 24.20 28.46 20.86 14.65 Ave 26.84 27.03 27.20 27.40 27.54 6.63 6.56 6.50 6.42 6.37 11.98 24.55 28.47 20.85 14.16 SD 0.03 0.03 0.03 0.03 0.03 0.01 0.01 0.01 0.01 0.01 0.20 0.28 0.37 0.28 0.29 % RSD 0.12 0.12 0.11 0.10 0.11 0.15 0.15 0.16 0.16 0.15 0.02 0.01 0.01 0.01 0.02 Basic Acidic Acidic1 Acidic2 It is important to note that it was possible to obtain over 100 separations prior to observing degradation of basic isoforms beginning around separation #120 for protein MAK33. This was accomplished by implementing extra capillary conditioning runs every 6 runs or with every buffer set increment. See figure 8.
Figure 8: Select electropherograms from a MAK-33 cief separation. Six separate 24 sample sequences using a new N-CHO capillary, conditioning runs every 6 separations and Sample Loading Solution as capillary cleaning solution. The figure is offset by 1.5 minutes and 0.013 AU to better illustrate resolution. Electropherograms 120 and 144 at the top of the figure indicate sub-optimal performance 5. Conclusion: This work illustrates improved capillary performance for application of cief using the Beckman Coulter commercially available N-CHO Capillary and Neutral Capillary. Simple method modification including replacement of the 4.3M Urea capillary cleaning solution with Sample Loading Solution (PN 608082) resulted in improved capillary performance and run life when used for separation of challenging MAbs. Furthermore, addition of a conditioning method with each buffer set increment every 6 runs contributed to the improved performance and increased assay efficiency. In summary, the alternative method described provides a solution for challenging cief separations resulting in longer capillary run life and yielding improved separation performance for at least 100 runs in a single N-CHO capillary with no loss of peak resolution or profile.
References: 1. I.D. Cruzado-Park, S. Mack, C.K. Ratnayake, Application bulletin A-11634 Identification of System Parameters Critical for High-Performance cief. 2. I.D. Cruzado-Park, S. Mack, C.K. Ratnayake, Application bulletin A-12015, A Robust cief method: Intermediate Precision for the ph 5-7 range. 3. I.D. Cruzado-Park, S. Mack, C.K. Ratnayake, Application bulletin A-12026, High-resolution cief of therapeutic monoclonal antibodies: A platform method covering ph 4-10 4. PA 800 plus Pharmaceutical Analysis System Capillary Isoelectric Focusing (cief) Analysis Application Guide, A78788. 5. Bonn, Ryan; Rampal, Sushma; Rae, Tracey; Fishpaugh, Jeffrey; Electrophoresis, 2013, 34, 825-32, 6. F Kilar and S. Hjerten; Electrophoresis, 1989, 10,23-9. All trademarks are the property of their respective owners. Beckman Coulter and the stylized logo are trademarks of Beckman Coulter, Inc. and are registered in the USPTO. For Beckman Coulter s worldwide office locations and phone numbers, please visit www.beckmancoulter.com/contact B2013-14118 IB-18071A www.beckmancoulter.com 2013 Beckman Coulter, Inc. PRINTED IN U.S.A.