March 15, 2011 (Vol. 31, No. 6)
Srinivasa Rao technical manager Dionex
MabPac Strong Cation-Exchanger Columns Designed to Extend Capabilities of mAb Analysis
Monoclonal antibodies (mAbs) have been successfully developed for the treatment of a number of serious diseases. The FDA has approved more than 20 mAbs, and an additional 150 mAbs are currently in preclinical or clinical trials, or awaiting FDA approval. In the last decade, mAbs have become the largest part of the growing biologics drug market and have transformed the biotechnology and biopharmaceutical industries by growing exponentially into annual multibillion dollar sales. One of the key challenges in the development of mAbs is the comprehensive analytical characterization required to demonstrate consistent product quality.
mAbs generally exhibit charge heterogeneity from C-terminal processing of lysine residues, deamidation, glycation, N-terminal pyroglutamate formation, and other modifications. Therefore, the manufacturing and testing procedures for mAbs involve the monitoring of impurities resulting from these modifications.
Weak cation-exchange chromatography is well suited for such applications, and over the years Dionex’ ProPac® WCX-10 columns, packed with nonporous polymeric particles with carboxylic acid functional groups, have been considered the gold standard for high-resolution analysis and characterization of acidic and basic variants using inert HPLC platforms.
In this article, we describe the MAbPac™ SCX-10 column with a new stationary phase with strong cation-exchange functionality, specifically developed for the characterization of heterogeneity of mAbs. This column complements the existing ProPac WCX-10 column, providing high resolution and orthogonal selectivity for mAb separations. We demonstrate selectivity differences in heterogeneity characterization of mAbs using both the ProPac WCX-10 and the MAbPac SCX-10 columns, emphasizing the application of these columns.
The MAbPac SCX-10 column is based on a 10 µm nonporous, highly cross-linked styrenic polymeric media with a uniform hydrophilic coating. Strong cation-exchange functionality is introduced by grafted polymeric chains with sulfonic acid groups. The ATRP grafting technique is used to control the degree of polymerization and graft density of these polymeric chains. As a result, this stationary phase exhibits stability over a wide pH range with high selectivity and minimal band spreading.
Figure 1 shows the separation of a protein mixture demonstrating the selectivity differences between the ProPac WCX-10 and MAbPac SCX-10 columns. Cytochrome C elutes first on ProPac WCX-10, followed by lysozyme and ribonuclease A. Under the same conditions, for the MAbPac SCX-10 column, the elution order is reversed and, in this case, ribonuclease A elutes first followed by cytochrome C and lysozyme, respectively. Such selectivity/elution differences are very beneficial in characterizing closely related protein variants with similar elution profiles during method development. Also, the peak capacity of MAbPac SCX-10 column is higher than the ProPac WCX-10 column.
Figure 1. Selectivity differences between ProPac WCX-10 (4×250 mm; A) and MAbPac SCX-10 (4×250 mm; B) columns: Eluents: A: 20 mM MES (pH 6.4); B: 20 mM MES (pH 6.4) + 1M NaCl; Gradient: 0-48%B in 20.25 min; Flow Rate = 1 mL/min; Sample mixture: Ribonuclease A (1 mg/mL), Cytochrome C (0.2 mg/mL) and Lysozyme 0.25 mg/mL. Injection volume = 10 µL; Detection; UV at 254 nm; Temperature = 30°C. Peaks: 1. Ribonuclease A; 2. Cytochrome C; 3. Lysozyme.
Figure 2 displays an example of a mAb separation exhibiting charge heterogeneity using the WCX and SCX columns. Both the acidic and basic variants could be resolved from three main peaks that display lysine truncation variation.
Figure 2. Monoclonal antibody characterization using cation-exchange columns: Comparison of ProPac WCX (4×250 mm; A) and MAbPac SCX-10 (4×250 mm; B) columns with same gradient slope.Eluents: A: 20 mM MES (pH 5.6) + 60 mM NaCl; B: 20 mM MES (pH 5.6) + 300 mM NaCl; Gradient for (A): 25-46.44B% in 50 min; for (B ): 15–36.44%B in 50 min; Flow Rate = 1 mL/min; Sample: 10 mg mAb/mL; Injection Volume = 5 µL; Detection; UV at 280 nm; Temperature = 30°C. Peaks 1– 5: Acidic variants; Peaks 6, 8, 11: C-terminal Lys truncation variants of main peak; Peaks 12-17: other basic peaks.
Figure 3 displays the characterization of the mAb lysine truncation variants at the C-terminal of the heavy chains by using the MAbPac SCX-10 column. This mAb sample was treated with carboxypeptidase B for three hours at 37°C to cleave C-terminal lysine residues. The treatment illustrates how different retention times of the three main peaks are related to the presence of C-terminal lysine.
A shallow gradient (1–35%B in 50 min) resolves three variant forms differing by the presence of lysine at the C-terminal of the heavy chains with either none (peak 6), one (peak 7), or two (peak 8) lysine residues. Further carboxypeptidase treatment of the mAb demonstrates the quantitative disappearance of peaks 7 and 8 containing 1 and 2 terminal lysine residues, respectively, on their heavy chains. The concomitant increase in area of peak 6 with no lysines demonstrates that the three main peaks are indeed due to lysine truncation variation.
In summary, we have demonstrated selectivity differences of a separation of a protein mixture and heterogeneity characterization of a mAb using a MAbPac SCX-10 column, and a ProPac WCX column emphasizing the utility and applications of the columns. The MAbPac SCX-10 column is rugged and is positioned as a complement to the ProPac WCX column for high-resolution mAb separations.
Figure 3. Characterization of lysine truncation variants of a mAb using MAbPac SCX-10 (4×250 mm) column. Samples: A. mAb 900 µg in 100 µL H2O (no carboxypeptidase); B. mAb 900 µg in 90 µL H2O + carboxypeptidase (CBP) 50 µg in 10 µL (incubation for 3 hours at 37°C). Eluents: A: 20 mM MES (pH 5.6) + 60 mM NaCl; B: 20 mM MES (pH 5.6) + 300 mM NaCl; Gradient: 1–35% B in 50 min; Flow Rate = 1 mL/min; Injection Volume = 5 µL; Detection; UV at 280 nm; Temperature = 30°C. (A) and (B) Peaks 1– 5: Acidic variants; (A) Peaks 6, 7, 8: C-terminal Lys truncation variant peaks; Lysine truncation variant peaks 7, 8 lose their terminal lysine and becomes peak 6 which has no lysines after CBP treatment. Similarly, a minor lysine truncation variant peaks of (A) 9, 10, 11 become 7 (B) after CBP treatment.
Srinivasa Rao (email@example.com) is technical manager, Yuanxue Hou is senior staff chemist, Yury Agroskin is director, and Chris Pohl is senior vp CSTO at Dionex. MAbPac is a trademark and ProPac is a registered trademark of Dionex.