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requires only nanolitre injection volumes and therefore minimises the consumption of high-value products. Furthermore, with minimal sample preparation requirements, this method represents a versatile separation platform for high-throughput screening of charge variants. If necessary, the electroosmotic flow can be minimised during capillary electrophoresis with the use of high-concentration zwitterionic buffer components such as epsilon amino caproic acid and specific capillary coating additives like triethylenetetramine [9].


In general, CZE enables analysis times of <10 minutes for next-generation protein therapeutics in their native forms. Studies have demonstrated excellent migration time and corrected peak area distribution reproducibility [10]. With minimal sample preparation for low-concentration samples (buffer exchange), CZE offers the ability for high-throughput analysis in all applications, including stability testing [11]. Figure 3 shows the charge variant analysis of the NIST mAB by CZE with great reproducibility. In addition to the main peak, 5 smaller peaks were readily identified in less than 4 min with the peak area distribution shown by the bar diagram.


CZE Rapid Charge Variant Analysis for Next-Generation Antibodies: Two Examples


To demonstrate the ultrafast charge variant analysis option of next-generation antibodies, the CZE Rapid Charge Variant Analysis Kit (SCIEX, Brea, CA) comprising CZE Rapid Charge Variant Separation Buffer, Acid Wash/ Regenerating Solution, CE Grade Water and Protein Test Mix was used for the separation of the bispecific antibody of Emicizumab (Genentech, South San Francisco, CA) and the BiTE of Blinatumomab (Amgen, Thousand Oaks, CA), new generation drugs for the treatment of haemophilia A, and leukemia [12], respectively. CZE was performed on the PA800 Plus Pharmaceutical Analysis System (SCIEX) using the EZ-CE Pre- Assembled Capillary Cartridge (SCIEX), with all data acquired and processed by the 32 Karat version 10.1 software package (SCIEX).


The Emicizumab sample was prepared by diluting the bispecific antibody (30 mg/ mL) with water to a concentration of 1 mg/ mL. Six consecutive runs were completed (Figure 4), each in under 8 minutes, to reveal six peaks (inset in Figure 4). Peak 3 was the main component in the sample. Peaks 1 and 2 were faster migrating, possibly basic species, while peaks 4-6 were slower


Figure 3: Reproducibility of Charge Variant Analysis Using of the NIST Monoclonal Antibody by CZE. Conditions: CZE Rapid Charge Variant Analysis Kit (SCIEX), 30 cm total capillary length (20 cm effective, 50 µm ID), applied electric field strength: 1000 V/cm, Separation temperature: 25°C. Pressure injection at 5 psi for 10 sec.


Figure 4: CZE Charge Heterogeneity Analysis of the Bispecific Antibody Emicizumab. Conditions were the same as in Figure 3.


migrating, possibly acidic species.


The 6 consecutive runs for the Emicizumab sample resulted in the average migration time and corrected peak area percent reproducibility of 0.2 and 1.55 percent


relative standard deviation (%RSD), respectively, as shown in Table 1.


The Blinatumomab sample contained a much lower concentration (0.2 mg/mL) of the BiTE active ingredient. Initially it was


Table 1: Peak area and migration time reproducibility values for the traces in Figure 4.


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