6 May / June 2021

cIEF runs take longer (typically take up to 30-40 minutes), it provides isoelectric point (PI) values. Figure 6 presents a comparison of the results obtained by using CZE and cIEF. It is clearly seen from this figure that the CZE technique can be readily applied to rapid, high throughout charge heterogeneity profiling. In the case of Emicizumab, CZE in fact provided results approximately five times faster than cIEF with apparently identical relative peak area for the main peak, 53% in both cases.

Figure 5: Effect of sample preparation on the charge heterogeneity analysis of Blinatumomab before (A) and after buffer exchange using a 10 kDa cutoff filter (B). Separation conditions were the same as in Figure 3, except pressure injection at 5 psi for 20 sec.

directly injected for CZE analysis, however, as can be seen in Trace A in Figure 5, no separation was achieved due to interference from the formulation buffer. Buffer exchange was attempted by washing the therapeutic protein sample three times with 100 µL of water through a 10 kDa cut-off centrifugation filter (Millipore, Billerica, MA) at 14 000 x g to eliminate these matrix-related components. The buffer-exchanged samples were subject to CZE analysis and resulted in the separation of five components in less than 6 minutes, as shown in Figure 5, Trace B. Peak 1 was a faster migrating, possibly basic ingredient, Peak 2 was the main component and Peaks 3-5 were slower migrating, possibly acidic species. The migration time and corrected peak area percent reproducibility were evaluated similarly to those discussed for the Emicizumab sample and determined to be 0.28% RSD and 1.58 % RSD, respectively

Many next-generation antibody compounds are produced at low concentration. Since each CZE run is typically <10 minutes in length, it is recommended for such low- concentration samples to first perform the analysis without any sample preparation. If insufficient separation is achieved, then

simple buffer exchange via washing with water can be performed to eliminate the matrix effects.

Applicability of Rapid CZE for Stability Studies

Monitoring the deamidation of therapeutic proteins provides crucial information about the stability of the product. To show the applicability of rapid CZE analysis for such important stability studies, a three-day forced degradation of Emicizumab was performed at pH 8.7 and 45 ºC. The sample was prepared by diluting it with water and 1 M Tris-HCl buffer to obtain a 1 mg/mL protein solution in 100 mM Tris-HCl buffer and then incubated in a heating block. The samples were buffer exchanged on 10 kDa cut-off filters prior to analysis. Figure 6 depicts the resulting data for the separated species using CZE and cIEF modes. CZE is a rapid screening option that can reveal changes in concentration of different components within a sample during forced degradation studies. Because of its mode of action, however, it cannot be used to identify which peaks are acidic and which are basic. For such a determination, capillary isoelectric focusing (cIEF) method can be used. Albeit,

Ultrafast CZE Option for Charge Heterogeneity Analysis of Next-Generation Antibodies

Capillary zone electrophoresis, with its unique mode of action, brings key benefits for charge variant analysis that are needed for novel antibody modalities but not provided by other methods.

CZE offers high-resolution, accurate, reproducible separation of structurally similar charge variants. High-concentration samples can simply be diluted with water prior to injection, while simple buffer exchange may be necessary to eliminate formulation matrix effects for low-concentration samples.

For different types of next-generation antibodies (a bispecific and a BiTE), separations were accomplished in <8 minutes with excellent migration time and corrected peak area percent distribution reproducibilities. In addition, CZE provided similar quantitative results to those obtained using the slower cIEF method when used to analyse samples collected during forced degradation studies.

As importantly, the CZE technique allows for true high-throughput screening and rapid analysis for development, in-process and product release testing applications. Overall, therefore, the CZE method described herein can be readily applied for high-throughput charge heterogeneity analysis of new therapeutic modalities.

It is also worth noting that capillary zone electrophoresis in combination with mass spectrometry enables the identification of modified peptides by separating them from their non-deamidated counterparts, which is not possible with LC due to the marginal mass differences of these molecules.


The technical help of Dr Zoltan Szabo is greatly appreciated.

Figure 6: CZE vs. cIEF analysis of the Forced Deamidation products of Emicizumab. Conditions: CZE Rapid Charge Variant Analysis Kit (SCIEX), cIEF: Advanced cIEF Starter Kit (SCIEX).

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