Rapid charge variant analysis of NANOBODY® using capillary zone electrophoresis (CZE)


Dries Dejaegere1, Gustavo Rivera1 and Stephen Lock2
1Sanofi Large Molecules Research, Belgium, 2SCIEX; United Kingdom.

Abstract


This technical note demonstrates the development of a rapid CZE method for charge variant analysis of NANOBODY samples. With a minimum method optimization using SCIEX Rapid Charge Variant Analysis kit, the developed CZE assay has been evaluated against conventional methods, such as capillary isoelectric focusing (cIEF) and cation exchange chromatography (CEX).

Introduction


Nanobody molecules are derived from a type of antibody that contains only heavy chains, rather than an antibody with both heavy and light chains. A single nanobody domain is approximately 10% of a standard monoclonal antibody (mAb) size. 1,2 By connecting individual nanobody domains together, multivalent nanobody molecules are produced that can bind to multiple targets, making this type of molecule a promising drug modality for complex disease pathology.

Charge variants are important product quality attributes for biological molecules, as they can influence the stability and biological activity of the biotherapeutics. Characterizing the charge heterogeneity of protein therapeutics is important for critical quality attribute (CQA) assessment to ensure drug safety, efficacy and potency.3 Differences in charge variants are often observed due to post-translational modifications, such as chemical modifications to select amino acid sites like deamidation, glycosylation, etc.4  

The rapid CZE assay can achieve the separation of parent intact species from deamidated impurities in ess than 12 minutes, significantly increase the throughput comparing to conventional cIEF and CEX methodology.

Key features of rapid CZE analysis of NANOBODY samples
 

  • Minimum method development: using SCIEX Rapid Charge Variant Analysis kit, limited method optimization was needed to develop a rapid CZE assay

  • Effective charge variant separation: The method is capable of separating charge variants found in a stressed sample using a separation time of less than 12 minutes (Figure 1)

  • Rapid analysis: Analysis times were faster using CZE than conventional methods. The CZE method achieved separations in 12 minutes, compared to 26 minutes for cIEF and 35 minutes for CEX

  • Seamless data process: Compatible with Chromeleon™ chromatography data software (CDS)

Figure 1. CZE separation of intact and deamidated NANOBODY samples. A comparison of an intact NANOBODY sample (black) overlayed with the same sample that had undergone a forced deamidation stress (blue). The figure highlights how a generic CZE method can separate charge isoforms of a NANOBODY sample with limited method optimization. The separation time was 12 minutes.

Methods


Sample preparation: 10 mg/mL NANOBODY samples (45–75 kDa), in formulation buffer (pH 6–7.4), were diluted 10-fold with 3 different types of buffers: 1) Milli-Q water, 2) CZE kit buffer 3) 20:80 (v/v) CZE buffer: Milli-Q water. Then, the prepared samples were injected onto mass spectrometry or stored in the autosampler (at 12°C) during a batch analysis for a maximum of 1 day.

CZE separation method: Samples were analyzed using a PA 800 Plus system (SCIEX). A bare fused silica capillary (length 30.2 cm, effective length 20 cm, 50 µm ID, SCIEX P/N: 338451) was conditioned with 0.1M HCl (5 min, 50 psi) and CZE buffer solution from the kit (5 min, 50 psi). Then, a voltage conditioning step was applied (30 min, 30 kV, normal polarity, 0.17-min ramp).

Samples were analyzed using the CZE Rapid Charge Variant Analysis kit (SCIEX P/N: C44790). The method began with a capillary equilibration step by rinsing with 0.1M HCl (5 min, 50 psi) and CZE buffer solution (3 min, 40 psi). The sample was then pressure injected (10 sec, 0.7 psi), followed by a post-injection of water (10 sec, 0.1 psi). The sample was separated using normal polarity (16 min, 30 kV, 1-min ramp) at 25°C.

At the end of a batch, a shutdown method was run. This method included a rinse with 0.1M HCl (5 min, 50 psi) and water (5 min, 50psi), followed by an additional water rinse (10 min, 100 psi).

CE detection: Detection was performed by UV absorbance at 214 nm at a data sampling rate of 4 Hz.

CE data processing: Data were collected using the PA 800 Plus system and 32 Karat software (SCIEX). The data were exported and processed in Thermo Scientific™ Chromeleon CDS.

CEX data were obtained using an in-house method developed on an Agilent 1200 HPLC system using a BioPro IEX SF column. The cIEF profile was obtained by cIEF analysis on a PA 800 Plus system. The same PA 800 Plus system was used following previously reported methods.4 The master mixture was prepared by mixing the sample with urea (3M), cIEF gel, Pharmalytes 3–10, cathodic stabilizer (L-arginine) and anodic stabilizer (iminodiacetic acid).

Results and discussion


This study aimed to achieve a rapid charge variant analysis for NANOBODY samples. A 12-minute CZE method was developed, significantly increasing the sample throughput compared to classical in-house approaches using CEX and cIEF, which require approximately 45 minutes to complete the data acquisition.

The separation as evaluated in the initial tests using 2 different NANOBODY molecules. The isoelectric point (pI) of sample B was 9.2 and the pI of sample C was 10.0. A series of 5 samples were then analyzed to test the desired throughput using a 16-minute method and 4 Hz sampling rate. Finally, the effect of the sample buffer was evaluated. Figure 2 compares the same nanobody molecule (sample B) diluted with 1) CZE buffer solution, 2) Milli-Q water and 3) 20:80(v/v) CZE buffer: Milli-Q water. Dilution with Milli-Q water yielded the best peak shape. This result was attributed to improved field amplified sample stacking compared to the other sample solvents5.

Figure 2. The effect of the sample buffer on the CZE electropherograms. Samples were diluted in CZE buffer (red), a mixture of CZE buffer and Milli-Q water (blue) or Milli-Q water (black).

To evaluate the reproducibility of the CZE assay, NANOBODY sample B was injected in 5 different days. The overlaid electropherograms demonstrate consistent results across the days (Figure 3).

Finally, this CZE method was compared against traditional inhouse cIEF and CEX approaches. An example of Nanobody sample C analyzed using the 3 methods is shown in Figure 4. This comparison (Figure 4) shows that the CZE method can separate and detect charge variants (for example, deamidated species) of a Nanobody sample in less than half the analysis time of the other 2 conventional techniques with very little up-front development time.

Figure 3. 5-day reproducibility study of NANOBODY CZE analysis

Figure 4. Comparison of intact Nanobody (black) and stressed NANOBODY (blue) samples separated using 3 methods. Samples were analyzed by CZE (A), a classic CEX method (B) and standard cIEF method (C) without optimization6 .

Conclusion
 

  • With limited method development and optimization time a rapid CZE method for the separation and detection of charge isoforms present in Nanobody samples was achieved using the SCIEX CZE Rapid Charge Variant Analysis kit

  • Different dilution buffers were evaluated to achieve the optimal peak shape and separation in the electrophoretic separation. Milli-Q water demonstrates the best peak shape.

  • Performance of this assay over 5 days illustrates consistent data quality and separation profile using the rapid 12-minute CZE method we developed. The throughput is twice that of current inhouse CEX and cIEF methods.

  • Seamless data processing using Chromeleon CDS facilitates data integrity.

References
 

  1. Jovčevska I and Muyldermans S (2020),The Therapeutic Potential of Nanobodies. BioDrugs 34:11–26.

  2. Ingram JR, Schmidt FI, Ploegh HL (2018), Exploiting Nanobodies’ Singular Traits. Ann Rev Immunol 36:695– 715.

  3. Liu H, et al. (2014). In vitro and in vivo modifications of recombinant and human IgG antibodies. MAbs. 2014;6(5):1145-54.

  4. Moritz, B. et al. (2015). Evaluation of capillary zone electrophoresis for charge heterogeneity testing of monoclonal antibodies, J. Chromatogr. B., 983-985, 101- 110.

  5. Zarad, W. et. Al (2021). Field amplified sample stacking and in-capillary derivatization for forensic analysis of morphine and morphine-6-glucuronide in human urine by capillary electrophoresis. Talanta Open 3, 100041.

  6. Ratnayake, C. et al. (2018). Achieving Robust cIEF Analysis of Monoclonal Antibodies While Increasing Capillary Run-Life and Maintaining High Resolution and Reproducibility. Sciex technical note RUO-MKT-02-7480- A.
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