Fast and Simple Charge Heterogeneity Analysis of Monoclonal Antibodies Using Whole-Column Detection Capillary Isoelectric Focusing

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  • Published: Dec 5, 2011
  • Author: Jiaqi Wu
  • Channels: Ion Chromatography

Fast and Simple Charge Heterogeneity Analysis of Monoclonal Antibodies
Using Whole-Column Detection Capillary Isoelectric Focusing (cIEF)


Jiaqi Wu
ProteinSimple
27 Coronet Road, Toronto, Ontario  M8Z 2L8
Canada


Common sources of charge heterogeneity in monoclonal antibodies include heavy chain C-terminal lysine heterogeneity, deamidation and glycosylation.  Quantitative charge heterogeneity analysis for monoclonal antibodies in the pharmaceutical industry is traditionally performed by ion exchange chromatography (IEC)1.  However, IEC method development often requires a significant time investment and the resolution or robustness of the resulting method is not always sufficient.  Capillary isoelectric focusing (cIEF) techniques are also used for the analysis of monoclonal antibody charge heterogeneity4, and imaged cIEF is a fast, robust, high-resolution alternative to IEC for this application.  The generic or platform methods used with imaged cIEF also allow a single method to be used for multiple molecules.  Imaged cIEF performs capillary IEF with whole-column detection, and is significantly different from traditional cIEF which requires a lengthy mobilization step.    The elimination of a mobilization step significantly increases sample throughput and reduces assay complexity.  For monoclonal antibodies, assay throughput is four injections per hour.

Monoclonal antibodies can be analyzed by the iCE3 system in native and denatured forms.  Figure 1 shows six consecutive injections of a monoclonal antibody using the iCE3 under native conditions.

Figure 1.  Overlaid electrophergrams for six consecutive injections of anti-alpha1-antitrypsin mAb.  Sample preparation: 0.2 mg/mL mAb in 0.35% methylcellulose, 1% pH 3-10 Pharmalytes, 3% pH 5-8 Pharmalytes and 4 M urea.  cIEF column:  5 cm, 0.1 mm ID capillary.  Anolyte and Catholyte: 0.08 M H3PO4 and 0.1 NaOH.  The sample is focused at 1.5 kV for 1 minute and then at 3 kV for 7 minutes.

Imaged cIEF assays provide excellent reproducibility.  Figure 1 demonstrates the charge heterogeneity of an mAb, and seven isoforms are observed.  The data obtained from the iCE3 can be quantitatively analyzed in exactly the same manner as IEC.   The pI values of the sample peaks are determined by calibration with internal pI marker peaks.  Figure 2 shows the pI value and peak area percentage for all seven mAb isoform peaks.

Figure 2.  pI value and peak area percentage for the seven anti-alpha1-antitrypsin mAb isoforms.
The first number listed is the pI value, the second is peak area percentage.

Common sources of charge heterogeneity with monoclonal antibodies include c-terminal lysine and glycosylation.  These peaks may be identified using cIEF in combination with an enzyme treatment such as carboxypeptidase to cut the C-terminal lysine, and sialidase to remove the sialic acid6.  Figure 3 is an example of imaged cIEF analysis for a monoclonal antibody under denatured (8 M urea) and reduced (DTT) conditions.  Under these conditions, disulfide bonds in the antibodies are reduced and the monoclonal antibodies are broken into heavy and light chains.  The heavy chain and light chain have different pI values, thus they can be separated by cIEF.  In this way, contributions from both the heavy chain and light chain on the antibodies' charge heterogeneity can be observed.  Figure 3 shows the analysis of the anti-alpha1-antitrypsin mAb under denatured and reduced conditions. The heavy chain and light chain of this mAb are well separated by imaged cIEF, and it can quickly be determined that the charge heterogeneity is mainly on the heavy chain.

 

Figure 3.  Electropherograms of three injections (a, b and c) of anti-alpha1-antitrypsin mAb under denatured conditions (8 M urea) only, and under denatured and reduced conditions (d, e and f).  Sample preparation, denatured condition:  0.1 mg/mL in 0.35% methylcellulose, 1% pH 3-10 Pharmalytes, 3% pH 5-8 Pharmalytes and 8 M urea.  Sample preparation, denatured and reduced conditions:  0.1 mg/mL in 0.35% methylcellulose, 1% pH 3-10 Pharmalytes, 3% pH 5-8 Pharmalytes, 8 M urea and 20 mM DTT.  cIEF column: 5 cm, 0.1 mm ID capillary.  Anolyte and Catholyte: 0.08 M H3PO4 and 0.1 NaOH.  The sample is focused at 1.5 kV for 1 minute and then at 3 kV for 7 minutes.

In cIEF the protein of interest is mixed with ampholytes and pI markers.  Because formulation buffers and salt can disrupt the cIEF separation, they must be minimized during sample preparation either by dilution or desalting.  In the absence of formulation buffer and in the presence of ampholytes proteins can exhibit stability issues, resulting in IEF profile changes over time.  Protein stability is a significant problem with traditional cIEF as it requires both a focusing and mobilization step.   Imaged cIEF does not require a mobilization step, and run times are 15 minutes per sample compared to 50 minutes per sample for traditional cIEF.    Traditional cIEF also requires a highly basic additive such as TEMED or arginine to block the basic end of the capillary during focusing.  The basic nature of these additives can induce changes to the protein sample.  Imaged cIEF uses whole column detection which eliminates the need for arginine or TEMED and provides better sample stability.   The short run time, higher throughput, and simple sample preparation of imaged cIEF reduces sample stability issues and also allows analysis of difficult proteins. 

In cases where the throughput of the iCE3 system alone cannot address labile protein needs, the system is equipped with automated, on-board sample preparation and mixing.   Protein samples stay in their formulations in individual vials in a cooled sample tray until just before injection, at which time they are mixed with IEF buffers.   The iCE3 can support up to 3 different buffers per run or batch for sample preparation.  For example, the pre-mixed IEF buffer that includes carrier ampholytes, pI markers and other necessary additives is added to a reagent vial.  Sample vials containing 20 microL protein samples in their formulation as well as IEF buffer vials are placed in a sample tray which is then cooled.  The system automatically adds 180 µl of IEF buffer to the sample vial and performs on-board mixing just prior to sample injection. 
To test the thoroughness of sample mixing, three injections were performed from the same sample vial after on-board mixing as shown in Figure 4.  The reproducibility of the electropherograms for each of the three injections shows that the sample is well mixed. The on-board sample preparation and mixing feature provided by the iCE3 allows analysis of labile proteins, and also results in more accurate and reproducible data by eliminating potential errors in manual sample preparation.
In conclusion, imaged cIEF with the iCE3 system is an easy-to-use and fast tool for charge heterogeneity analysis of monoclonal antibodies.

Figure 4.  Electropherograms of three injections from one sample vial of anti-alpha1-antitrypsin mAb after automatic sample preparation and on-board mixing with IEF buffer.  IEF buffer:  0.35% methylcellulose, 1% pH 3-10 Pharmalyte, 3% pH 5-8 Pharmalytes, 4 M urea and two pI markers.  Sample:  20 microL of 2mg/mL  anti-alpha1-antitrypsin mAb.  180 microL of IEF buffer was added to the sample solution and mixed.  cIEF column:  5 cm, 0.1 mm ID capillary.  Anolyte and Catholyte:  0.08 M H3PO4 and 0.1 NaOH.  The sample is focused at 1.5 kV for 1 minute and then at 3 kV for 7 minutes.



References

  1. C. Quan, et al, Anal. Biochem., 373, 179-191 (2008).
  2. Z. Sosic, et al, Electrophoresis, 29, 4368-4376 (2008).
  3. X. Chen, et al, mAbs, 1, 563-571 (2009).
  4. S. Hjertén, et al, J. Chromatogr., 346, 265-270 (1985).
  5. J. Wu, et al, J. Chromatogr. B, 714, 113-118 (1998).
  6. M. Hsieh, et al, a poster presentation at CEPharm2006.

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