Residual Protein A Protocol

Introduction

During bioprocessing, most therapeutic monoclonal antibodies (mAbs) are isolated from cell culture media using Protein A chromatography.¹ During purification, Protein A can leach from the solid phase, ending up in the purified product. This can be problematic as regulatory guidelines dictate that detection and reduction of residual Protein A are necessary to obviate potential immunogenic consequences in vivo.² Thus, there is pressing need to rapidly detect residual Protein A contamination during product characterization with high sensitivity and throughput. ELISA-based methods are time-consuming and labor-intensive, requiring extensive hands-on setup, followed by manual washing and incubation steps at carefully timed intervals. Furthermore, the sensitivity of ELISA varies depending on the antibody and protein antigen, and may not be suitable to detect very low levels of protein (<1 ng/mL). These shortcomings are compounded by matrix interference effects that often confound ELISA results. 

ProteinSimple’s Simple Western™ provides a powerful solution by bringing immunoassay into a capillary electrophoresis format. Its operation is fully automated, requiring no user intervention after a simple setup, thereby greatly reducing time and labor while increasing precision and reproducibility. It minimizes matrix interference effects so you can be confident the quantification of Protein A in your sample is accurate. In addition, you gain molecular weight information of your Protein A contaminant. Here, we show you how Simple Western can quickly detect Protein A with high sensitivity, down to 28 pg/mL, without the caveats of ELISA.

Materials

ProteinSimple - A Bio-Techne Brand

• Instrument: Jess™, Abby™, or Wes™
• 12–230 kDa Separation Module (PN SM-W004)
• No Secondary Detection Module (PN DM-003)

Residual Protein A reagents

• Recombinant Protein A short form (Novus Biologicals, a Bio-Techne Brand, NBP2-34964)
• Recombinant Protein A long form (Novus Biologicals, a Bio-Techne Brand, NBP2-34966)
• Anti-Protein A antibody (R&D Systems, a Bio-Techne Brand, MAB99761)

Other reagents

• Donkey anti-Rat HRP (Jackson Immunoresearch, 712-035- 153)
• CHO HCP Antigen Concentrate, 3G (Cygnus Technologies, F553X)

Methods

1. Prepare 0.1X Sample Buffer by diluting the 10X Sample Buffer 1:100 in water.
2. Dilute the anti-Protein A antibody to 1.2 µg/mL in Antibody Diluent 2 (PN 042-203).
3. Dilute the Donkey anti-Rat HRP secondary antibody to 13.5 µg/mL in Antibody Diluent 2.
4. To determine the LOD and LOQ, reconstitute the short and long forms of recombinant Protein A antigens per the manufacturer’s recommendations and then create a serial dilution series of the antigens in 0.1X Sample Buffer (In this example, a 2-fold dilution series from 400 pg/mL to 25 pg/mL was created.)
5. To determine the LOD and LOQ in the presence of CHO HCP, dilute the CHO HCP stock in 0.1X Sample Buffer to 0.1 mg/mL and then create a serial dilution series of the antigens in 0.1X Sample Buffer and 0.1 mg/mL CHO HCP.
6. Follow the default sample preparation and assay conditions described in the Product Insert of the 12-230 kDa Separation Module.

Results

To establish the assay range, titrations of the Protein A short and long forms were evaluated with Simple Western. Figure 1 shows the electropherograms of the Protein A antigen titrations, Figure 2 shows the linearity of the Protein A antigen titrations, and Figure 3 shows the LOD and LOQ.

The Protein A antibody shows slight cross reactivity with the 230 kDa internal fluorescent standard. This binding will not impact quantitation as Protein A short and long forms have apparent molecular weights of ~42 and ~55 kDa and are well-separated from the 230 kDa peak. To avoid this cross reactivity, 2–40 kDa fluorescent master mix (Fluorescent 5x Master Mix 5, PN PS-FL05-8) may be used in place of the standard 12–230 kDa master mix.

To reflect a real-world scenario, the Protein A short form was spiked into a lysate background. We used the Cygnus CHO HCP Antigen Concentrate (0.1 mg/mL) and compared the LOD/LOQ in the absence and presence of CHO HCP lysate (Figure 4). The linearity comparison shows a minor change in detection of Protein A in the presence of CHO HCP. Additionally, the LOD/LOQ for Protein A increased in the presence of CHO HCP.

Conclusion

Using off-the-shelf antibodies and default Simple Western settings, we developed a quick and robust assay to detect Protein A. Unlike ELISA, no user intervention is required after initial setup, and sensitivity can be achieved that is comparable to or surpasses ELISA-based methods. As little as 28.1 pg/mL was detected, and even in the dense and complex CHO HCP lysate solution, a lower limit of detection of Protein A was achieved at 35.3 pg/mL. For these reasons, Simple Western makes a powerful solution to streamline workflows to meet regulatory standards in the product development pipeline of monoclonal antibodies.

References

1. Protein A chromatography for antibody purification, S Hober, K Nord and M Linhult, Journal of Chromatography B, 2007; 848:40-47.
2. Points to consider in the manufacture and testing of monoclonal antibody products for human use, U.S. Food and Drug Administration, 1997.