What's Old is New: Building Better Antibodies

New developments in science can have unexpected consequences, including the beneficial ability to take something old and make it new again. Such is the case with antibodies: recent discoveries in the human genome have helped rejuvenate the promise of monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) as the foundation of new-generation therapeutics.

Both mAbs (around since the 1970s) and pAbs (developed in the 1890s) have strengths that make them attractive as therapeutics. Polyclonals are very versatile and have a high affinity for numerous antigen epitopes, making them a powerful weapon against diseases that are capable of rapid mutation and evolution. Monoclonals have high levels of specificity and homogeneity, letting them target specific structures on molecules in disease cells.

While definitely appealing, however, both were originally based on non-human proteins, so they shared the problem of immune-reactivity side effects when used in humans.

Testing to minimize adverse effects

Reducing or eliminating these adverse reactions depends on the ability to create antibodies that closely resemble human proteins. By applying knowledge about the human genome and proteome, it is now possible to create mAbs and pAbs from proteins that are more humanlike, if not fully human. However, the process is difficult with mAbs and is even more complex with pAbs.

In both cases, successful production of antibody-based therapeutics requires precise testing and analysis of any and all proteins in the compound. The key steps include identification of all proteins present, quantitation of those proteins (size, concentration) and monitoring of purity and stability.

Assessing the current measurement standard

SDS-PAGE has earned its place as the standard for protein measurement and analysis, but it does have some significant disadvantages:

• Variability of results (usually as a result of imprecise sample preparation)
• Loading problems
• Variability in staining
• Modest throughput rate of samples
• Long turnaround time of samples
• Significant amounts of liquid hazardous waste

Any alternative to SDS-PAGE must be comparable or superior when measured against attributes such as precision, accuracy and reproducibility. Additional advantages such as speed and ease of use can provide productivity improvements that address the pragmatic, business need to accelerate time-to-market of more-effective therapeutics.

Delivering comparable results in less time

Capillary gel electrophoresis (CGE) is a well-known alternative to SDS-PAGE. In use since the early 1990s, CGE offers two important advantages for protein analysis: improved automation and faster sample throughput. Agilent has extended these advantages even further by transferring traditional CGE principles into glass chip format. This technology is embodied in two products:

• Agilent 2100 bioanalyzer: Developed in collaboration with Caliper Technologies, the bioanalyzer is a compact lab-on-a-chip system for rapid and automated analysis of protein, DNA, RNA and cells. Lab-on-a-chip technology integrates multiple experimental procedures such as sample handling, separation staining, detection and analysis in a single process.
• Protein 200 Plus LabChip^® kit: This fast and reliable assay is capable of analyzing a multitude of different protein samples. Compared to the previous version, the Protein 200 Plus LabChip kit provides significant enhancements in capabilities and ease of use, performing automated analysis of ten 4-µl samples in under 30 minutes.

When used together, the 2100 bioanalyzer and LabChip kit can size and analyze proteins of 14 to 200 kDa. In addition, it's possible to analyze relative quantitation based on internal standards in each sample, and absolute quantitation based on user-defined calibration standards.

Comparing results, weighing the advantages

Recently, researchers have compared the Agilent solution with SDS-PAGE (and other methods) in applications such as therapeutic recombinant humanized mAbs, half-antibody molecules in IgG4 and protein sizing and quantitation. (Please click the preceding links to access the conference posters.)

The comparisons highlight several strengths of the Agilent solution. Foremost is evidence that accuracy and precision are directly comparable to SDS-PAGE. The 2100 bioanalyzer and LabChip kit also improve productivity by providing easier handling, faster turnaround time and increased throughput of samples. And because the results are captured and preserved as digital data, analysis is also streamlined. What's more, working at the microchip scale produces a much lower volume of liquid hazardous waste.

Specific to the analysis of antibodies, the Agilent solution provides additional advantages:

• Single-chip analysis under both reducing and non-reducing conditions (SDS-PAGE requires two separate gels).
• Absolute quantitation through a software-based calibration capability (instead of SDS-PAGE plus an additional protein quantitation method).
• Wider linear dynamic range than SDS-PAGE.

For a detailed discussion, the Agilent application note "Comparison of different protein quantitation methods" compares results from the 2100 bioanalyzer/LabChip combination with SDS-PAGE as well as the Lowry and Bradford batch-processed methods.

Antibody-based therapies will continue to evolve, but the need for precise, accurate and repeatable testing will remain constant. In all cases, faster identification and quantitation plus dependable monitoring will be part of the future success of these old treatments that are being made new again.

For more information

Working in concert, the Agilent 2100 bioanalyzer and Protein 200 Plus LabChip kit provide a powerful, automated solution for identifying proteins and monitoring the purity and stability of recombinant proteins. To learn more about these and other Agilent life sciences products and resources, please visit the main page of the Life Sciences/Chemical Analysis section of our Web site.

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