Treatment of cancer has been one of the challenges faced by researchers and doctors across the globe. Drug discovery is aimed to find drugs where there is a wide difference between the effective dose and the dose where serious side effects are observed. This ensures that the treatment is effective while the side effects are minimum. Researchers across the globe strive to make the drugs as specific as possible. Many effective small-molecule drugs are known, but it is difficult to discover highly specific small-molecule drugs. On the other hand, biologics can be developed with exquisite selectivity.
Protein biopharmaceuticals, based on monoclonal antibodies, have emerged as an effective approach to cancer treatment due to their binding specificity. Drugs based on monoclonal antibodies (mAbs), known as immunotherapeutic drugs, are designed to boost the body's own immune system to fight the disease. Monoclonal antibody based drugs provide necessary specificity to kill the cancer cells without affecting the normal host cells.
Recent cancer research is focused on new targeted therapeutics called “antibody-drug conjugates” (ADCs). Antibody drug conjugates are monoclonal antibodies covalently conjugated to cytotoxic drugs through either lysine or cysteine residues. The antibody brings specificity to drug delivery by binding to epitopes on the surface of cells responsible for a disease state (for example, cancer). Once the ADC reaches its target, the conjugated drug is released creating a high local concentration of cytotoxic drug. The specificity of the mAb towards the cancer cells helps in effective targeting of the tumor cells while the bound cytotoxic drug when released at site can bring more effective destruction of the diseased cell. This approach is found to reduce side effects of the small molecule drug. Antibody-drug conjugates are extremely complex and the characterization of ADCs presents a great analytical challenge. The conjugation introduces heterogeneity to the already heterogeneous mAb and additional analytical methods are required for efficient characterization of these molecules.
Agilent provides complete workflow solution that works together to unlock complex analytical challenges such as Intact ADC drug antibody ratio (DAR) determination; Processed ADC DAR characterization; and ADC payload localization.
Intact ADCs drug antibody ratio (DAR) determination
Drug antibody ratio is an important parameter to characterize the antibody-drug conjugates (ADCs). Drug antibody ratio values must be determined and monitored throughout the discovery process. Low value of DAR suggests poor efficacy of ADC, while high value suggest undesired toxicity and poor pharmacokinetic properties. To determine the DAR of an ADC, LCMS analysis of the intact ADC is done. Separation by liquid chromatography is the first step and a variety of columns can be selected here. Agilent TOF and Q-TOF instruments are generally used to analyze intact ADCs. These instruments provide high resolution and accurate mass measurements that are required for DAR calculation. The raw mass spectra can easily be deconvoluted with Agilent MassHunter BioConfirm software to obtain accurate intact protein molecular weight and from this the DAR can be calculated. Due to complexity of the deconvoluted mass spectrum of ADC, interpretation becomes challenging. To overcome this difficulty a novel DAR calculator software solution has been used to automatically determine the DAR with the data reporting feature. The generated report aids in data driven decision making for the scientist
Processed ADCs DAR characterization
To further characterize the ADCs, we can chemically or enzymatically convert these molecules to simpler forms. ADCs can be deglycosylated using deglycosylating enzyme to remove glycans, this helps in reducing the complexity of the mass spectrum for accurate DAR calculations. The ADCs can also be reduced with reducing agents to yield light and heavy chains. Analysis of these fragments also greatly simplifies the mass spectrum. These sample preparations can be done manually or performed on the Agilent AssayMAP Bravo automated sample preparation platform. The AssayMAP Bravo combines unparalleled reproducibility, labour savings, scalability (8 to 96 samples can be run simultaneously), simple person-to-person and site-to-site method transfer, and minimization of human error. Additionally, AssayMAP Bravo comes with protein sample prep workbench software, a simple user interface that allows non-automation experts to take advantage of the power of automation in days rather than months. In addition to automating the deglycosylation and reduction of purified ADCs, AssayMAP Bravo can also be used to purify ADCs from complex matrices such as serum.
ADC payload localization
It is also important to determine the site of conjugation or location of the drug on ADC. This analysis can be either qualitative to simply determine the sites of conjugation or quantitative to assess the percent occupancy of the conjugation sites. Quantitative analysis helps in better understanding the structure activity relation due to conjugation. In this workflow the ADCs are digested with protease and then analyzed using LC-MS/MS to generated peptide map.
The sample preparation steps for generating peptide map can also be automated using Agilent AssayMAP Bravo automated sample preparation platform (fig 3). Reversed-phase LC separation is performed give rapid and high resolution separation of peptides which are then are analyzed by a high resolution and accurate mass Q-TOF system. Finally, data are evaluated with MassHunter BioConfirm software to determine which peptides are conjugated to the drug.
Antibody drug conjugates are seen as an effective approach to cancer treatment as they minimize side effects by targeting the drug to diseased cell based on the specificity of the monoclonal antibodies. Interest in ADCs is high and growing. While the concept seems straightforward, these conjugates are analytically challenging due to their complexity and heterogeneity, and only a few providers across the globe have the technical expertise to develop them. Agilent Technologies provides a broad portfolio of products and solutions like automated sample preparation, liquid chromatography (LC) and mass spectrometry (MS), and comprehensive software solutions to interpret the data that is generated. These solutions work in harmony to advance new biotherapeutic drug candidate research from conception to reality.
(Author is lead scientist – Biopharma Agilent Technologies)