Metabolism and pharmacokinetics (DMPK) analysis services from BOC Sciences can propose personalized antibody-drug conjugates (ADCs) drug pharmacokinetic research strategies and specific experimental plans based on the needs of customer projects to accelerate the development and registration application of ADC drugs. We have successfully built an integrated bioanalytical platform for ADC drugs (LBA, LC-MS/MS and LC-HRMS), and developed and verified multi-dimensional bioanalytical methods to evaluate the pharmacokinetics of ADC.
Compared with traditional antibody drugs or small molecule drugs, ADC drugs have complex structures, and many factors will affect their efficacy. At the same time, the dynamic processes of ADC drugs in vivo are diverse. Evaluating the exposure-efficacy and exposure-safety relationships of ADC drugs from distribution, uncoupling, metabolism and elimination is crucial for drug design and subsequent development. The absorption, distribution, metabolism and excretion (ADME) of ADC drugs in various tissues directly affect the efficacy and safety of ADC, which is more complex and challenging than other drugs. In addition, ADC drugs need to analyze not only macromolecules (total antibodies and conjugated antibodies) but also small molecules (free load and conjugated load).
Fig. 1. DMPK research strategy for ADC drugs.
Therefore, during the different development stages of ADC drugs, bioanalysis is essential to help drug developers understand their properties. Analyzing ADC drugs usually requires the collaboration of multiple analysis platforms, combining classic small molecules/macromolecules and fusion analysis methods, which puts forward the concept of integrated bioanalysis. The DMPK research of ADC drugs runs through all stages of ADC drug early discovery/screening, preclinical and clinical, with different research focuses at different stages. Each stage usually includes quantitative analysis of total antibody, conjugated antibody (ADC), free payload, and possible metabolites. In the drug screening stage, in vitro stability, drug-antibody ratio (DAR) and in vitro biotransformation identification are usually added. In preclinical and clinical stage evaluations, immunogenicity assessments are usually included.
ADC drug research usually requires the integration of small molecule and large molecule research strategies, and different strategies can provide unique information. For example, metabolite identification studies based on high-resolution mass spectrometry can help understand drug metabolism and help determine potential payload metabolites. The small molecule analysis platform LC/MS can help quantify the payload or payload-related metabolites released by ADC drugs. Quantitative analysis of the total antibody fraction of ADC drugs or conjugated antibodies is usually completed using the macromolecule analysis platform Ligand Binding Assay (LBA). The DAR evaluation of ADC drugs and the overall stability evaluation of ADC rely on the integration of the LBA-LC/MS platform. The combination of different strategies helps drug developers understand the metabolic characteristics of ADC drugs and their relationship with the drug's efficacy and toxicity.
|Antibodies with DAR≥0
|Requires ADC naked antibody bridging total antibody method
|Conjugated Antibody (ADC)
|Antibodies with DAR≥1
|If there are anti-payload antibodies, LBA platform analysis is recommended. If not, depending on the ADC drug structure, characteristic peptides, intact proteins or cleaved payload quantification can be used
|Toxic small molecules that bind to antibodies
|For cleavable linkers, the payload can be quantified after cleaving it. For non-cleavable linkers, the approach of characteristic peptide segments can be considered
|Toxic small molecules released
|Routine small molecule analysis may require high sensitivity
|Payload Related Products
|Main metabolites or active/toxic ingredients
|It is recommended to perform LC-HRMS metabolite identification first
|Antidrug Antibodies (ADA)
|PC is usually prepared from the ADC drug itself
The LBA method is the most commonly used strategy to quantify the antibody portion of ADC drugs, including quantification of total antibody and conjugated antibody. This technology has the characteristics of high sensitivity, high throughput and stability at relatively low experimental and instrument costs, and the corresponding regulatory system for reference is complete. LBA can meet different testing needs by designing specific assay formats.
Triple quadrupole low-resolution LC/MS technology has revolutionized small molecule drug development over the past 20 years as a powerful method for qualitative and quantitative analysis. It has a wide range of applications and is currently the "gold standard" for bioanalysis of small molecules and peptides. The selected reaction monitoring/multiple reaction monitoring (SRM/MRM) analysis mode with excellent selectivity and sensitivity and the analysis function of multiple ion channels are its great advantages. The LC/MS method can efficiently quantify the free load, load-related metabolites, and antibody characteristic peptides of ADC drugs in in vivo/in vitro experiments. Macromolecules are challenging to detect using this technology due to their large molecular weight, difficulty in ionization, complex structure, and strong heterogeneity.
LC/HRMS platforms usually refer to LC/MS technologies with higher resolution, and common mass spectrometry platforms are based on Orbitrap or time-of-flight (TOF) detectors. Its multi-level fragmentation function based on MS2 to MSn and accurate m/z mass-to-charge ratio determination of fragment ions can effectively identify the relevant structures and sequences of antibodies, peptides or small molecular parts with the help of existing databases or de-novo sequencing software. The LC/HRMS method exhibits extremely high selectivity because it can accurately measure the mass of compounds. This technique is often used for the identification of metabolites when studying the biotransformation of small molecule ADC moieties.
LBA-(HR)LC/MS is considered to a certain extent as the fusion of two gold standard technologies for the analysis of large molecules (LBA) and small molecules (LC/MS). First, LBA technology is used to affinity capture or enrich the target analyte (ADC), and then the complex mixture is separated by liquid chromatography. Finally, mass spectrometry is used to qualitatively and quantitatively detect different molecular species, analyze their structure or study their biological transformation, etc. The enriched ADC can be enzymatically digested to quantitatively detect characteristic peptides, or it can be quantitatively detected through intact protein analysis. The advantage of this technology lies in the simultaneous acquisition of rich ADC drug structure-related information in qualitative/quantitative analysis, including dynamic evaluation of DAR, biotransformation of drugs, and simultaneous monitoring of multiple molecular species.
BOC Sciences has extensive experience in preclinical bioanalysis. We can help customers quickly complete the establishment and verification of preclinical bioanalytical methods to meet the FDA/NMPA/TGA IND filing requirements for pharmacokinetics. Currently, the ADC analysis and characterization platform we have established can help ADC drug researchers more efficiently/accurately evaluate ADC in vivo/in vitro and ADME characteristics at different research stages.