【 Macropharma 】 Structural characterization of ADC drugs and case sharing
Hongren Biopharmaceutical
Antibody-drug conjugates (ADCs) consist of three main components: Antibodies that selectively recognize antigens on the surface of cancer cells, drug payloads that kill cancer cells, and linkers that connect antibodies and payloads are one of the fastest growing drug classes in oncology in recent years. The ways of ADCs coupling small molecules mainly include lysine coupling. Cysteine coupling, site-specific coupling, etc. (Figure 1).
Figure 1: Different ways of coupling ADCs
The typical model of ADC action assumes the following: ADC drugs rely on the specificity and targeting of monoclonal antibodies to tumor cell-associated antigens to reach tumor cells, and enter the cells through endocytosis. The conjugated chain breaks at low PH or lysosomal protein in the cell, releasing cytotoxic drugs that lead to tumor cell death (Figure 2).
Figure 2: ADC mechanism of action
Because of the payload and linker coupling, ADC drugs are structurally more complex and heterogeneous than conventional antibody drugs. To ensure drug safety and efficacy, in-depth characterization of ADCs is critical in their development. In addition to analyzing individual antibodies and small molecule drugs, ADC drug quality research and structural characterization should also focus on characterizing the specific properties of ADC coupling, such as drug-antibody ratio DAR, drug distribution, proportion of uncoupled antibodies, and coupling sites. The characterization of naked resistance, small molecule drugs and ADC itself is as follows:
The comprehensive characterization analysis of ADC should adopt appropriate and advanced analytical instruments and technologies to conduct comprehensive and detailed analysis from various perspectives such as physicochemical properties, immunological properties, biological activity and impurities, and fully understand the relevant characteristic changes before and after coupling in combination with the characteristic analysis of naked antibody, so as to provide as detailed information as possible to reflect the quality attributes of the final product.
The analytical methods involved include LC-MS, Edman, SEC-MALS, CD, DSC, Elisa, SEC, HIC, HPLC, CE-SDS, iCIEF, CZE, etc. Refer to the following table for details:
method | peculiarity |
Liquid mass （LC-MS） | > A powerful tool for quality attribute research > Complete molecular/subunit/peptide analysis at multiple levels > DAR, conjugated sites, proportion of unconjugated antibodies, etc > Evaluation of product heterogeneity and process consistency of ADC |
Protein sequencing | > Direct method for studying N-terminal amino acid sequence > The results can be compared with those of N-terminal sequence analysis by mass spectrometry |
SEC-MALS | > A useful tool for studying apparent molecular weight > The molecular weight distribution and oligomerization state of the product can be analyzed > Protein conjugate can be characterized |
Circular dichroism | > Study the secondary structure and advanced structure of the product |
Differential scanning calorimetry | > The thermal stability and thermodynamic characteristics were studied > It can be used for formulation screening |
Dynamic light scattering | > The protein particle size and thermal stability of the product were studied > It can be used for formulation screening |
Enzyme-linked immunosorbent assay (ELISA) | > Common methods of binding activity detection > It can be used for ADC product binding activity determination, ADC product concentration determination, total antibody concentration determination, free drug concentration determination, etc > It can be used to investigate the difference of antigen binding power before and after coupling |
Molecular exclusion Chromatography (SEC) | > Apparent molecular weight size > Common methods for studying molecular size variants |
Hydrophobic chromatography （HIC） | > Relative hydrophobic groups on the molecular weight surface interact with the medium > ADC molecules basically maintain the natural configuration > An efficient method for calculating DAR values > An effective method for calculating the proportion of uncoupled antibody molecules |
Reversed phase Chromatography (RPLC) | >Denaturation analysis method > Applicable for release > Peptide map analysis, small molecule residue analysis > Cys-ADC analysis summary has a wide range of applications (coupling location, proportion of uncoupled antibodies, DAR, etc.) |
Hydrophilic Chromatography (HILIC) | > Complementary analytical method with RPLC > Common methods of glycogram analysis |
Ion Exchange Chromatography (IEC) | > Non-denaturation analysis method > Most common charge heterogeneity analysis methods (intact molecules/subunits) > Salt ion gradient /Ph gradient |
Sds-capillary Electrophoresis (CE-SDS) | > An efficient tool for studying molecular size heterogeneity > An effective tool for studying drug distribution > Investigation of first order fragments of polymers after coupling > And HIC, RP and other methods complement and confirm each other |
Capillary isoelectric Focusing electrophoresis (CIEF/iCIEF) | > An effective tool for studying the heterogeneity of molecular charge > It can provide product molecular characteristic map information > The results of iCIEF, LC-MS and HIC are mutually confirmed |
Capillary Zone electrophoresis (CZE) | > Platforming and rapid charge heterogeneity analysis method > It is related to the heterogeneity of molecular charge and the hydrodynamic radius > Complementary to IEC and Icief (complete level) > Can be used as a supplement to RPLC (peptide level) |
Hongren Biologics quality analysis platform can provide comprehensive characterization analysis services for "molecular weight, primary structure and advanced structure confirmation of ADC drugs". Specific analysis cases are as follows:
1. Molecular weight and DAR value
Complete molecular weight determination: At the complete molecular weight level of the protein, its molecular weight, DAR value distribution, qualitative and quantitative composition are confirmed. The determination methods of complete molecular weight LC-MS/MS can be divided into variable property spectrum and non-variable property spectrum (native MS) method.
Since the disulfide bond between the cysteine-coupled ADC drug chains is opened, the light/heavy chains are only bound by non-covalent bonds, which will dissociate into antibody fragments under traditional variable property spectrum conditions, and the molecular weight of the complete protein cannot be determined. The complete molecular weight of the protein can be determined by using non-variable property spectroscopy (Native MS) while maintaining the non-covalent force.
The following is a case of DAR value detection.
Figure 4: Molecular weight detection results of ADC drug TDM1
2. Peptide map analysis
Peptide map analysis is an extremely important key technology in biologic drug analysis, which provides the most comprehensive information of biologic drug characterization. Amino acid sequence, post-translational modification, disulfide bond form, NC terminal, drug coupling site and so on can be confirmed by peptide map analysis. In this case, Trypsin was used to identify the conjugate site of ADC drug (TDM1) and the pairing form of disulfide bond.
l Conjugate site analysis of ADC drugs
Figure 5: TDM1 small molecule drug structure and fragment form
Figure 6: Results of identification of ADC drug TDM1 coupling site
l Disulfide bond analysis of ADC drugs
Figure 7: Results of identification of ADC drug TDM1 disulfide bond
3, the size of the heterogeneous
Molecular weight size variation (polymers, particles, fragments) can directly affect the effectiveness or safety of protein therapeutics. Aggregation is the most common way of protein physical degradation. Due to the presence of drugs/linkers, they are more hydrophobic around the coupling site, causing ADCs to aggregate more easily than parent antibodies.
Common methods for the analysis of large and small heterogeneous bodies include: Molecular exclusion chromatography (SEC-HPLC), non-reduced and reduced sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE), sodium dodecyl sulfonate-polyacrylamide capillary gel electrophoresis (CE-SDS), dimensional exclusion chromatography combined with multi-angle Light scattering (SEC-MALS), and analytical ultracentrifugation (AUC) The molecular size variants (i.e., polymers and fragments) of ADCs are appropriately identified.
The MALS technique is based on the direct correlation between the molecular weight of protein and the intensity of light scattering to determine the absolute molecular weight of protein, without the use of standard protein, independent of elution volume. After separation by Size Exclusion Chromatography (SEC), each component enters the multi-angle light scattering detector. Light scattering occurs when laser irradiation is applied to the analyte, and the intensity of the scattered light is measured simultaneously by MALS from multiple angles. The intensity of scattered light is proportional to the square of molar mass, concentration and refractive index increment, and the absolute molecular weight distribution and polymerization degree of the analyte can be directly calculated.
Figure 8: ADC drug TDM1 SEC-MALS detection results
4. Charge anisoplast
The charge heterogeneity of protein therapy is an important quality property with potential effects on stability and biological activity. For monoclonal antibodies, the charged exosomes are usually determined by appropriate methods such as capillary zone electrophoresis (CZE), ion exchange high performance liquid chromatography (IEX-HPLC), capillary isoelectric focusing electrophoresis (CIEF), or imaging capillary isoelectric focusing electrophoresis (iCIEF). The suitability of these methods for ADC analysis depends on the properties of the drug-linker (especially the charge) and the choice of binding sites (e.g., lysine, interchain sulfhydryl, carbohydrate, etc.).
At present, imaging capillary isoelectric focusing electrophoresis (iCIEF) is commonly used to characterize the charge anisoplasms and isoelectric points of ADC and other samples with relatively complex charge.
Figure 9: ADC drug TDM1 iCIEF detection results
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