抗体药物偶联物即ADC类药物的生物分析
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ADAs could develop against protein components of either modality, the linker and/or the small-molecule drug may act as a hapten once conjugated to form the ADC. Due to the unique feature, should domain specificity experiments be developed? Additionally, technical challenges, e.g. sensitivity, matrix effect and drug ห้องสมุดไป่ตู้olerance.
Challenges from ADA testing for ADC No specific guidelines for emerging biotherapeutic modalities. Evaluation of a potential humoral response against an ADC is more complex than for mAb therapeutics:
Recommended multi-tiered strategies from ADA testing for ADC
*different from other biotherapeutics
ADA detection format：Bridge ELISA
advantages：economy, cost saving disadvantages：low sensitivity, poor drug tolerance
Validation parameters:
Screening cut point Specific cut point Sensitivity QC acceptance criteria Selectivity/interference Precision Robustness Stability
Bioanalysis of antibody–drug conjugates
总结与评论专员 章登吉
Bioanalytical challenges from ADC
multicomponent structure(Mixtures)
inherently heterogeneous
dynamic nature
PK/TK Assay validation for ADC General Bioanalytical guidelines for bioanalysis can be used for ADC PK/TK Assays:
生物样品定量分析方法验证指导原则(2015版药典） Guidance for Industry: Bioanalytical Method Validation(FDA,2013) Guideline on bioanalytical method validation (EMA,2011)
Total antibody from one dose group in TK study
160000 140000 120000 100000 80000 60000 40000 20000 0 15min 24h 72h 168h 336h 504h
系列1 系列2 系列3 系列4 系列5 系列6 系列7 系列8
Although there are no specific guidelines for validating LBA- or LC-MS/MS-based PK/TK assays for ADC, there is strong scientific rationale to use the general guidelines therapeutic protein and small molecule assay validation as much as possible.
One Example : improve Assay drug tolerance for one ADC
Y: S/N; X: Positive ADA control concentration According to the data, the drug tolerance is improved to 2µg/mL when ADA is 62.5ng/mL after acidification, the method is bridge ELISA.
Therefore, multiple analytes are utilized to determine PK and fate of ADCs in vivo
Analytes commonly assessed for ADC
*DAR: Drug to Antibody Ratio Linker and linker-small molecule should also be considered based on your case.
Rapid clearance, altering the pharmacokinetic (PK) and/or pharmacodynamic profile of the drug loss of efficacy due to neutralization of drug generalized immune effects, such as anaphylaxis, serum sickness or hypersensitivity Neutralization of the endogenous human protein, causing deficiency syndrome, with fatal outcomes
ADA status of the animal model is required for interpretation of the pharmacology and toxicology data Monitoring of immunogenicity is required at all stages of biotherapeutic drug development for evaluation of safety and efficacy and is a key component of regulatory filings
Validation parameters
Standard curve LLOQ/ULOQ Accuracy and Precision Selectivity Specificity Dilution linearity Parallelism Stability
Example: TK assays(EIA methods) for one ADC perfo
180000 160000 140000 120000 100000 80000 60000 40000 20000 0 15min STD1:total antibody STD2:intact ADC Good correlation from the two assay curves 160000.0 140000.0 120000.0 100000.0 80000.0 60000.0 40000.0 20000.0 0.0 15min 24h 72h 168h 336h 504h Good correlation between in tact ADC with total mAb, which also showed that good performance of our assays intact ADC total mAb 24h 72h 168h 336h 504h 系列1 系列2 系列3 系列4 系列5 系列6 系列7 系列8
Intact ADC from one dose group in TK study
Reasons for ADA testing The development of anti-drug antibodies (ADA) to protein therapeutics can result in:
assessed for ADC
PK/TK Assay strategies Platforms commonly used for ADC: Ligand binding assay: ELISA or other methods LC-MS/MS Assay format: Intact ADC: Target human tumor antigen or anti-drug Ab as capturing reagents Total mAb: Target human tumor antigen as capturing reagents usually
Therapeutic Protein Products. (FDA draft guideline,2016) Guideline on Immunogenicity assessment of biotechnology-derived therapeutic proteins.(EMA draft guideline, 2015)
Favored ADA Detection Format：ECL Method
Advantages: high sensitivity, good drug tolerance Disadvantages: high cost
ADA Assay validation for ADC Refer to general guideline temporarily: Assay Development and Validation for Immunogenicity Testing of
Challenges from ADA testing for ADC No specific guidelines for emerging biotherapeutic modalities. Evaluation of a potential humoral response against an ADC is more complex than for mAb therapeutics:
Recommended multi-tiered strategies from ADA testing for ADC
*different from other biotherapeutics
ADA detection format：Bridge ELISA
advantages：economy, cost saving disadvantages：low sensitivity, poor drug tolerance
Validation parameters:
Screening cut point Specific cut point Sensitivity QC acceptance criteria Selectivity/interference Precision Robustness Stability
Bioanalysis of antibody–drug conjugates
总结与评论专员 章登吉
Bioanalytical challenges from ADC
multicomponent structure(Mixtures)
inherently heterogeneous
dynamic nature
PK/TK Assay validation for ADC General Bioanalytical guidelines for bioanalysis can be used for ADC PK/TK Assays:
生物样品定量分析方法验证指导原则(2015版药典） Guidance for Industry: Bioanalytical Method Validation(FDA,2013) Guideline on bioanalytical method validation (EMA,2011)
Total antibody from one dose group in TK study
160000 140000 120000 100000 80000 60000 40000 20000 0 15min 24h 72h 168h 336h 504h
系列1 系列2 系列3 系列4 系列5 系列6 系列7 系列8
Although there are no specific guidelines for validating LBA- or LC-MS/MS-based PK/TK assays for ADC, there is strong scientific rationale to use the general guidelines therapeutic protein and small molecule assay validation as much as possible.
One Example : improve Assay drug tolerance for one ADC
Y: S/N; X: Positive ADA control concentration According to the data, the drug tolerance is improved to 2µg/mL when ADA is 62.5ng/mL after acidification, the method is bridge ELISA.
Therefore, multiple analytes are utilized to determine PK and fate of ADCs in vivo
Analytes commonly assessed for ADC
*DAR: Drug to Antibody Ratio Linker and linker-small molecule should also be considered based on your case.
Rapid clearance, altering the pharmacokinetic (PK) and/or pharmacodynamic profile of the drug loss of efficacy due to neutralization of drug generalized immune effects, such as anaphylaxis, serum sickness or hypersensitivity Neutralization of the endogenous human protein, causing deficiency syndrome, with fatal outcomes
ADA status of the animal model is required for interpretation of the pharmacology and toxicology data Monitoring of immunogenicity is required at all stages of biotherapeutic drug development for evaluation of safety and efficacy and is a key component of regulatory filings
Validation parameters
Standard curve LLOQ/ULOQ Accuracy and Precision Selectivity Specificity Dilution linearity Parallelism Stability
Example: TK assays(EIA methods) for one ADC perfo
180000 160000 140000 120000 100000 80000 60000 40000 20000 0 15min STD1:total antibody STD2:intact ADC Good correlation from the two assay curves 160000.0 140000.0 120000.0 100000.0 80000.0 60000.0 40000.0 20000.0 0.0 15min 24h 72h 168h 336h 504h Good correlation between in tact ADC with total mAb, which also showed that good performance of our assays intact ADC total mAb 24h 72h 168h 336h 504h 系列1 系列2 系列3 系列4 系列5 系列6 系列7 系列8
Intact ADC from one dose group in TK study
Reasons for ADA testing The development of anti-drug antibodies (ADA) to protein therapeutics can result in:
assessed for ADC
PK/TK Assay strategies Platforms commonly used for ADC: Ligand binding assay: ELISA or other methods LC-MS/MS Assay format: Intact ADC: Target human tumor antigen or anti-drug Ab as capturing reagents Total mAb: Target human tumor antigen as capturing reagents usually
Therapeutic Protein Products. (FDA draft guideline,2016) Guideline on Immunogenicity assessment of biotechnology-derived therapeutic proteins.(EMA draft guideline, 2015)
Favored ADA Detection Format：ECL Method
Advantages: high sensitivity, good drug tolerance Disadvantages: high cost
ADA Assay validation for ADC Refer to general guideline temporarily: Assay Development and Validation for Immunogenicity Testing of