Eurofins Advinus provides a one stop solution for biologics/ biosimilar testing which includes Toxicology, TK and Immunogenicity assessment. Eurofins Advinus has evaluated more than a dozen biologics/ biosimilar for toxicology studies.
Eurofins Advinus offers the following Biologics/Biosimilar services.
- Toxicology studies (GLP)
- Single dose Toxicity study in most relevant species (mice/rats/dogs)
- Repeat Dose Toxicity study with Recovery & Immunogenicity (mice/rats/dogs)
- Local Tolerance Study in rabbits
- Genotoxicity (GLP)—–For Novel vaccine adjuvants and additives
- Ames Test
- In vitro chromosomal aberration test in Human Peripheral Blood Lymphocytes (MUT-CHAB/HPBL)
- Micronucleus test (MNT) in mice/rats
- Safety Pharmacology (GLP)
- hERG assay
- Pulmonary functions (rats)
- Modified Irwin Test/Functional Observation Battery in rats (FOB-rats)
- CVS function assessment using telemetered dogs.
- Toxicology studies (GLP)
- Single dose Toxicity study in most relevant species (mice/rats/dogs)
- Repeat Dose Toxicity study with Recovery & Immunogenicity (mice/rats/dogs)
- Carcinogenicity Studies in rats or mice or Transgenic mice—For Novel vaccine adjuvants and additives
- Local Tolerance Study in rabbits
- Juvenile toxicity studies in rodents
- Humoral or Cell Mediated Immune (CMI) Response Assessmen
- Reproduction studies (GLP)
- Fertility and early embryonic development to implantation (SEG-I)
- Prenatal developmental study (SEG-II)
- Peri-natal and post-natal developmental study (SEG-III)
Biologics are complex entities produced by living systems and are used currently in various therapies. Their analysis is more complex and challenging than traditional small molecules. These biologics can generate immune responses that can affect its efficacy and also cause severe allergic and anaphylactic responses. Eurofins Advinus has wide experience in developing and validating sensitive and specific analytical/immunogenicity methods for all your biologics analysis needs. The methods are designed and optimized specifically to the requirements of your biologic product such as peptides, enzymes, monoclonal antibodies, vaccines, and gene therapy products. Our team is experienced in different modes of execution for biologics services: end-to-end development, validation and sample analysis; method transfer and validation; optimization of commercial kits and sample analysis etc.
- Generation of specific critical reagents in-house (polyclonal antibody)
- Assessment of pharmacokinetic parameters using accurate and sensitive methods validated as per latest guidelines
- Immunogenicity assessment by multi-tiered approach
- Anti-drug-antibody (ADA) screening assay
- Confirmatory assay
- Titre estimation
- Neutralizing antibody assay by ELISA or cell-based assays
- Antibody isotyping
- Functional bioassays
- Biomarker analysis such as Cytokines
- Well defined strategies for specific assays
| PK/ TK Assay | ADA Assay | |
| ELISA format | Sandwich ELISA preferred | Bridging ELISA preferred |
| Key Requirement | Good dynamic range to cover the Cmin and Cmax | High sensitivity to detect low binding ADA (at least 100 ng/mL) |
| Key Challenges | Demonstrate dilution linearity and parallelism | Detect ADA in presence of high levels of circulating drug concentrations and Drug–ADA complex |
List of available methods for different Biologics/Biosimilars
 LC-MS method for quantification of Etanercept in plasma |
 LC-MS method for quantification of Insulin Glargine in rat plasma |
 LC-MS method for quantification of Cetuximab in plasma |
|
Rituximab:
| ADA ELISA – Immunoassay for detection of antibodies to Rituximab in human serum | PK ELISA – Bioanalytical method for quantification of Rituximab in human serum | ||
|---|---|---|---|
| Parameters | Method Validation Results | Parameters | Method Validation Results |
| Sensitivity | 8 ng/mL | Linearity Range | 8 to 256 ng/mL |
| Precision | Global %CV of positive control (inter & intra) ≤ 20% | Dilution Linearity | 40,000 fold |
| Selectivity | 10 serum samples (6 normal, 2 lipemic, 2 hemolytic) met pre-defined acceptance criteria | Assay Range | Up to 256 ng/mL in serum samples |
| Robustness | Demonstrated across different analysts and over different sample incubation durations | Limit of Quantification | 8 ng/mL |
| Free Drug Tolerance | 25 ng/mL of Rituximab at 10 ng/mL of ADA | Accuracy | 87.9 to 99.82% (at all QC levels, n=25) |
| Precision | Global %CV ≤ 20% (at all QC levels, n=25) | ||
Bevacizumab:
| ADA ELISA – Immunoassay for detection of antibodies to Bevacizumab in human serum | PK ELISA – Bioanalytical method for quantification of Bevacizumab in human serum | ||
|---|---|---|---|
| Parameters | Method Validation Results | Parameters | Method Validation Results |
| Sensitivity | 187.5 ng/mL | Linearity Range | 60 to 2000 ng/mL |
| Precision | Global %CV of positive control from all precision batches (inter & intra) ≤ 20% | Dilution Linearity | 40,000 fold |
| Selectivity | 5 serum samples (3 normal, 1 lipemic, 1 hemolytic) met pre-defined acceptance criteria | Selectivity | 10 serum samples (6 normal, 2 lipemic, 2 hemolytic) met pre-defined acceptance criteria |
| Robustness | Demonstrated across different analysts and over different sample incubation durations | Assay Range | Up to 2000 ng/mL in human serum samples |
| Free Drug Tolerance | 3.125 ng/mL of Bevacizumab at 100 ng/mL of ADA | Limit of Quantification | 60 ng/mL |
| Accuracy | 83.41 to 113.25% (at all QC levels, n=25) | ||
| Precision | Global %CV ≤ 20% (at all QC levels, n=25) | ||
Aflibercept:
| PK ELISA – Bioanalytical method for quantification of Aflibercept in plasma | |
|---|---|
| Parameters | Method Validation Results |
| Linearity Range | 50 to 2000 ng/mL |
| Dilution Linearity | 8000 fold |
| Selectivity | 6 plasma samples met pre-defined acceptance criteria |
| Assay Range | Up to 2000 ng/mL |
| Limit of Quantification | 50 ng/mL |
| Accuracy | 76.44 to 104.99% (at all QC levels, n=12) |
| Precision | Global %CV ≤ 20% (at all QC levels, n=12) |
- Experience in various biologics/biosimilars such as Etanercept, Insulin Glargine, Rituximab, Bevacizumab, Trastuzumab, Aflibercept, Glatiramer acetate, etc.
- In vivo efficacy assays
- In vivo assessment of pharmacokinetic and pharmacodynamics parameters for biosimilar and comparison with innovator molecule
- In vitro pharmacological functional assays
- In vivo immunogenicity assessment for biosimilar and innovator molecule by multi-tiered approach-
- Anti-drug-antibody (ADA) screening assay
- Confirmatory assay
- Titer estimation
- Neutralizing antibody assay by ELISA or cell based assays
- Antibody isotyping
- Biomarker analysis
| PK/ TK Strategies | Immunogenicity Testing Strategies | |
| Type of ELISA | Sandwich ELISA preferred | Bridging ELISA preferred |
| Biosimilar comparability | Demonstration of equivalence with reference | Demonstration of equivalence with reference |
| Assay requirement | Assay with high sensitivity to cover the Cmin and Cmax | High sensitivity to detect very low levels of low affinity ADAs |
| Challenges with the Assay | Demonstrate dilution linearity and parallelism | Detect ADA in presence of high circulating drug concentrations and Drug–ADA complex |
- Ligand binding assays or Enzyme Linked Immunosorbent Assays (ELISA)
- Sandwich ELISA
- Bridging ELISA
- Direct/Indirect ELISA
- Competitive ELISA
- LC-MS/MS platform like API-6500+
- Immuno pull down, tryptic digestion and signature peptide identification approaches
- Cell-based assays using flow cytometry, fluorescence, luminescence readouts
- Critical Reagent generation and development
- Polyclonal antibody generation in rat, rabbit, guinea pig and chicken
- Protein A/G and Antigen specific purification
- Biotin, HRP and AP labelling
Anti-Drug Antibody (ADA) Development
- Polyclonal antibody generation
- In rat, rabbit, guinea pig and chicken
- Antigen: Biotherapeutic, full-length or truncated antibody, peptide
- Purification
- Protein A/G
- Antigen specific
- Labelling
- HRP, AP, Biotin and Digoxigenin
- ELISA development and validation for use in PK and Immunogenicity studies
Quantification of Biological Molecules by LC-MS/MS
- Assays can be developed and validated on LC-MS/MS platform like API-6500+
- Method development & optimization for the compound using immuno pull down, tryptic digestion and signature peptide identification approach
- Pre-study method validation
- Analysis of study samples for pharmacokinetics (PK), toxicokinetics (TK), pharmacodynamics (PD) and vaccine efficacy studies for quantification of proteins, peptides and monoclonal antibody based drugs