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)
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
- Cell-based assays (flow cytometry, fluorescence, luminescence, etc.)
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