info@advinus.com

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 Etanercept in plasma
LC-MS method for quantification of Insulin Glargine in rat plasma
LC-MS method for quantification of Insulin Glargine in rat plasma
LC-MS method for quantification of Cetuximab in 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
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