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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
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 serumPK ELISA – Bioanalytical method for quantification of Rituximab in human serum
ParametersMethod Validation ResultsParametersMethod Validation Results
Sensitivity8 ng/mLLinearity Range8 to 256 ng/mL
PrecisionGlobal %CV of positive control (inter & intra) ≤ 20%Dilution Linearity40,000 fold
Selectivity10 serum samples (6 normal, 2 lipemic, 2 hemolytic) met pre-defined acceptance criteriaAssay RangeUp to 256 ng/mL in serum samples
RobustnessDemonstrated across different analysts and over different sample incubation durationsLimit of Quantification8 ng/mL
Free Drug Tolerance25 ng/mL of Rituximab at 10 ng/mL of ADAAccuracy87.9 to 99.82% (at all QC levels, n=25)
PrecisionGlobal %CV ≤ 20% (at all QC levels, n=25)

Bevacizumab:

ADA ELISA – Immunoassay for detection of antibodies to Bevacizumab in human serumPK ELISA – Bioanalytical method for quantification of Bevacizumab in human serum
ParametersMethod Validation ResultsParametersMethod Validation Results
Sensitivity187.5 ng/mLLinearity Range60 to 2000 ng/mL
PrecisionGlobal %CV of positive control from all precision batches (inter & intra) ≤ 20%Dilution Linearity40,000 fold
Selectivity5 serum samples (3 normal, 1 lipemic, 1 hemolytic) met pre-defined acceptance criteriaSelectivity10 serum samples (6 normal, 2 lipemic, 2 hemolytic) met pre-defined acceptance criteria
RobustnessDemonstrated across different analysts and over different sample incubation durationsAssay RangeUp to 2000 ng/mL in human serum samples
Free Drug Tolerance3.125 ng/mL of Bevacizumab at 100 ng/mL of ADALimit of Quantification60 ng/mL
Accuracy83.41 to 113.25% (at all QC levels, n=25)
PrecisionGlobal %CV ≤ 20% (at all QC levels, n=25)

Aflibercept:

PK ELISA – Bioanalytical method for quantification of Aflibercept in plasma
ParametersMethod Validation Results
Linearity Range50 to 2000 ng/mL
Dilution Linearity8000 fold
Selectivity6 plasma samples met pre-defined acceptance criteria
Assay RangeUp to 2000 ng/mL
Limit of Quantification50 ng/mL
Accuracy76.44 to 104.99% (at all QC levels, n=12)
PrecisionGlobal %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 StrategiesImmunogenicity Testing Strategies
Type of ELISASandwich ELISA preferredBridging ELISA preferred
Biosimilar comparabilityDemonstration of equivalence with referenceDemonstration of equivalence with reference
Assay requirementAssay with high sensitivity to cover the Cmin and CmaxHigh sensitivity to detect very low levels of low affinity ADAs
Challenges with the AssayDemonstrate dilution linearity  and parallelismDetect 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