Biopharmaceutical Characterization

Biosimilars are complex protein structures that require characterization to demonstrate comparability to the original biologic. Owing to their large structure and their production and purification from biological systems they are prone to deviate from the structures that are defined as drug. Characterization of the biologics therefore needs special emphasis. Shantani offer a comprehensive array of assays and services to support your Biosimilar Development Program.

Drug Substance and Drug Product Characterization Workflow

Client engagement and project deliveries

  • Supported over 20 biosimilar development projects
  • Worked with more than 6 biosimilar developers worldwide
  • Industry leading portfolio of off-the-shelf cell based bioassays and binding assays
  • Combined functional and physicochemical/structural analysis

Successfully Characterized Biosimilars includes

  • Transtuzamab
  • Dengue mAb
  • Rituximab
  • Bevacizumab
  • EGFR
  • Erythropoietin
  • FHA
  • CRM197
  • Diphtheria Toxin and Toxoid
  • Tetanus Toxin and Toxoid
  • Pertactin and Pertussis Toxins
  • Hepatitis-B
  • HPV6 / HPV11 / HPV16 / HPV18
  • Bacillus Calmette–Guérin (BCG)
  • Interferon–beta (INF-β) / PEGylated INF-β

Intact Mass Analysis

  • Typically carried out using ESI-Q-TOF (Agilent 6540 UHDA)
  • Standard and well-established protocols for Biotherapeutics
    1. Underivatized / Derivatized
    2. Glycosylated / De-glycosylated
    3. Reduced / Non-reduced

Peptide Mapping

  • Standard and well-established protocols for single and multi-protease, in-gel or in-solution, digestions of proteins
  • Peptides are separated using RPLC and infused into ESI-QTOF set up for MS and MS/MS analysis
  • Obtained masses are compared with the theoretical digest of the proteins using bioinformatic tools and peptide-maps are prepared

Glycan Profiling

  • Standard protocols for N and O Glycan profiling
  • Extracted N and O glycans are labelled with 2-AB and analyzed using LC-Flu based workflow and Glycans are identified using mass-spectrometry
  • Fluorescence labelled glycans were analyzed using Fluorophore Assisted Carbohydrate Electrophoresis (FACS)

Charge Variant Analysis

  • Charge Variants are analyzed using HPLC-strong-cation exchange chromatography (SCX) and UV/Fluorescence Detectors
  • Linear pH gradient is the key to success and well-optimized protocol is utilized for the purpose

Circular Dichroism and Fluorescence Spectroscopy (Secondary and Tertiary Structure Analysis)

  • Samples are de-formulated using Gel-Filtration-Chromatography and then analyzed using Fluorescence and CD Spectroscopy
  • Tryptophan Fluorescence Quenching experiments are performed for in-depth analysis

Disulfide Bond Analysis

  • mAbs are digested with proteases in reduced and non-reduced conditions and obtained peptides are analyzed using LC-UV or LC-MS based workflows.

Cytotoxicity Assays

  • Redox indicator dye is used to study cytotoxicity levels in different type of cell lines.
  • End point measurements are done using both colorimetry and fluorimeter.
  • The assay is scalable and can be performed in different plate (6/12/24/96/384 wells) formats.

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