Conjugate Sites Analysis

With our state-of-the-art analytical mass spectroscopy facility, Creative biolabs provides our global customers with customized in-depth conjugate site analysis of antibody-drug conjugates (ADCs). We guarantee top-quality analysis featuring comprehensive sequence coverage and accurate peptide identification.

Antibody-drug conjugates employ the specific monoclonal antibodies (mAbs) to achieve targeted delivery of the conjugated cytotoxic molecules to tumor cells. This delicate design increases the payload efficacy against tumor cells and in the meantime, reduces the cytotoxicity of the otherwise highly toxic payload to normal tissues comparing to traditional chemotherapeutic treatments. However, the ADCs are more complex and heterogeneous compared to the unmodified therapeutic mAbs. This is mainly due to a wide distribution of drug conjugation sites, the unique characteristics of the conjugation sites, and different types of conjugation chemistries. As a result, the effects of ADCs are remarkably different.

As one important aspect in ADC characterization, the identification of the conjugate sites provides an accurate map of the payload positions on a mAb. This set of results is extremely helpful in the prediction/validation of the effects on antigen binding activities induced by the conjugation process and in identification of bio-similarity ADC products. Conjugate sites analysis and determination is achieved by peptide mapping using highly sensitive and accurate LC-MS/MS.

After years of accumulative work, Creative biolabs has established a cutting-edge analytical mass spectroscopy platform to perform comprehensive analysis of the ADC conjugate sites. Generally, our analysis follows this peptide mapping workflow:

  • Sample preparation
    • De-glycosylation: the ADC samples are treated with PNGase F to remove the N-glycans to reduce the complexity of the ADC molecules and reduce the special hindrance for enzymatic degradation.
    • Reduction and alkylation: the de-glycosylated ADC samples are further reduced by DTT and then blocked using Iodoacetamide to facilitate digestion.
  • Protease digestion: the blocked ADC protein fragments are cleaved into smaller peptides by two or more proteases to increase the sequence coverage. We offer a verity of proteases, including trypsin, chymotrypsin, Lys-C, Glu-C, Asp-N, Arg-C….
  • LC/MS analysis
    We offer both standard flow UV LC-MS/MS system to analyze large sample amounts (>20 µg per analysis) and nano-flow LC-MS/MS that is suitable for small quantity (in the low µg range) analysis. Our platform is equipped with various MS instrument, including QTOF, MALDI MS/MS, and Tripple TOF… to analyze versatile samples.
  • Data analysis
    The specialists at Creative biolabs will analyze the mapping data and provide information regarding:
    (1) The amino acid sequences of heavy chain and light chain;
    (2) Verification of the chemical nature of the conjugate sites (Lys, Cys…);
    (3) Identification of conjugated vs unconjugated amino acids;
    (4) Quantification of conjugated vs unconjugated amino acids.

Conjugate Sites Analysis A schematic demonstration of ADC conjugate site analysis (left) and a sample report of the de-convoluted fragmentation spectra of peptides containing DM1 conjugated lysine (mAbs., 2016).

Creative biolabs is a pioneer in the development of antibody-drug conjugates. Based on years of efforts in therapeutic antibody discovery and synthetic organic chemistry, our Ph.D. level scientists are committed to provide our customers with one-stop services of their ADC development projects. Please contact us for more information and a detailed quote.


  1. Wakankar, A.; et al. Analytical methods for physicochemical characterization of antibody drug conjugates. mAbs. 2011, 3(2): 161-172.
  2. Chen, L.; et al. In-depth structural characterization of Kadcyla(R) (ado-trastuzumab emtansine) and its biosimilar candidate. mAbs. 2016, 8(7): 1210-1223.

For Research Use Only. NOT FOR CLINICAL USE.

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