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AqT™ Antibody SN38 Conjugate Evaluation Kit

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SDS USER MANUAL CM81380 and CM81380.1

The AqueaTether™ (AqT™) antibody SN38 conjugate evaluation kit is one of the few ADC kits currently available for customers to perform conjugations in-house and evaluate the benefit of AqT™ linkers for ADC on a small scale. The kit uses SN38 modified with a super-hydrophilic, water-soluble, and charge neutral AqueaTether™ (AqT™) linker to improve its water solubility and decrease its non-specific interactions with neighboring drugs after conjugation. The end result is an ADC with exceptionally high loading with no or minimum aggregation. 

SN38 is loaded onto the antibody through an amide bond via the surface amines of the antibody. After AqT™ ADC reaches the target, the hydrophilic AqT™ linker can be separated from SN38 via its releasable ester bond. This tactic (traceless release) allows: 1) efficient transportation of the ADC in the biological media with minimum aggregation and toxicity, 2) preservation of the bystander killer effect of SN38.

The kit is configured for labeling either one 1 mg (Cat#: CM81380.1) or 3 mg antibody sample (Cat#: CM81380).

Key Features of this AqT™ ADC evaluation kit

  • Proprietary super-hydrophilic and water-soluble linkage
  • High loading with average 5 to 7 SN38 per antibody with minimum added aggregation
  • Sugar alcohol-based AqT™ linker preserves the properties of the antibody
  • Releasable linkage with fully released SN38 (traceless release) to maximize the efficacy
  • Fast and easy preparation: 4 h preparation and <1 h hands-on time

Comparison of ADC made by CM81380 and CM11408


ADC by CM81380 (AqT™ Ab SN38 Evaluation Kit)

ADC by CM11408 (Ab SN38 Conjugation Kit with Classical Linker)


Yes (composition of matter protected by AqT™ molecules)

No, if your antibody is generic

Linker Properties

Proprietary super-hydrophilic and water soluble AqT™ linker

Classic hydrophobic ethylene-type linker


Flexibile long spacer (>20 atoms, the exact length is not disclosed here)

Short spacer (4 atoms)

Releasable Chemistry

Same (ester bond)

Same (ester bond)


High: 5−7 SN38 per antibody

Low: 2−4 SN38 per antibody


Slight changes in hydrophobicity

Dramatically increases hydrophobicity

Aggregation by SEC

No or less than 2% added aggregation

Average 10−50% added aggregation

Heterogenicity by HIC

Less heterogenicity

Highly heterogenous product


Stable even with high loading: easily stored frozen without any stabilizer

Unstable even with low loading

 Workflow for preparing AqT™ antibody-SN38 conjugates


AqT™ SN38 Acid (C18 and HIC HPLC Profiles)

SN38 is an inhibitor of topoisomerase I that eventually leads to inhibition of both DNA replication and transcription. On April 7, 2021, the FDA granted regular approval for sacituzumab govitecan (Trodelvy, Immunomedics, Inc.), an ADC that consists of SN-38 linked with a humanized IgG antibody targeted against TROP-2, for the treatment of patients with metastatic triple-negative breast cancer (mTNBC) following at least two prior therapies for metastatic disease. TROP-2 is a cell-surface glycoprotein expressed in more than 90% of TNBCs. Unlike highly toxic chemicals, such as DM1 and MMAE, ADCs with chemotherapeutic drugs may need much higher loading to achieve a therapeutic index.

SN38 is a very hydrophobic molecule. Antibodies labeled with SN38 with classical linkers tend to aggregate and may precipitate out from solution over time. SN38 with AqT™ linker greatly increases the hydrophilicity and water solubility of SN38. The hydroxy (-OH) groups of AqT™ form a network of hydrogen bonds (H-bond) with neighboring water and create a microenvironment that shields neighboring SN38 from stacking or interacting with one another. This AqT™ enhanced H-bond network also protects the antibody from enzymatic degradation and retains its activities.


Overlay of C18 reversed phase HPLC analysis of SN38 acid and AqT™ SN38 acid

AqT SN38 acid has Rt of 1.6 minutes less than SN38 acid with classical linker (11.9% less acetonitrile to elute out)

 Buffer A: 0.1% TFA in water; Buffer B: 0.1% TFA in acetonitrile


Overlay of HIC HPLC analysis of SN38 acid and AqT™ SN38 acid

Column-bound SN38 acid need additional 1M (NH4)2SO4 decreases to elute out compared to AqT™ SN38 acid

Buffer A:  0.1M Sodium phosphate buffer, 1.8 M (NH4)2SO4, pH 7.0; Buffer B: 0.1M Sodium phosphate buffer, pH 7.0


AqT-SN38 ADC (SEC HPLC: aggregation profile)


SEC separates the conjugates by apparent MW or size. The larger MW of the conjugate, the earlier it elutes out. Aggregates usually appear to the left side of the product. For SN38 ADC, there is usually 5 to 50% aggregation for a typical ADC with a loading of 2 to 4. For AqT™ SN38 ADC with a DAR of 6.1, less than 1.2% aggregation was observed.

AqT SN38 ADC (HIC HPLC, hydrophilicity and heterogenicity profile)


Due to the highly heterogeneous nature of surface amine labeling, antibody loaded with the same number of drugs (same DAR) may have slightly different hydrophobicity. So, for ADC labeled via surface amine chemistry, a broad peak will be seen in a typical HIC analysis without clear separation of the peaks.

SN38 ADC is highly hydrophobic. After labeling with an average 4.7 SN38 per antibody, the peak retention time of the antibody increases 1.7 minutes (0.36 minutes per SN38). SN38 antibody is also highly heterogeneous with an HIC peak spanning more than 5.2 minutes (1.1 minutes per SN38).

However, for AqT™ SN38 ADC with an average 6.1 AqT™ SN38 per antibody, the increase in hydrophobicity is much smaller (0.1 minutes per AqT™ SN38). AqT™ SN38 labeled antibody is also less heterogeneous with an increase in the peak width of only 0.49 minutes per AqT™ SN38. 

Other Details

Scale of the Reaction:
1 or 3 mg
Antibody Labeling Sites:
Surface amines
Antibody Drug Linkage:
releasable super hydrophilic AqT™ spacer

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