CASE STUDYCASE STUDYAdvanced Antibody Drug Conjugate

characterization

HIGHLIGHTS> ADC characterization using LC-MSE allows the analysis of

multiple attributes in one analysis run> LC-MSE allows the detection of post-translational

modifications, disulfide bridges and conjugation sites

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The challengeThe challenge

Antibody Drug Conjugates (ADCs) are composed of a payload (small molecule) linked to a monoclonal antibody (biomolecule) via a linker. ADCs are heterogeneous compounds, and more complex than unmodified monoclonal antibodies: to run analyses, a cross-functional understanding of both biologicals and small molecules is needed. Then, the appropriate analytical equipment - such as a mass spectrometer - is required to allow advanced characterization of these bioconjugates.With 90% in-house analysis, Novasep’s QC experts have developed an internal platform method for fast ADC characterization. The method, presented in this case study, is now used for customer projects.

The solutionThe solution

TECHNOLOGY: LC-MSE

ANALYTE: Cysteine-based ADCPROJECT SCOPE: Characterization of

an Antibody Drug ConjugateSPECIFICATIONS: ICH Q6B

For ADCs the ICH guideline Q6B “Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products” requires advanced characterization of the ADC molecule including amino acid sequence confirmation, peptide mapping and disulfide bridges.The use of an LC-MSE system allows the analysis of several ADC characteristics such as peptide mapping and conjugation site confirmation in a single run. LC-MS analysis is thus a multi-attribute method.Novasep’s analytical team used the following methods for this ADC characterization:

Peptide mapping under reducing conditions using LC-MSE for amino acid sequence confirmation, conjugation site confirmation and post-translational modification detection of the antibody

Peptide mapping under non-reducing conditions using LC-MSE for disulfide bridge confirmation

Peptide mapping under reducing conditions using LC-MSE for amino acid sequence confirmation, conjugation site confirmation and post-translational modification detection of the antibody

Peptide mapping under non-reducing conditions using LC-MSE for disulfide bridge confirmation

In Figure 2, the cysteine conjugated ADC was analyzed to determine the disulfide bonds remaining in the ADC. The analytical team was able to confirm that only the intrachain disulfide bonds and part of the heavy chain interchain disulfide bonds were still present. This was coherent with the former data and with a cysteine conjugated ADC with a DAR of 4.

As expected for a cysteine conjugated ADC, the drug linker was found on cysteine residues of the antibody. The analytical team also confirmed that the most conjugated sites were the light chain and heavy chain interchain cysteines followed by the heavy chain interchain cysteines. A minority of drug-linker was found on cysteine residue of one of the intrachain disulfide bridges. The detected conjugation sites were coherent with the Drug-to-Antibody Ratio (DAR) of 4 for this ADC.

Figure 1: Peptide Coverage Map of a cysteine conjugated ADC showing the identified peptides (green) and the modified amino acids including post-translational modifications of the antibody and the conjugated toxin-linker.

Figure 2: Peptide Coverage Map of a cysteine conjugated ADC showing the identified disulfide containing peptides (blue) and examples of peptide fragmentation

PEPTIDE COVERAGE MAP

Identified sequenceModified amino acids

PAYLOAD CONJUGATION MAP

Drug-conjugated peptidesDrug position

Peptide mapping under reducing conditions using LC-MSE

Peptide mapping under reduced conditions allows amino acid sequence confirmation, and therefore confirmation of the identity of the antibody used for the ADC manufacturing. To perform this analytical method, a digestion of the ADC is first performed under reducing conditions to obtain a mixture of peptides. The digestion is followed by a separation of the peptides using a liquid chromatography system under reversed phase conditions. The peptides are then identified using LC-MSE allowing the measurement of the mass of the intact and fragmented peptide, simultaneously. The LC-MSE equipment software then summarizes the data in a peptide coverage map. For example, in Figure 1, a peptide coverage map of the ADC amino acid sequence was obtained, the post-translational modifications of the amino acids were confirmed and conjugation sites were identified.

Peptide mapping under non-reducing conditions using LC-MSE

Peptide mapping under non-reducing conditions allows disulfide bond detection. To perform this analytical method, first a digestion of the ADC is performed under non-reducing conditions to obtain a mixture of peptides still containing their disulfide bonds. The digestion is followed by a separation of the peptides using a liquid chromatography system under reversed phase conditions. The peptides are then identified using LC-MSE allowing the measurement of the mass of the intact and fragmented peptide, simultaneously. The LC-MSE equipment software then summarizes the data in a peptide coverage map which can be filtered for peptides containing a disulfide bond.

Peptide mapping under reducing conditions using LC-MSE Peptide mapping under non-reducing conditions using LC-MSE

Many analytical methods for ADCs have been developed internally at Novasep, to limit outsourcing and increase reactivity. With 90% in-house analysis for ADC characterization, Novasep has the versatility to adapt to client’s analytical protocols.

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© 2019 Groupe Novasep SAS. Groupe Novasep SAS and/or its affiliates (hereafter Groupe Novasep) own or license the copyrights, trademarks, names, logos, and other rights to the information in this brochure. No right or license is granted to any other losses for access or reliance and not from access. Any unauthorized use without the express prior written consent of Groupe Novasep is prohibited. Disclaimers: The information contained in this brochure are provided “as is”, for informational purposes only, without any representation or warranty of accuracy or completeness. In no event will Groupe Novasep be liable to any party for any damages or any other losses from access or reliance upon any information contained in this brochure.

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THE OUTCOME> Peptide mapping allows to confirm several ADC characteristics

in one run > LC-MSE can be used for the characterization of ADC reference

standards according to ICH Q6B

AsiaNovasep Asia+86 21 6045 1600

EuropeNovasep+33 437 282 030

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Rest of the worldNovasep+33 437 282 030

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