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Qualitative analysis of antibody-drug conjugates (ADCs): an experimental comparison of analytical techniques of cysteine-linked ADCs.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Analytisk kemi. Recipharm OT Chem AB, Uppsala, Sweden. (Bergquist)
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Preparativ läkemedelskemi.ORCID-id: 0000-0002-5520-0179
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Analytisk kemi. (Bergquist)
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Analytisk kemi. (Lind)ORCID-id: 0000-0002-9510-3816
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2018 (Engelska)Ingår i: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 143, nr 22, s. 5487-5496Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Antibody-drug conjugates (ADCs) are an emerging type of biotherapeutics that utilize multiple tissue-specific antibodies combined with a range of linker designs to enable the transportation and selective release of cytotoxic drugs in close proximity to tumours. Consisting of antibodies conjugated to small drug molecules through a variety of linkers, ADCs are chemically complex analytes. Here we present a unique experimental comparison of four techniques for ADC analysis: hydrophobic interaction chromatography (HIC-UV/Vis), reversed phase liquid chromatography mass spectrometry (RPLC-MS), using either a QToF or an Orbitrap analyser, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Four different ADCs consisting of Trastuzumab, monomethyl auristatin E (MMAE) and a peptidic linker moiety differing in their respective stoichiometric ratios in regard to drug-to-antibody ratio (DAR) were used for the comparison. We found that the determined DAR from all techniques was comparable, while the accuracy of the molecular weights for the conjugated light and heavy chain differed more extensively. This indicates that the choice of a mass analyser is more crucial for determining the accurate weights of the light and heavy chains than to evaluate the DAR of a given batch. However, ambiguous DAR assignment in HIC-UV/Vis or bias for either the light or heavy chain fragments in the mass spectrometry-based techniques can influence the obtained average DAR value and the use of complementary techniques is advisable. Out of the four techniques evaluated, HIC-UV/Vis and MALDI required less time to obtain an average DAR value and would therefore be good for initial screenings in the early stages of the discovery phase of new ADCs.

Ort, förlag, år, upplaga, sidor
2018. Vol. 143, nr 22, s. 5487-5496
Nationell ämneskategori
Analytisk kemi
Forskningsämne
Kemi med inriktning mot analytisk kemi
Identifikatorer
URN: urn:nbn:se:uu:diva-371857DOI: 10.1039/c8an01178hISI: 000456684800019PubMedID: 30289422OAI: oai:DiVA.org:uu-371857DiVA, id: diva2:1274725
Forskningsfinansiär
Stiftelsen för strategisk forskning (SSF), ID14-0081Carl Tryggers stiftelse för vetenskaplig forskning , CTS 15:57Vetenskapsrådet, SRC 2015-4870Tillgänglig från: 2019-01-02 Skapad: 2019-01-02 Senast uppdaterad: 2020-01-31Bibliografiskt granskad
Ingår i avhandling
1. Development and evaluation of analytical techniques for antibodies and antibody-drug conjugates: From verification of conjugation to stability testing
Öppna denna publikation i ny flik eller fönster >>Development and evaluation of analytical techniques for antibodies and antibody-drug conjugates: From verification of conjugation to stability testing
2020 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Antibody-drug conjugates (ADCs) are becoming increasingly important in oncology. ADCs are inherently heterogeneous analytes. For a successful drug candidate to go through to the clinic, extensive characterization and evaluation of the constructs are necessary. The drug-to-antibody ratio (DAR) directly determines the toxicity and efficacy of the product and has to be closely monitored during the entire process.

In this thesis, several analytical techniques suitable for DAR determination in early-stage development have been evaluated against two more readily available options. The main focus in all studies has been on cysteine-linked ADCs, due to their prevalence in successful designs and their higher demand on the analytical set-up.

The comparisons showed that mass spectrometry (MS) derived DAR values did conform well with hydrophobic interaction chromatography derived values, irrespective of which MS instrument was used. For MS instruments, overall the desolvation was found to have a bigger impact on the apparent DAR values than the resolution of the instrument and low-resolution instruments, such as triple quadrupoles, can be viable options for DAR determination in early-stage development. Furthermore, it also could be concluded that MS-derived DAR values are susceptible to alterations in the sample preparation workflow.

Stability testing is vital to ensure a safe product. In this thesis, different compositions of mobile phases in size exclusion chromatographic (SEC) were tested for their ability to elute ADC aggregates. It was concluded that sufficient ionic strength to elute ADC aggregates from a SEC column purely by adding ammonium acetate cannot be achieved without exceeding salt concentrations compatlible with MS sources.

Finally, two new analytical workflows have been designed by applying a proteomic desalting protocol and supercharging reagents to mAb and ADC samples. Both techniques showed promise; the magnetic beads as a more flexible desalting alternative for mAbs and ADCs and the addition of selective supercharging reagents for improved sensitivity and peak shapes in MS spectra (without significant alterations of the derived DAR values).

Taken together, this thesis provides guidance on many aspects of ADC analysis from DAR determination to aggregate detection. The gathered knowledge can help setting up faster or more reliable quality checks for new candidates.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2020. s. 83
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1901
Nyckelord
Antibody-drug conjugates, drug-to-antibody ratio, monoclonal antibody, mass spectrometry, hydrophobic interaction chromatography, sample preparation, aggregation
Nationell ämneskategori
Analytisk kemi
Forskningsämne
Kemi med inriktning mot analytisk kemi
Identifikatorer
urn:nbn:se:uu:diva-403473 (URN)978-91-513-0867-8 (ISBN)
Disputation
2020-03-20, Room A1:111a, BMC, Husargatan 3, Uppsala, 09:15 (Engelska)
Opponent
Handledare
Tillgänglig från: 2020-02-26 Skapad: 2020-01-31 Senast uppdaterad: 2020-02-26

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