Direct Bioanalysis of ADCs using Affinity Capture-LC-HRAMS Techniques for Characterization and ...

50 Pages·2015·4.96 MB·English

Direct Bioanalysis of ADCs using
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Techniques for Characterization
and Quantification—a Progress
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Rand Jenkins et al
EBF 8th Open Symposium
BBaarcceelloonnaa SSppaaiinn, 1188 NNoov 22001155
Overview
1 Biotherapeutics diversity
22 Whhy new BA approachhes are needdedd
33 LLCC-MMSS pprrootteeiinn bbiiooaannaallyyssiiss wwoorrkkfflloowwss
44 AADDCC cchhaarraacctteerriizzaattiioonn
55 IInnttaacctt AADDCC qquuaannttiiffiiccaattiioonn
6 Oppen qquestions & final thougghts
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Biotherappeutics diversityy
ppeeppttiiddeess
mAbs
bispecifics (bsAbs, DART®s, BiTE®s)
AADDCCss
prodrug/carrier conjugates (albumin, PEG)
ffusiion protteiins
messenger RNA therapeutics™
oligonucleotides (ssDNA, siRNA, mRNA)
CCAARRss
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Alternative formats for bispecifics
CChhriisttophhSSpiiess, QQiianttiingZZhhaii, PPaull JJ. CCartter, AAlltternattiive mollecullar fformatts andd tthherapeuttiic
applications for bispecific antibodies,Molecular Immunology 67 (2015) 95–106
4
Antibodyy-Drugg Conjjuggates
KKaur SS et all. BBiioanallysiis, ((22001133)) 55((22)), 220011–222266
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GlyycoConnect ADC: a new gglyycan conjjuggation idea
Remon van Geel et al, Chemoenzyymatic Conjjuggation of Toxic Payyloads to the Globallyy Conserved N-Glyycan of Native
mAbs Provides Homogeneous and Highly Efficacious Antibody−Drug Conjugates, BioconjugateChem.2015, in press
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Whyy new BA apppproaches are needed
++ BBiiootthheerraappeeuuttiiccss iinnnnoovvaattiioonn iiss pprroodduucciinngg iinnccrreeaassiinnggllyy ccoommpplleexx
molecular constructs
+ Their inherent heterogeneity and potential to undergo
stability- and/or catabolism-related changes are driving the
need for greater characterization
++ CCoonnvveennttiioonnaall LLiiggaanndd BBiinnddiinngg AAssssaayyss ((LLBBAA)) aarree lliimmiitteedd iinn tthheeiirr
ability to provide data reflecting molecular structure (e.g.
Drugg Antibodyy Ratio ((DAR)) distribution for ADCs))
+ MS-based methods, including affinity capture-liquid
chhromattographhy-hhiighh resolluttiion accuratte mass specttromettry
(AC-LC-HRAMS), are emerging as versatile tools
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LLCC-MMSS pprrootteeiinn bbiiooaannaallyyssiiss wwoorrkkfflloowwss
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Protein LC-MS//MS Bioanalyysis General Strateggyy
Protein/Peptide in Biomatrix Sample
MW < ~10 kDa MW > ~10 kDa
Direct Measurement  Indirect Measurement
((IInnttaacctt AAnnaallyyttee)) ((PPrrootteeoollyyttiicc PPeeppttiiddeess))
Extraction//Enrichment Extraction//Enrichment
(Chemical (e.g. PPT, SPE)  (Chemical (e.g. PPT)
or Affinity Capture)  or Affinity Capture)
Clean up 
Proteolytic
LC‐MS/MS
(SPE or 
DDiiggeessttiioonn
Peptide AC)
LC‐MS/MS
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Protein LC-HRAMS Bioanalyysis Alternative Strateggyy
Protein/Peptide in Biomatrix Sample
MW < ??? kDa MW > ~10 kDa
Direct Measurement  Indirect Measurement
((IInnttaacctt AAnnaallyyttee)) ((PPrrootteeoollyyttiicc PPeeppttiiddeess))
Enrichment Extraction//Enrichment
Affinity Capture (Chemical (e.g. PPT)
or Affinity Capture)
LC‐HRAMS
Clean up 
Proteolytic
(SPE or 
DDiiggeessttiioonn
Peptide AC)
LC‐HRAMS
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