Determination of thiol-to-protein ratio and drug-to-antibody ratio by in-line size exclusion chromatography with post-column reaction.
Anal Biochem
; 527: 33-44, 2017 06 15.
Article
em En
| MEDLINE
| ID: mdl-28432000
ABSTRACT
An in-line size-exclusion (SE) ultra-high-performance liquid chromatography (UHPLC)- 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) method to quantify thiols in monoclonal antibodies (mAb) when manufacturing antibody-drug conjugates (ADCs) was developed. The mAbs are separated on an SE-UHPLC column and monitored with a UV detector at a wavelength of 280 nm. Eluents are channeled into a reaction coil and mixed with DTNB to form 5-thio-2-nitrobenzoic acid (TNB). Thiol concentration is calculated using absorption at 412 nm. Using optimized conditions, partially reduced mAbs can be separated from low-molecular weight contaminants and undergo the DTNB reaction. The standard curve of L-cysteine had good linearity between 100 and 1000 µM. The selectivity, linearity, repeatability, and robustness of this method were evaluated. The calculated free-SHprotein ratios of partially reduced mAbs were consistent between in-line SE-UHPLC-DTNB and conventional methods. The SE-UHPLC-DTNB method showed time- and temperature-dependent changes in the free-SHprotein ratio of mAbs during reduction. The changes in drug-antibody ratio (DAR) of ADCs during the conjugation reaction were also evaluated. This method is an inexpensive and versatile alternative to conventional methods of estimating the free-SHprotein ratio of mAbs and the DAR of ADCs. This method also minimizes assay time.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Oligopeptídeos
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Compostos de Sulfidrila
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Cromatografia em Gel
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Imunoconjugados
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Ácido Ditionitrobenzoico
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Aminobenzoatos
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Anticorpos Monoclonais
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
2017
Tipo de documento:
Article