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Monolith affinity chromatography for the rapid quantification of a single-chain variable fragment immunotoxin

Satzer, Peter ; Sommer, Ralf ; Paulsson, Johanna LU ; Rodler, Agnes ; Zehetner, Romana ; Hofstädter, Klaus ; Klade, Christoph and Jungbauer, Alois (2018) In Journal of Separation Science 41(15). p.3051-3059
Abstract

We developed a novel analytical method for concentration determination of tandem single-chain antibody diphtheria toxin (immunotoxin). The method is based on polymethacrylate monoliths with Protein L ligands as the binding moiety. Different buffers were tested for elution of the Protein L-bound immunotoxin and 4.5 M guanidinium hydrochloride performed best. We optimized the elution conditions and the method sequence resulting in a fast and robust method with a runtime <10 min. Fast determination of immunotoxin is critical if any process decisions rely on this data. We determined method performance and a lower limit of detection of 27 μg/mL and a lower limit of quantification of 90 μg/mL was achieved. The validity of the method in... (More)

We developed a novel analytical method for concentration determination of tandem single-chain antibody diphtheria toxin (immunotoxin). The method is based on polymethacrylate monoliths with Protein L ligands as the binding moiety. Different buffers were tested for elution of the Protein L-bound immunotoxin and 4.5 M guanidinium hydrochloride performed best. We optimized the elution conditions and the method sequence resulting in a fast and robust method with a runtime <10 min. Fast determination of immunotoxin is critical if any process decisions rely on this data. We determined method performance and a lower limit of detection of 27 μg/mL and a lower limit of quantification of 90 μg/mL was achieved. The validity of the method in terms of residual analysis, precision, and repeatability was proven in a range from 100 to 375 μg/mL. The short runtime and ease of use of a high-performance liquid chromatography method is especially useful for a process analytical tool approach. Bioprocesses related to immunotoxin where fermentation or other process parameters can be adjusted in accordance to the immunotoxin levels will be benefited from this method to achieve the highest possible purity and productivity.

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author
; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
affinity chromatography, antibody-drug conjugates, immunotoxins, monoliths, process analytical tools
in
Journal of Separation Science
volume
41
issue
15
pages
9 pages
publisher
John Wiley and Sons Inc.
external identifiers
  • pmid:29873445
  • scopus:85050611247
ISSN
1615-9306
DOI
10.1002/jssc.201800257
language
English
LU publication?
yes
id
a7de60ec-7def-491a-9597-c6315e18263f
date added to LUP
2018-10-01 09:51:47
date last changed
2020-09-30 05:43:12
@article{a7de60ec-7def-491a-9597-c6315e18263f,
  abstract     = {<p>We developed a novel analytical method for concentration determination of tandem single-chain antibody diphtheria toxin (immunotoxin). The method is based on polymethacrylate monoliths with Protein L ligands as the binding moiety. Different buffers were tested for elution of the Protein L-bound immunotoxin and 4.5 M guanidinium hydrochloride performed best. We optimized the elution conditions and the method sequence resulting in a fast and robust method with a runtime &lt;10 min. Fast determination of immunotoxin is critical if any process decisions rely on this data. We determined method performance and a lower limit of detection of 27 μg/mL and a lower limit of quantification of 90 μg/mL was achieved. The validity of the method in terms of residual analysis, precision, and repeatability was proven in a range from 100 to 375 μg/mL. The short runtime and ease of use of a high-performance liquid chromatography method is especially useful for a process analytical tool approach. Bioprocesses related to immunotoxin where fermentation or other process parameters can be adjusted in accordance to the immunotoxin levels will be benefited from this method to achieve the highest possible purity and productivity.</p>},
  author       = {Satzer, Peter and Sommer, Ralf and Paulsson, Johanna and Rodler, Agnes and Zehetner, Romana and Hofstädter, Klaus and Klade, Christoph and Jungbauer, Alois},
  issn         = {1615-9306},
  language     = {eng},
  month        = {08},
  number       = {15},
  pages        = {3051--3059},
  publisher    = {John Wiley and Sons Inc.},
  series       = {Journal of Separation Science},
  title        = {Monolith affinity chromatography for the rapid quantification of a single-chain variable fragment immunotoxin},
  url          = {http://dx.doi.org/10.1002/jssc.201800257},
  doi          = {10.1002/jssc.201800257},
  volume       = {41},
  year         = {2018},
}