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Development of a Type IIs Cloning Strategy and Subsequent Production of Monoclonal Antibodies from PCR Amplification, Donor Vectors & Synthesized DNA Fragments

Jönsson, Malin LU (2018) KIMM05 20181
Department of Immunotechnology
Abstract
Alongside the emerging field of immunotherapy, with monoclonal antibody drugs at the forefront, radically reshaping medicine, the need for process and technology improvements that can lower drug development costs and bring better drugs to the market faster is increasing. One such process improvement being fast and efficient cloning strategies which enable moving into high-throughput formats. Traditional cloning methods does not allow for high throughput cloning strategies to be implemented due to the multiple rounds of restriction enzyme digestion and loss of DNA during numerous purification steps that are involved in the cloning procedure. Presented here is an improved strategy for production of monoclonal antibodies using a Type... (More)
Alongside the emerging field of immunotherapy, with monoclonal antibody drugs at the forefront, radically reshaping medicine, the need for process and technology improvements that can lower drug development costs and bring better drugs to the market faster is increasing. One such process improvement being fast and efficient cloning strategies which enable moving into high-throughput formats. Traditional cloning methods does not allow for high throughput cloning strategies to be implemented due to the multiple rounds of restriction enzyme digestion and loss of DNA during numerous purification steps that are involved in the cloning procedure. Presented here is an improved strategy for production of monoclonal antibodies using a Type IIs-mediated assembly. This is shown to result in > 90 % cloning efficiency when utilizing either PCR fragments, donor vectors or synthesized DNA fragments and validated by cloning of five proteins of biopharmaceutical value. Further, the improved expression protocol demonstrated here generates 2-4 times higher protein yields without repercussions on the high product quality. This workflow facilitates utilization of high throughput strategies, enabling performance of larger screenings and decreases time and effort demanded by traditional cloning methods by elimination of bottlenecks within these processes. (Less)
Please use this url to cite or link to this publication:
author
Jönsson, Malin LU
supervisor
organization
course
KIMM05 20181
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
8951739
date added to LUP
2018-06-20 14:35:24
date last changed
2018-06-20 14:35:24
@misc{8951739,
  abstract     = {{Alongside the emerging field of immunotherapy, with monoclonal antibody drugs at the forefront, radically reshaping medicine, the need for process and technology improvements that can lower drug development costs and bring better drugs to the market faster is increasing. One such process improvement being fast and efficient cloning strategies which enable moving into high-throughput formats. Traditional cloning methods does not allow for high throughput cloning strategies to be implemented due to the multiple rounds of restriction enzyme digestion and loss of DNA during numerous purification steps that are involved in the cloning procedure. Presented here is an improved strategy for production of monoclonal antibodies using a Type IIs-mediated assembly. This is shown to result in > 90 % cloning efficiency when utilizing either PCR fragments, donor vectors or synthesized DNA fragments and validated by cloning of five proteins of biopharmaceutical value. Further, the improved expression protocol demonstrated here generates 2-4 times higher protein yields without repercussions on the high product quality. This workflow facilitates utilization of high throughput strategies, enabling performance of larger screenings and decreases time and effort demanded by traditional cloning methods by elimination of bottlenecks within these processes.}},
  author       = {{Jönsson, Malin}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Development of a Type IIs Cloning Strategy and Subsequent Production of Monoclonal Antibodies from PCR Amplification, Donor Vectors & Synthesized DNA Fragments}},
  year         = {{2018}},
}