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Establishing a pipeline for production of recombinant antibodies utilizing Extensible Mammalian Modular Assembly (EMMA) kit

Lipsanen, Riikka (2025) MOBN03 20242
Degree Projects in Molecular Biology
Popular Abstract
Production of Recombinant Antibodies Using Modular Cloning

Antibodies are a part of the immune system tasked in recognizing and binding foreign substances, antigens, and recruiting other parts of the immune system to neutralize them. Antibodies are Y-shaped proteins consisting of two heavy and two light chains. Both light and heavy chains contribute to the variable and constant regions of the antibody. The variable region binds to the antigens. Antibodies have been widely utilized in research and diagnostics and have emerged as therapeutic agents in recent years. The growing interest in utilizing antibodies has led to the development of many cell culture-produced recombinant antibodies. These are synthesized in cells that do not... (More)
Production of Recombinant Antibodies Using Modular Cloning

Antibodies are a part of the immune system tasked in recognizing and binding foreign substances, antigens, and recruiting other parts of the immune system to neutralize them. Antibodies are Y-shaped proteins consisting of two heavy and two light chains. Both light and heavy chains contribute to the variable and constant regions of the antibody. The variable region binds to the antigens. Antibodies have been widely utilized in research and diagnostics and have emerged as therapeutic agents in recent years. The growing interest in utilizing antibodies has led to the development of many cell culture-produced recombinant antibodies. These are synthesized in cells that do not naturally produce antibodies. The cells are genetically modified to produce antibodies of interest.

Modular cloning is a method of plasmid construction, where each component is cloned as a separate unit. The plasmid is a circular DNA sequence used to genetically modify cells. Components of a plasmid include for example the coding sequence of the gene of interest (GOI), the promoter that induces the translation of the GOI, and the modulators of gene expression that improve the yield of antibody produced. The modular cloning allows the different components to be exchanged easily, allowing the testing of multiple combinations to produce different antibodies.

The aim of this study was to set up a pipeline for producing recombinant antibodies in mammalian cell culture. The laboratory has an established protocol for producing antibodies by hybridoma clones and for producing recombinant proteins in mammalian cells. Hybridomas are immortalized B immune cells producing antibodies without genetic engineering, and the DNA sequence of a variable region of an antibody was obtained from them. This sequence was then cloned into a plasmid together with an antibody constant region. Alternatively, a variable region sequence was acquired from the literature and cloned into a plasmid with an antibody constant region. The formed fusion antibodies were expressed in mammalian cells and purified to test their binding.

Lastly, the antibody with a known variable region was produced and purified successfully. The ability bind target antigen was also tested. The antibodies synthesized from hybridomas were produced successfully, although the purification was unsuccessful. We believe that the purification problems are due to the incorrect folding of the recombinant antibody. The 3D structure of the antibody is crucial for the purification method used and misfolding is one of the most common problems with recombinant antibody production. Nevertheless, we conclude that the pipeline for producing recombinant antibodies in mammalian cells was established successfully.

Master’s Degree Project in Molecular Biology 60 credits 2025
Department of Biology, Lund University
Advisor: Peter Garred, MD, DMSci, Professor of Clinical Molecular Medicine
Department of Clinical Immunology, Rigshospital, Copenhagen University (Less)
Please use this url to cite or link to this publication:
author
Lipsanen, Riikka
supervisor
organization
course
MOBN03 20242
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9214692
date added to LUP
2025-10-30 13:59:53
date last changed
2025-10-30 13:59:53
@misc{9214692,
  author       = {{Lipsanen, Riikka}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Establishing a pipeline for production of recombinant antibodies utilizing Extensible Mammalian Modular Assembly (EMMA) kit}},
  year         = {{2025}},
}