Method development for improving monoclonal antibody production

Abstract

Biopharmaceuticals are a fast-growing niche in the pharmaceutical industry compared to small molecules. Even though a lot of progress has been made in recent years, the process to take a drug candidate to clinical trials can be lengthy and the selection process of high producing stable clones can be an arduous process.

In this master thesis the design of new expression vectors for the production of monoclonal antibodies have been theoretically evaluated and the transfection protocol using electroporation has been optimized, with the goal to increase monoclonal antibody expression and cell line stability. The suggested expression vector consists of two different promoters, CMV for the control of the light and heavy chain genes, and SV40 for the selection gene GS. The restriction site Eco31I will be used for the insertion of the expression cassettes for both the heavy and light chain genes. The expression cassettes contains 5'UTR, signal peptide, the chain, 3'UTR and a polyA tail. The expression vector will be surrounded by the insulators cHS4. The bacterial selection marker Amp will be used for the vector production.

To optimize the electroporation a multivariate approach was taken to evaluate the effect of pulse duration, strength and number of pulses and their interaction. The electroporation was evaluated based on viability and recombinant protein expression. It was concluded that a square wave pulse of 350 V for a pulse duration of 15 ms divided into three pulses is of interest and should be further investigated. By combining the optimized transfection protocol and the new expression vector yields could be increased and thereby reducing production costs and the timeline to take biopharmaceutical candidates to clinical trials.

Popular Abstract

Did you know that many pharmaceuticals are proteins that are produced by genetically modified organisms? Genetically modified organisms can be used to produce pharmaceuticals (biopharmaceuticals) for the treatment of various diseases as well as for the production of vaccines. This report will focus on how to increase the production of a desired protein in a genetically modified organism. This to decrease the production time and cost, which in turn will decrease the time to take the drug candidate to the market.

Genes encodes proteins that are needed by the cells. By introducing a new fragment containing the gene and other components to cells it's possible to make cells produce a new protein, which is not essential for the cells survival. The fragment has to contain more than the gene for the protein to be expressed, and its design can be optimized to instruct the cell to produce more of a specific protein over an extended period of time. Over time the cells production of the desired protein often declines, and this could be prevented by protecting the gene fragment with insulators. Optimization of the fragment is done by changes to the fragment, such as changing the promoter for the gene. These changes to the gene fragment as well as its integration into the cell is the focus of this master thesis.

The introduction of the gene fragment coding for the protein of interest could be performed in different ways. This report will focus on a physical method, electroporation, which uses electrical pulses. The electrical pulse form pores in the cell membrane and allow the gene of interest to enter the cell. By varying the length, strength and number of electrical pulses to the cell it is possible to affect how much of the gene enters the cell and how well the cells will survive the electrical pulses. By increasing the amount of gene fragment that is introduced into the cell, the likelihood of the fragment being integrated into the genome increases.

This thesis determined that it was beneficial for the protein production to divide the electrical pulse into two or three shorter pulses, instead of using a single long pulse. It was also determined that an increased pulse strength increased the expression of the protein. The newly designed gene fragment has been designed in a way to increase the cells production of the protein as well as to decrease the risk of a decrees in production over time.

Biopharmaceuticals are crucial for the treatment of many diseases such as cancers, autoimmune diseases and chronic inflammatory diseases. This optimized method for introducing a gene fragment as well as the newly designed fragment can lead to a reduced time line to take a new biopharmaceutical to clinic.

• author: Blomberg, Sofie
• supervisor: Mats Ohlin
• organization: Department of Immunotechnology
• course: KIMM05 20211
• year: 2021
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9060688
• date added to LUP: 2021-09-09 17:19:59
• date last changed: 2021-09-09 17:19:59