Variations in critical quality attributes (CQAs) on therapeutic monoclonal antibodies (mAbs) due to cultivation conditions, storage conditions and external stress-factors
(2024) In Journal of Immunology KIMM05 20241Department of Immunotechnology
- Abstract
- Ensuring close control of critical quality attributes (CQAs), such as post-translational modifications (PTMs), in therapeutic monoclonal antibodies (mAbs) is essential for maintaining function and batch homogeneity in large-scale production. Common PTMs include deamidation, fragmentation, oxidation, charge variants, and glycosylations. This study aimed to evaluate CQA variations and correlate the findings to cultivation conditions, post-collection storage, and three external stress factors. Samples were collected after 7-14 days of cultivation from four batches, with specific conditions, and evaluated by orthogonal methods. Batch 1 (50 L) was stored in a freezer; batch 2 (1000 L), and batches 3 and 4 (5 L each) were stored in a fridge.... (More)
- Ensuring close control of critical quality attributes (CQAs), such as post-translational modifications (PTMs), in therapeutic monoclonal antibodies (mAbs) is essential for maintaining function and batch homogeneity in large-scale production. Common PTMs include deamidation, fragmentation, oxidation, charge variants, and glycosylations. This study aimed to evaluate CQA variations and correlate the findings to cultivation conditions, post-collection storage, and three external stress factors. Samples were collected after 7-14 days of cultivation from four batches, with specific conditions, and evaluated by orthogonal methods. Batch 1 (50 L) was stored in a freezer; batch 2 (1000 L), and batches 3 and 4 (5 L each) were stored in a fridge. Three stressors were applied: 1) high pH + 37°C, 2) hydrogen peroxide, and 3) mechanical stress. Analysis methods included Size Exclusion High-Performance Liquid Chromatography, Capillary Electrophoresis Sodium Dodecyl Sulfate, Capillary Isoelectric Focusing, Hydrophilic Interaction Liquid Chromatography, Liquid Chromatography Mass Spectrometry, and Backgrounded Membrane Imaging. Results indicated that earlier sample collection promoted better CQAs, suggesting earlier harvest. The study did not investigate mAb yield, necessary to weigh quality against product outcome. All stressors affected the CQAs differently depending on the stressor, and should all be avoided if possible. Batch 1, with 50 L cultivation volume, freezer storage, and cell separation directly after collection, showed the best retained CQAs and the most robust mAbs. The study concluded that batch 1's conditions were optimal, but further investigation including triplicates, statistical analysis, and consistent measurement points is required to confirm the conclusion. (Less)
- Popular Abstract
- Production of high quality, potentially cancer curing antibodies and their production and storage conditions
10 million people die from cancer yearly and the urgent development of new treatments is of great importance [1]. Innovative companies like BioInvent International AB offer a promising solution to the riddle of this devastating disease by their expertise in immunology, cancer- and antibody biology. A crucial part of therapeutic antibody development is finding the optimal process condition and creating a robust antibody.
Antibodies are naturally made from the body’s own immune system to fight off bacteria, viruses and cancerous cells. Antibodies can be engineered to have specific binding abilities and effects, enabling them to... (More) - Production of high quality, potentially cancer curing antibodies and their production and storage conditions
10 million people die from cancer yearly and the urgent development of new treatments is of great importance [1]. Innovative companies like BioInvent International AB offer a promising solution to the riddle of this devastating disease by their expertise in immunology, cancer- and antibody biology. A crucial part of therapeutic antibody development is finding the optimal process condition and creating a robust antibody.
Antibodies are naturally made from the body’s own immune system to fight off bacteria, viruses and cancerous cells. Antibodies can be engineered to have specific binding abilities and effects, enabling them to target specific cancer cells. The antibodies are produced in cells which work as antibody-producing factories. When growing the antibody-factory cells, and producing the antibodies, there are important parameters that can affect the antibodies' effects in the body. Some changes, even if they are very small, may even be harmful to us. These changes of the antibody may be introduced by both the production conditions, which are the conditions of the antibody-factory cells, as well as the storage conditions, after the production. It is important to examine which parameters, such as production volume and storage temperature, that affect the antibodies and in what way, to make sure the most suitable conditions are used. As a result, the antibody will have the wanted effects, which means that it efficiently and safely can attack cancer cells in our body. To exemplify the changes that can occur to the antibodies, they can cluster together, aggregate, break into smaller pieces, fragmentate, and various chemical changes can lead to the antibodies becoming non-natural. All of these changes to the antibody are undesirable and need to be held under control, for our safety. The study showed that storing the antibodies in a freezer, instead of a fridge, was beneficial and caused fewer changes in the critical quality attributes. Furthermore, it seemed to be optimal to use a production volume of 50L, instead of a larger one of 1000L or a smaller one of 5L, as they both seemed to cause the producing cells to become stressed, hence leading to changes of the antibody. In addition, it is important to examine how well the antibodies respond to stress, as they will need to have some robustness in order to withstand transportation from the production site to the site of usage, storage and administration into the body. The administration is often intravenous which involves passing the antibodies through a thin needle, which may cause mechanical stress to the antibody. The antibodies were subjected to induced stress by storing them at high pH, in an oxidative environment (hydrogen peroxide) and by mechanically stressing them with a needle and syringe. By analyzing antibodies, it became clear that mechanical stress caused them to aggregate and form small particles, which may cause harmful effects in the body. The high pH and the hydrogen peroxide both led to changes of the antibodies. Although further studies are needed to confirm these results, these analyses are important to understanding and finding optimal conditions for the antibody-factories and their storage after production, as well as creating robust and safe antibodies, to fight off cancer and improve the lives of millions of people.
1. Cancer Research UK. “Worldwide Cancer Statistics.” Cancer Research UK, CRUK, 12 Mar. 2019, www.cancerresearchuk.org/health-professional/cancer-statistics/worldwide-cancer. Accessed 15 Apr. 2024. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9170581
- author
- Nash, Felicia LU and Sannum, Emma LU
- supervisor
- organization
- course
- KIMM05 20241
- year
- 2024
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- monoclonal antibodies, post-harvest conditions, production conditions, external stressors, critical quality attributes
- publication/series
- Journal of Immunology
- language
- English
- id
- 9170581
- date added to LUP
- 2024-07-11 09:39:46
- date last changed
- 2024-07-11 09:46:12
@misc{9170581,
abstract = {{Ensuring close control of critical quality attributes (CQAs), such as post-translational modifications (PTMs), in therapeutic monoclonal antibodies (mAbs) is essential for maintaining function and batch homogeneity in large-scale production. Common PTMs include deamidation, fragmentation, oxidation, charge variants, and glycosylations. This study aimed to evaluate CQA variations and correlate the findings to cultivation conditions, post-collection storage, and three external stress factors. Samples were collected after 7-14 days of cultivation from four batches, with specific conditions, and evaluated by orthogonal methods. Batch 1 (50 L) was stored in a freezer; batch 2 (1000 L), and batches 3 and 4 (5 L each) were stored in a fridge. Three stressors were applied: 1) high pH + 37°C, 2) hydrogen peroxide, and 3) mechanical stress. Analysis methods included Size Exclusion High-Performance Liquid Chromatography, Capillary Electrophoresis Sodium Dodecyl Sulfate, Capillary Isoelectric Focusing, Hydrophilic Interaction Liquid Chromatography, Liquid Chromatography Mass Spectrometry, and Backgrounded Membrane Imaging. Results indicated that earlier sample collection promoted better CQAs, suggesting earlier harvest. The study did not investigate mAb yield, necessary to weigh quality against product outcome. All stressors affected the CQAs differently depending on the stressor, and should all be avoided if possible. Batch 1, with 50 L cultivation volume, freezer storage, and cell separation directly after collection, showed the best retained CQAs and the most robust mAbs. The study concluded that batch 1's conditions were optimal, but further investigation including triplicates, statistical analysis, and consistent measurement points is required to confirm the conclusion.}},
author = {{Nash, Felicia and Sannum, Emma}},
language = {{eng}},
note = {{Student Paper}},
series = {{Journal of Immunology}},
title = {{Variations in critical quality attributes (CQAs) on therapeutic monoclonal antibodies (mAbs) due to cultivation conditions, storage conditions and external stress-factors}},
year = {{2024}},
}