Phase behaviour of solutions of monoclonal antibodies
(2025) KEMR45 20251Department of Chemistry
- Abstract
- Our work is to study the phase behaviour of monoclonal antibodies (mAbs). Subcutaneous injection of monoclonal antibodies is the preferred administration, which requires high concentration formulations. However, monoclonal antibodies may undergo aggregation, reversible self-assembly and liquid-liquid phase separation at high concentrations which lead to problems in the formulation and may trigger adverse reaction when injected. Our work has focused on the characterization of liquid-liquid phase separation in solutions of a monoclonal antibody (called mAb-01) and the corresponding driving forces. Electrophoretic light scattering and static and dynamic light scattering were applied to characterize interparticle interactions. An optical... (More)
- Our work is to study the phase behaviour of monoclonal antibodies (mAbs). Subcutaneous injection of monoclonal antibodies is the preferred administration, which requires high concentration formulations. However, monoclonal antibodies may undergo aggregation, reversible self-assembly and liquid-liquid phase separation at high concentrations which lead to problems in the formulation and may trigger adverse reaction when injected. Our work has focused on the characterization of liquid-liquid phase separation in solutions of a monoclonal antibody (called mAb-01) and the corresponding driving forces. Electrophoretic light scattering and static and dynamic light scattering were applied to characterize interparticle interactions. An optical transmission setup was made to obtain the phase boundary for liquid-liquid phase separation (LLPS) and a special filtration setup was made to carry out light scattering measurements at very low concentrations. The antibody solutions undergo phase separation upon an increase of the temperature at higher concentrations, exhibiting a so called lower critical solution temperature (LCST), which is unusual for globular proteins. The phase boundary moves to higher temperatures at higher ionic strength, indicating the importance of attractive electrostatic interactions. Dynamic light scattering experiments confirm these findings through measurements of the diffusion interaction parameter kd as a function of ionic strength and temperature. We demonstrate the occurrence of LLPS with a lower critical solution temperature for the mAb investigated in this study. Electrophoretic light scattering indicates a complex ionic strength dependence of the net charge, and light scattering confirms the importance of attractive electrostatic interactions caused by the heterogeneous charge distribution on the mAb surface that results in large oppositely charged patches. (Less)
- Popular Abstract
- Monoclonal antibodies are large, Y-shaped proteins engineered in laboratory, targeting on diseases such as cancers, cardiovascular diseases and autoimmune diseases. To use these antibodies in an effective and practical way, ideally via subcutaneous self-administration of the patient, one needs to prepare aqueous solutions containing large amounts, i.e. high concentrations, of these proteins. Such solutions are then called ‘formulations’ and are ideally translucent and easily flowing. For the antibody solution we studied in the project, at high concentrations, we have observed that antibodies gathered together to form large droplets when the temperature is increasing and approaches the body temperature. These large droplets lead to a turbid... (More)
- Monoclonal antibodies are large, Y-shaped proteins engineered in laboratory, targeting on diseases such as cancers, cardiovascular diseases and autoimmune diseases. To use these antibodies in an effective and practical way, ideally via subcutaneous self-administration of the patient, one needs to prepare aqueous solutions containing large amounts, i.e. high concentrations, of these proteins. Such solutions are then called ‘formulations’ and are ideally translucent and easily flowing. For the antibody solution we studied in the project, at high concentrations, we have observed that antibodies gathered together to form large droplets when the temperature is increasing and approaches the body temperature. These large droplets lead to a turbid appearance of the solution, which is an adverse and unwanted effect, prohibiting good, stable formulations and safe delivery.
In the project, we have applied Electrophoretic light scattering technique to determine the charge on the antibodies and to find out how strong the salts are involved in hindering other antibodies. We also built up an optical transmission setup to determine at which temperature we could see antibodies start gathering. In addition, we have performed static/dynamic light scattering and obtain the information about how the interactions between antibodies change with different salt, the amount of salt added and different temperatures.
The combined results of our experiments tell us that our antibodies were prone to stay alone at low temperature but gather together to form large droplets at high temperatures. This occurs when the solution is heated up. By adding salt, antibodies will stay well distributed even at temperatures where solutions without salt are already turbid. However, further increasing the temperature by ca 10 degrees will lead to turbid solutions as well, just like in the case without adding salt. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9204358
- author
- Chen, Xinyu LU
- supervisor
- organization
- course
- KEMR45 20251
- year
- 2025
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- monoclonal antibody, light scattering, liquid-liquid phase separation, protein-protein interaction, physical chemistry
- language
- English
- id
- 9204358
- date added to LUP
- 2025-06-23 11:49:48
- date last changed
- 2025-06-23 11:49:48
@misc{9204358,
abstract = {{Our work is to study the phase behaviour of monoclonal antibodies (mAbs). Subcutaneous injection of monoclonal antibodies is the preferred administration, which requires high concentration formulations. However, monoclonal antibodies may undergo aggregation, reversible self-assembly and liquid-liquid phase separation at high concentrations which lead to problems in the formulation and may trigger adverse reaction when injected. Our work has focused on the characterization of liquid-liquid phase separation in solutions of a monoclonal antibody (called mAb-01) and the corresponding driving forces. Electrophoretic light scattering and static and dynamic light scattering were applied to characterize interparticle interactions. An optical transmission setup was made to obtain the phase boundary for liquid-liquid phase separation (LLPS) and a special filtration setup was made to carry out light scattering measurements at very low concentrations. The antibody solutions undergo phase separation upon an increase of the temperature at higher concentrations, exhibiting a so called lower critical solution temperature (LCST), which is unusual for globular proteins. The phase boundary moves to higher temperatures at higher ionic strength, indicating the importance of attractive electrostatic interactions. Dynamic light scattering experiments confirm these findings through measurements of the diffusion interaction parameter kd as a function of ionic strength and temperature. We demonstrate the occurrence of LLPS with a lower critical solution temperature for the mAb investigated in this study. Electrophoretic light scattering indicates a complex ionic strength dependence of the net charge, and light scattering confirms the importance of attractive electrostatic interactions caused by the heterogeneous charge distribution on the mAb surface that results in large oppositely charged patches.}},
author = {{Chen, Xinyu}},
language = {{eng}},
note = {{Student Paper}},
title = {{Phase behaviour of solutions of monoclonal antibodies}},
year = {{2025}},
}