Differences in the production methods of therapeutic antibodies can lead to changes in their structure, depending on the chosen recombinant procedure. Discrimination, which is based on several types of glycosylation, even affects the stability of antibodies. This was the result of a highly accurate comparison of the properties with respect to the isotopes of antibodies prepared in cell cultures.
The study was conducted by the University of Natural Sciences and Life Sciences (Vienna), where ultramodern mass spectrometers were used for this purpose to identify the minimal differences in glycosylation of immunoglobulins.
Antibodies are one of the most accurate forms of drugs and are increasingly being used to fight cancer and other ailments. They are often produced by recombination, with a number of different production processes. Each procedure creates an identical protein scaffold in the antibody, but there are differences in what is known as glycosylation or modification by the addition of certain carbohydrates.
Previously, little was known about how these differences and the form they are getting. Determining these subtle but potentially medically important discriminations requires an extremely complex analysis, which is only possible with the latest ultra-modern mass spectrometer. The Vienna research team had access to such equipment located in the University’s EQ BOKU section and revealed some amazing results.
The team headed by Professor Richard Strasser is essentially the first to recognize the exact differences in glycosylation patterns of immunoglobulin A produced either in human cell cultures (HEK293) or in plant systems (Nicotiana benthamiana). Professor Strasser, a member of the Department of Applied Genetics and Cell Biology, commented: “We are surprised how great the differences are. There were strong contradictions between the two systems regarding the structure of carbohydrates used for glycosylation and their position in proteins.
With the help of state-of-the-art techniques called capillary reversed-phase chromatography and electrospray mass spectrometry provided by EQ, the team has been able to analyze glycosylation in each system by discovering particular details. They found that immunoglobulin A produced in the HEK293 cell culture had many more and more complex N-glycans – a group of carbohydrates linked to specific nitrogen atoms – than that produced in mammalian cell cultures. Immunoglobulin A produced in plants also had a significantly smaller range of structures. This was mainly due to the fact that the plants do not have any of the metabolic pathways required for the glycosylation of mammals. “But we also saw glycosylation in antibodies produced in plants that can only appear on plants,” Professor Strasser added.
Although glycosylations in the antibodies produced in the Nicotiana benthamiana plant may differ from the normal method, the antibodies exhibited the same binding properties for antigens as those made using human cells. This shows that with regard to therapeutic applications, the choice of the production system has no difference. However, further analysis by Professor Strasser’s team has revealed that the stability of immunoglobulin A varies according to the production method – a factor that can have a decisive effect on treatment use.
The assay included the thermal stability test of the antibodies, which was found to be lower in plant-derived immunoglobulin A. “We need to carry out further tests to find out how important this is for the application of these antibodies to treatment,” explains Professor Strasser.