Multifaceted Evaluation of IgG Glycan Profiles Considering the Components in Glycosyltransferase Reactions

Importance of the Topic

N linked glycans have critical influence on antibody efficacy and safety in biopharmaceuticals. Control of glycan patterns is essential for optimizing antibody dependent cellular cytotoxicity and complement dependent cytotoxicity in therapeutic antibodies. Multifaceted analysis of substrate precursors, cofactors and sugar nucleotides enables strategic development of culture conditions for desired glycoforms.

Objectives and Overview of the Study

This study aims to combine liquid chromatography mass spectrometry and inductively coupled plasma mass spectrometry techniques to evaluate key components involved in glycosyltransferase reactions. Using CHO K1 cell fed batch cultures producing immunoglobulin G, the work evaluates variations in sugar precursor concentration, metal ion cofactor uptake and nucleotide sugar substrates, and correlates these parameters with resulting antibody glycan profiles under different medium supplements.

Methodology

Cell culture was performed in flask fed batch format with CHO K1 cells seeding at 2.0 x 10^5 cells per mL under three conditions: control, manganese supplemented, and manganese plus galactose supplemented. Samples of supernatant and cell pellets were collected at regular intervals for multicomponent analysis.

• Metal ion analysis: Supernatant samples diluted in nitric and hydrochloric acid mix and measured by ICPMS 2050 with helium hydrogen collision gas.
• Sugar analysis in supernatant: Proteins removed by acetonitrile precipitation, diluted sample analyzed by LCMS 8060NX using a sugars and sugar nucleotide method package.
• Intracellular sugar and sugar nucleotide analysis: Cell pellets quenched in cold solvent, extracted, dried and reconstituted for LCMS 8060NX analysis.
• Glycan profiling: Antibodies purified from supernatant, released glycans labeled with 2 AB using a commercial kit, and separated on a metal free amide column by HPLC with fluorescence detection.

Used Instrumentation

• LCMS 8060NX triple quadrupole mass spectrometer with Sugars and Sugar Nucleotide Method Package.
• Nexera XR high performance liquid chromatograph with Shim pack GIST HP Amide column and fluorescence detector RF 20Axs.
• ICPMS 2050 inductively coupled plasma mass spectrometer for quantification of metal ions.
• Vi CELL BLU automated cell analyzer for viability and cell concentration assessment.

Main Results and Discussion

• Manganese uptake: Cells under manganese supplemented conditions exhibited higher Mn uptake rates compared to control, confirming efficient cofactor incorporation.
• Sugar and UDP galactose dynamics: Extracellular galactose was consumed over culture time and converted to intracellular UDP galactose, demonstrating active substrate flux.
• Glycan profile shifts: The ratio of mono and di galactosylated fucosylated glycoforms increased in the order control less than manganese less than manganese plus galactose, with sustained galactosylation in stationary phase under co supplementation.

Benefits and Practical Applications

Strategic analysis of glycosyltransferase reaction components allows targeted manipulation of cell culture supplements to achieve desired antibody glycoforms. This approach supports process development in biopharmaceutical manufacturing and quality control under critical quality attribute frameworks.

Future Trends and Potential Applications

Integration of comprehensive metabolomics and glycomics profiling is expected to further optimize glycoengineering strategies. Advanced method packages for cell culture profiling and primary metabolites can expand insights into energy metabolism and pentose phosphate pathway interactions. Application of machine learning to multivariate data sets may enhance predictive control of glycan patterns.

Conclusion

By combining ICP MS metal analysis, LC MS MS sugar and sugar nucleotide quantification, and HPLC RF glycan profiling, this study demonstrates a robust platform for multifaceted evaluation of glycosyltransferase reaction components. The findings support rational design of culture conditions to modulate antibody glycosylation profiles.

References

• Wang Z, Zhu J, Lu H. Antibody Glycosylation Impact on Antibody Drug Characteristics and Quality Control. Applied Microbiology and Biotechnology. 2020;104(5):1905-1914.
• Shimadzu Application News 01-00712 Analysis of Metal Elements in Culture Medium Using ICPMS 2050. 2024.