Profiling antibody drug conjugate positional isomers: a system-of-equations approach.

Antibody drug conjugates enable the targeted delivery of potent chemotherapeutic agents directly to cancerous cells. They are made by the chemical conjugation of cytotoxins to monoclonal antibodies, which can be achieved by first reducing interchain disulfide bonds followed by conjugation of the resulting free thiols with drugs. This process yields a controlled, but heterogeneous, population of conjugated products that contains species with various numbers of drugs linked to different former interchain disulfide cysteine residues on the antibodies.

Culture temperature modulates aggregation of recombinant antibody in cho cells.

During production of therapeutic monoclonal antibodies (mAb), it is highly desirable to remove and control antibody aggregates in the manufacturing process to minimize the potential risk of immunogenicity to patients. During process development for the production of a recombinant IgG in a CHO cell line, we observed atypical high variability from 1 to 20% mAb aggregates formed during cell culture that negatively impacted antibody purification. Analytical characterization revealed the IgG aggregates were mediated by hydrophobic interactions likely caused by misfolded antibody during intracellular processing.

Effect of temperature, pH, dissolved oxygen, and hydrolysate on the formation of triple light chain antibodies in cell culture.

THIOMABs are recombinant antibodies with reactive cysteine residues used for forming THIOMAB-drug conjugates (TDCs). We recently reported a new impurity associated with THIOMABs: one of the engineered cysteines forms a disulfide bond with an extra light chain (LC) to generate a triple light chain antibody (3LC). In our previous investigations, increased LC expression increased 3LC levels, whereas increased glutathione (GSH) production decreased 3LC levels.

Triple light chain antibodies: factors that influence its formation in cell culture.

THIOMABs are recombinant antibodies engineered with reactive cysteines, which can be covalently conjugated to drugs of interest to generate targeted therapeutics. During the analysis of THIOMABs secreted by stably transfected Chinese Hamster Ovary (CHO) cells, we discovered the existence of a new species--Triple Light Chain Antibody (3LC). This 3LC species is the product of a disulfide bond formed between an extra light chain and one of the engineered cysteines on the THIOMAB.

Reversible self-association increases the viscosity of a concentrated monoclonal antibody in aqueous solution.

This study was conducted to investigate the effect of reversible protein self-association on the viscosity of concentrated monoclonal antibody solutions. The viscosities of the monoclonal antibody solutions were measured by either a capillary viscometer or a cone-plate rheometer at different protein concentrations, pH, and ionic strength. Soluble aggregates were determined by size exclusion chromatography, light scattering, and analytical ultracentrifugation.