Characterization of recombinant glycoproteins from Chinese hamster ovary cells

Production of recombinant proteins may be accomplished in a variety of cellular expression systems. Bacterial expression is often preferred because yields are high and manipulation of the genetics of bacteria is relatively easy. However, when the goal is to produce proteins that are as nearly identical as possible to the non-recombinant protein, this may not always be the best choice, since bacterial systems do not usually form the correct disulfide pairs nor add carbohydrate. If the desire is to produce a protein with glycosylation that is similar to the human protein, expression of proteins in tissue culture from Chinese hamster ovary cells is one method of choice, since a great body of knowledge exists for this cell system. Traditional, non-mass spectrometric methods for characterization of carbohydrates are often quite involved and can require large quantities of sample. Many methods have recently evolved which take advantage of mass spectrometry to provide a maximum of information with a much lower requirement for sample. Additionally, information can be obtained on glycosylated and non-glycosylated peptides in the same experiment. This paper will focus on the application of electrospray mass spectrometry for the analysis of recombinant proteins produced in mammalian cell culture systems. Emphasis will be placed on strategies to optimize data obtained while minimizing sample preparation and isolation efforts. Examples will include confirmation of expected carbohydrate structures, as well as the use of ESI-MS in identifying unexpected structures. Also we will present results using a modified, miniaturized electrospray source which enhances quality of the data signal with lower sample amounts.