In-process glycoprofiling during recombinant therapeutic glycoprotein production

DESCRIPTION (provided by applicant): The biologics market is the most rapidly growing sector of the pharmaceutical industry, and the US Food and Drug Administration (FDA), as well as the European Medicines Agency (EMEA), requires that the glycoprofiles ofall such therapeutic glycoproteins and antibodies be extensively characterized and fall within the specifications defined at the time of licensure. Currently, glycoprofiling of cell culture material involves extensive sample purification followed by massspectrometric (MS) analysis, often requiring one or more weeks for completion. These long lead times make corrective action during cell cultivation impossible and the only recourse to specification failures is lot rejection, costing between 5- 50M. The availability of robust, rapid, and simple analytical methods for glycoprofiling would fundamentally alter biopharmaceutical RandD and commercial manufacturing. We will develop an innovative approach capable of in-process glycoprofiling based on combining multiplexed suspension array (MSA) technology with glycan-specific reagents. Our GlycoProf MSATM approach will provide a rapid, cost-effective, and easy to operate method for monitoring protein terminal glycosylation patterns. It will serve as a critical early warning system to protect against lot rejection. In addition, it will assist in the optimization of process development conditions. In the GlycoProf MSATM approach, flow cytometry is used to detect binding between glycans and glycan- specific reagents that are conjugated to spectrally-unique microspheres. By combining the individual reagents into a multiplexed array, the entire analysis can be obtained in less than a minute on a basic cytometer. The glycoprofile is provided by the relative fluorescence intensities for each specific glycan-MSA interaction. The GlycoProf MSATM approach will not supplant full characterization of a final product, but will address the unmet need for rapid glycoprofiling during production. We will establish optimal performance metrics for the GlycoProf MSATM method using glycans that display eukaryotic terminal sequences. Additionally, we will establish the ability of the GlycoProf MSATM method to reproduce the glycoprofiles for glycoproteins in which the glycan chains have beenengineered to exhibit uniform glycosylation patterns. Finally, we will determine the glycoprofile of a therapeutic glycoproteins and compare to the results from an independent analysis. PUBLIC HEALTH RELEVANCE: Therapeutic glycoproteins (biologics)such as antibodies address critical health care needs that are currently unmet by small molecule drugs, and they constitute the most rapidly growing sector of the pharmaceutical industry. Both the protein and glycan content of biologics must be extensively characterized according to FDA guidelines. To overcome the laborious and time-consuming methods currently employed for glycoprofiling, we are developing a rapid, robust, and cost-effective method that has the advantage of being suitable for application during biologic production.