See also Methods used to separate antibody glycovariants See also Mass Spectrometry (MALDI TOF MS)

Detection and Characterization of product associated variants during the product of polypeptides (e.g., antibodies) is important because profiling of impurities is a regulatory requirement of the FDA. Product assocaited variants include not only truncated or elongated peptides but also peptides have different glycosylation than the desired glycosylation. Proudct assocaited variants may exhibit alterations in one or mroe of molecular mass (detected by size exclusion chromatgoraph), isoelectric point (.e.g, detected by isoelectric focusing), electrophoretic mobility (e.g., detected by gel electrophoresis), phosphorylation state (e.g., detected by mass spectrometry), charge to mass ration (e.g., detected by mass spectrometry), mass or identity of proteolytic fragments (e.g., detected by mass spectrometry or gell electrophoresis), hydrophobicity (e.g., detected by HPLC), charge (e.g., detected by ion exchange chromatgaphy), affinity (e.g., in the case of an antibody, detected by binding to protein A, protein G and/or an peitope to which the desired antibody binds) and glycosylation state (e.g., detected by lectin binding affinity). (Allison, US 14.215370).

Chromatography

Affinity chromatography

–Lectin Affinity Chromatography:

Allison (US 14/215370) discloses culturing yeast cells to epxress a desired recombinant polypeptide, periodically obtaining one or more samples of the fermentation medium and detecting the amount and/or type of glycosylated impurities in the sample using a lectin that binds to the glycosylated impurities (e.g., ConA, LCH, GNA, DC-SIGN) and based on the amount of detected glycosylated impurities modifying one or more of the oeprating paraters or conditions of the fermentation and finally pooling different samples, eluates or fractions containing the desired recombinant antibody polypeptide from the same or different fermenation processes.  In one embodiment, the lectin is conjugated to a probe such as biotein, alkaline phosphatase or horseradish peroxidase and then immobilized to a support such as avidin. Standard protein-protein interaction monitoring process such as (e.g., surface plasmon resonance, ELISA dynamic light scattering) can be used to analyze the interaction between lectin and glycosylation impurities in samples from various steps of the purificaiton process.

HPLC: 

Normal pahse HPLC is an established technique for separating mixtures of oligosaccharides. The earliest methdos sued columns with diethylaminoethyl functional groups to separate neutral and acidic oligosaccharides. More recently, colums with imide and amide functional groups ahve been used. Oligosaccharides applied to a silica matrix with amide functional groups have been successively and predictably eluted with acetonitrile water gradients buffered with volatile sales. These solvent systems exploit the subtle differences in hydrophilicity between individual sugars and thus their affinity for the column matrix produces high resolution. (Guile, Analytical Biochemistry 210-220, 1996).

Mass Spectrometry

Electrospray-ionization mass spectrometry (ESI-MS): is a well established tool for biotherapeutic analysis. It draws intact proteins or peptide ions into the vacuum of a mass spectrometery, wehre the ion mass is measured. ESI-IMS introduces ions into a low pressure gas, wehre the effects of aerodynamic drag reveal their shape. One promising application for ESI-IMS is to generate biphysical “fingerprints” for comparing biosimilars with innovator drugs. 

MALDI TOF MS

Structural characterization of individual oligosaccharides has been proven to be much more challenging than for other biopolymers due to its branched and isomeric nature. Techniques based on mass spectrometry (MS) have been used extensively and successfuly for the characterization of glycans. Matrix-assisted laster desorption/ionization time-of-flight (MS) (MALDI TOF MS) has been the tool of choice because it generally produces singly charged ions, yielding simple spectra and making interpretation straightforward. (Qian, Anal. Biochem. 364 (2007) 8-18).