See also Lectin Affinity Chromatography under Affinity Chromatography See also antibody glycosylation in “biochemistry” under “glycosylation” See also Characterization and Detection of Glycosylation
The potential for significant changes in the biological activities of recombinant glycoproteins dpending on the system used for their expression is a real concern from a pharmacological standpoint. Changes in the glycosylation pattern may adversely affect the activity, serum half-life or immunogenicity of the recombinant protein. (Corai, Biotechnol. Prog. 27(1) 220-231 (2011)
A glycated variant is an antibody to which a sugar, such as glucose, has been covalentlly attached. This addition can occur by reaction of glucose with a lysine residue on the protein (e.g., in cell culture media). A glycated variant can be identified by mass spectrometry analysis of the reduced antibody evaluating the increase in mass of heavy or light chains. A glycated variant can also be quantified by boronate chromatgoraphy. A glycated variant differs form a glycosylation variant (Harris, US2009/0202546).
A glycosylation variant is an antibody with one or mroe carbohydrate moeities attached thereto which differ from one or more carbohydrate moieties attached to a main species antibody. (Harris, US2009/0202546).
IgGs are required to be N-glycosylated in the CH2 domain of the Fc to exhibit effector functions including ADCC and CDC. This is because fc glycosylation impacts antibody binding to Fc recepetors and complement activating protein, C1q. (Nti-Gyabaah, US 14/355014)
Methods Used to Separate Glycoforms
Use of Fc receptors for isolating isoforms:
Bolton (US2013/0084648) discloses methods for seperating polypeptide glycoforms using a meidum that includes an Fc receptor such as an extracellular protion of an Fc gamma RIII receptor. The invention is based on the fact that certain antibdy glycoforms have been observed to have a higher affinity for Fc receptors on leukocytes such as Fc gamma RI, Fc gamma RII, Fc gamma RIII, and C1q, which in turn can alter effector functions. Antibodies with oligomannose type ogligosaccharides display enhanced ADCC and reduced C1q binding. Terminal sialic acids have been shown to reduce the affinity of antibodies for Fc gamma recptors. Antibody forms lacking fucose on the primary core N-acetylglucosamine have increased affinity for Fc gamma RIIIa as compared to core-fucosylated forms and also have an incdreased ability to trigger ADCC. Afucosylated forms have comparable affinity for antigen, C1q, Fc gamma RI, the neonatal Fc recptor (FcRn) and slightly higher affinity for Fc gamma RIIa and Fc gamma RIIb, as compared to fucosylated forms. In some embodiments, oligosaccharides on the polypeptides are analyzed after separation.
(Freimoser-Grundschober, US14/352411) disclose the use of immobilized Fc receptors such as FcRyIIIa to separate differently fucosylated antibodies from an antibody pool.
Lectin Affinity Chromatography: See also affinity chromatography
Naso (US2010/0172911) discloses enrichment of sialyated forms of Fc-containing proteins by passing sublots of a particular Fc containing protein that differs in sialic acid content and passing it over a column containing an immobilized lectin that has differential affinity for sialyated and asialylated oligosaccharides. The nonbinidng flow through or the column unbound fraction can be separated form the bound fraction and the latter collected by passing elution buffer through the column. Depending on the lectin used, the nonbinding fraction may have a higher or lower sialic acid content than the fraction that binds. Examples of lectins that may be used to enrich for sialyated or askylated Fc containing proteins are the lectin from Maackia amurensis (MAAA) which specifically binds oligosaccharides with terminal sialic acid and the lecitn what germ agglutnin (WGA) which specifically binds oligosaccharides with either terminal sialic acid or terminal N-acetylglucosamine (GlcNAC). Another example is the lectin Ricin I (RCA) which binds ogligosaccharides with terminal galactose.
Allison (US14/215370) discloses a method or purifying recombinant polypeptides such as antibodies which have been produced in yeast or filamentous fungal cells using chromatography techniques such as Affintiy-Mixed Mode-HIC which includes lectin binding assays (using a lectin that binds to the glycosylated impurities such as a glycovariant of the antibody) to monitor for glycosylated impurities.
Tojo (Biol. Pharm. Bull. 32(9) 1604-1608 (2009) disclses a method of purifying antibody fractions containing predominantly non-fucosylated species by combining two column chromatography techniques, using columns of Concanavalin A (Con A) which preferentially binds to high mannose oligosaccharides and LCA, which prreferentially binds to fucose containing oligosaccharides.
Glycan targeting antibodies
Glycan targeting antibodies recognizing a specific carbohydrate structure have been used to enrich for specific glycan structure. (Freimoser-Grundschober, US14/352411)
Overload (“displacement chromatography”):
Ladiwala (US13/885446 or US1013/0331554) discloses the use of a strong anion exchange such as TMAE for the enrichment of sialic acid levels in an Fc biological product. Higher sialyated glycoroms have a higher net negative charge and binding affinity to the TMAE and compete for binding sites with the lower affinity lesser sialyated and non-sialyated glycoroms, thus displacing these lower affinity species. Subsequently, the column is eluted to recvoer the enriched higher sialyated glycoforms in the product pool