What is a Glycoprotein?
Complex biopharmaceuticals drugs such as antibodies and enzymes are proteins and can contain 2 to 30% carbohydrate moiety attached to the protein via asparagine residues (N-linked) or serine/threonine residues (O-linked). The process of glycosylation is a complex post-translation modification process and multiple types of sugars are covalently bound, with multiple attachment sites and depending on the cells/clones and cell culture conditions, these can result in subtle differences in the glycosylation patterns. Glycoproteins have a high degree of heterogeneity and micro heterogeneity and exist in subsets of glycoforms. Glycoforms have different physico-chemical properties and may have biochemical and functional diversity.
Purification of Glycoproteins.
m-Aminophenyl boronic acid coupled to agarose base matrix is the most widely used adsorbent for the purification of Glycoproteins. Biological compounds with sugar moieties such as glycoproteins bind to the ligand and are recovered using appropriate elution conditions. Applications of Boronate affinity chromatography adsorbent include purification of nucleosides, nucleotides, catecholamine, t-RNA and envelop viruses.
The most important application the adsorbent is as a general method for separation and purification of glycosylated from non-glycosylated proteins and often from other forms of glycosylated proteins. For optimal performance, the chemi-selective interaction of glycosylated proteins should be optimised while minimising any non-selective interaction with the adsorbent by careful control of electrostatic and cis-diol interactions. Specific binding step is carried out at high pH and moderately high ionic strength to suppress the ion-exchange properties of negatively charged boronate moiety of the ligand. Use of too high ionic strength may increase hydrophobic interactions between the target protein and the phenyl group of the phenylboronic acid ligand. The use of boric acid buffers and or addition of magnesium cation at low concentrations in the binding buffer to suppress the boronate charge is an alternative means for reducing the non-specific hydrophobic interactions.
Prometic Bioseparations’ Aminophenylboronate affinity products have been optimised for use in various bioseparation applications. All products share the same robust ligand and coupling chemistry. Aminophenylboronate products are available based on two different beaded agarose materials. Aminophenylboronate P6XL utilises PBL’s proprietary Purabead® P6XL near-monodisperse® agarose beads to produce a highly robust cross-linked agarose beads. Purabead products have very uniform particle size which enables reproducible column packing and high flow rates at low back-pressure. Consequently, Aminophenylboronate P6XL is the adsorbent of choice for bioprocess applications. Aminophenylboronate A6XL is PBL’s original product with a wider particle size distribution. This product is better suited to small scale or diagnostic applications.
Key features of Aminophenylboronate:
- Affinity purification and removal of glycoproteins and glycans
- High purity m-aminophenylboronic acid ligand
- Ligand binds selectively to carbohydrate groups with cis-diols
- High dynamic binding capacity
- Robust, long life adsorbent
- Sanitisable with NaOH allowing Multiple cycles
- Highly reproducible batch-to-batch manufacture to ISO 9001 standard
- Supported with comprehensive Regulatory Support Files
- Multiple end users in regulated bio- pharmaceutical manufacturing and medical diagnostics