One of the biggest challenges that protein chemists face when performing protein LC separations is protein adsorption—the adherence of proteins to the surface of column components, such as frits and hardware. Protein adsorption can lead to decreased protein sensitivity, recovery issues, and high LC column backpressure.
If you suspect that protein adsorption is affecting your protein LC separation, try a column cleaning procedure in your HPLC protein purification protocol or peptide purification reversed phase HPLC method.
Start by flushing out buffer with 95-100 % water, then clean out the hydrophobic proteins with a gradient from 95 % water/5 % acetonitrile up to 5 % water/95 % acetonitrile over 3-5 column volumes.
The best way to eliminate protein adsorption is to use biocompatible UHPLC/HPLC column hardware. bioZen UHPLC/HPLC columns consist of a new titanium infused biocompatible hardware and frit, which eliminates unwanted secondary interactions that can cause carryover or recovery inconsistency.
Analysis of Adalimumab
Determination of monomer, dimer, multimer, and high order structure (quaternary structural determination of a mAb) is crucial to aggregate analysis and quantitation. Size exclusion chromatography (SEC) is the preferred technique for aggregate analysis.
One of the best ways to make sure that you have optimized your SEC method for mAbs is to make sure that you are using a well salted buffer to help prevent non-specific secondary interactions. A good starting point for SEC method development is to use a 50 mM monopotassium, 250 mM potassium chloride, and pH 6.8 buffer.
As a rule of thumb, make sure you are using an SEC particle with tight pore size distribution. bioZen SEC-2, and bioZen SEC-3 thermally modified fully porous particles are extremely inert and offer high efficiencies for UHPLC SEC separations. .
Herceptin—vcMMAE using bioZen 3.6 μm Intact XB-C8
The average drug antibody ratio (DAR) distribution can have implications on antibody-drug conjugates (ADC) efficiency, safety clearance, and pharmacokinetics. It is also necessary to characterize DAR as an "intentional chemical modification" performed on the protein.
Though different chromatography modes can be employed to determine DAR, reversed phase chromatography has proven to be highly effective. bioZen Intact XB-C8 is a large pore size, core-shell particle that allows for intact ADC to interact with the moderately retentive stationary phase while providing increased efficiency.
We thank ADC Biotechnology LTD for providing ADC samples for this application
2-AB Labeled Glycans from Standard Solution
Glycosylation of proteins—specifically monoclonal antibodies—have indications in protein structure, as well as efficacy, clearance, and immunogenicity.
Determination of glycoforms (glycolsylated proteins) can be performed either on the intact protein or enzymatically digested fragments (e.g. Fc/Fab) using HILIC-FLR or HILIC MS. bioZen Glycan provides higher order separation and faster elution windows for released and labeled glycans across HPLC and UHPLC systems.
Intact Mass Spectra NIST mAb
Intact mass analysis involves using high resolution accurate mass (HRAM) instruments to confirm sequence and identify post-translational modifications..
bioZen Intact C4 and Intact XB-C8 provide tight peak shapes and fast run times for intact mass, which provide indications not only of relative abundance of glycoforms, but also stability, as degraded mAbs will not give good charge envelope by ESI-MS.
Intact Analysis Trastuzumab at 70, 80, and 90 °C
Observation of intact protein and fragmented protein during impurity profiling is essential in biologic development. However, impurity profiling and characterization of intact biologic fragments is a challenging undertaking because of the need to identify very small differences between variants.
Using a reversed phase large pore core-shell particle, such as bioZen 3.6 μm Intact XB-C8 can provide narrow peak shapes and high resolution between the target HC/LC, Fc/Fab, or isoforms.
Peptide Map Cetuximab
Peptide mapping is commonly used for identification of non-enzymatic, spontaneous post translational modifications, such asdeamidation, oxidation, succinimide formation, pyroglutamate formation, and others.
Digested mAbs or ADCs, typically include a large body of compounds, which are crucial to understanding post translation modifications. Reversed phase is typically used to confirm the primary sequence and identify post translational modification.
Successful peptide mapping requires the use of reversed phase columns that can provide unique elution profiles based on post translational modifications. bioZen peptide columns are packed with thermally modified fully porous or core-shell particles, which provides high selectivity and sensitivity for post translational modifications.
Kadcyla (4 Signature Peptides)
Infliximab (3 Signature Peptides)
Peptide quantitation is used to deconvolute complex chromatograms by searching for specific peptide sequences. It can also analyze the peptides with high sensitivity and specificity, even at low levels of peptide.
When quantitating signature peptides from biological matrices, sharp peak shape and sufficient retention of hydrophilic peptides is necessary to prevent any signal loss from matrix suppression regions.
bioZenPeptide columns were developed to deliver excellent selectivity for even the closely related peptides, as well as basic peptides..