Waters Protein-Pak Hi Res HIC Column and HIC Protein Standard - Manual

[ CARE AND USE MANUAL ]
CONT ENTS
I. INT RODUCTION
a. Mobile Phase
b. Flow Direction
II. CONNECTING COLUMN TO LC SYST EM
III. COLUMN SPECIFICATIONS AND USE
a. Shipping Solvent
b. Max. Flow Rate
c. Max. Pressure
d. pH Range
e. Salt Concentration
f. Organic Concentration
g. Temperature
h. Column Washing and Regeneration
i. Column Storage
j. Column Protection
IV. USEFUL FUNCTIONAL T EST
a. Sample Preparation
b: Representative Chromatogram
Protein-Pak Hi Res HIC Column and HIC Protein Standard
I. INT RODUCTION
This offering contains non-porous, polymethacrylate-based
particles (2.5 μm) functionalized with a butyl ligand coating
and is well suited for the characterization of proteins and
biotherapeutics including monoclonal antibodies (mAb) and
antibody drug conjugates (ADC).
While reversed-phase chromatography is a frequently used
bioanalytical technique, HIC offers attractive orthogonal separation
advantages. In reversed-phase LC, proteins are retained by
hydrophobic interaction with alkyl groups (e.g., C18) on the packing
material. However, the butyl ligand density on Waters Protein-Pak
Hi Res HIC Column is comparatively less resulting in fewer protein-
ligand hyrdophobic interactions. Consequently, HIC-based elution
is possible using gradients of decreasing salt concentration at
physiological pH values. Use of denaturing organic solvent eluents
(e.g., acetonitrile in 0.1% TFA) thus allowing biotherapeutics (e.g.,
acid labile, cysteine-linked ADCs) to be analyzed in
non-denaturing conditions.
In addition, Waters has developed HIC Protein Standard Test
Mix (that we include with each shipped column) designed for
user verification of HPLC/UPLC instrument and Protein-Pak
Hi Res HIC column performance prior to sample analyses.
This HIC-based intact protein validation mix, when used on a
regular basis, helps monitor system and column performance
and is also highly valuable in method development and /or
troubleshooting. As indicated in Figure 1, the HIC protein
standard mix contains a carefully chosen set of six proteins
that provide good chromatographic representation using a
gradient of decreasing salt concentration.
.