MATERIALS AND METHODS
Materials
The three resins evaluated in this study (MabSelect, IgSelect, and Prosep-vA High Capacity) were purchased from their respective
vendors. All resins were packed to 20 cm bed height in 1 cm I.D. columns made by GE Healthcare. The four test proteins—Fc-fusion
proteins (A and B) and MAbs (C and D)—were expressed in Chinese hamster ovary (CHO) cells and produced at Bristol-Myers Squibb
(Syracuse, NY). Model proteins such as horse cytochrome c and human serum albumin (HSA) samples were purchased from Sigma-Aldrich
(St. Louis, MO). All other chemicals were purchased from Mallinckrodt Baker (Phillipsburg, NJ).
All chromatography experiments were carried out on an AKTAexplorer chromatographic system from GE Healthcare. High Performance
Liquid Chromatography (HPLC) analysis was performed using Waters (Milford, MA) 2695 Separation Module and Waters 2996 Photodiode
Array Detector. An Orbital Shaker 100 from ArmaLab (Bethesda, MD) was used for batch-adsorption experiments.
Methods
The elution pH of the various proteins was obtained by linear gradient experiments under analytical conditions using pulse
injection of the samples. A gradient of pH was run from 6.5 to 2.5 over 10-column volumes in citrate buffer. The elution pH
at peak maxima was calculated from the gradient and further verified from the effluent pH trace obtained from the online monitor
pH/C-900 that is part of the AKTA system. In the binding mechanism exploration test, different mobile phase modifiers were
added to citrate buffers at both pH values and an identical pH gradient was run for comparison with the original pH gradient.
Dynamic binding capacities were determined by performing breakthrough experiments at six minutes residence time. The column
was equilibrated and regenerated using typical Protein A process conditions. Adsorption isotherms for the proteins on the
various stationary phases were determined using batch experiments.
Selectivity of the three resins was compared under preparative condition using cell culture harvest material. Each resin was
loaded to ~80% of its dynamic binding capacity for the respective protein. Typical Protein A equilibration, elution, and regeneration
conditions were used for these experiments. Sample protein concentration was determined using an analytical Protein A assay.
Host cell protein levels in the samples from the preparative experiments were determined using an in-house host cell protein
ELISA assay.
THEORY
A Langmuir Isotherm, as defined by the following equation, was used to compare the binding thermodynamics of the three resins
in this study:
in which Q is the equilibrium concentration of the solute on the stationary phase (expressed in mg solute per mL column volume), C is the mobile phase solute concentration at equilibrium, Q
max is the maximum static binding capacity, and K is the affinity binding constant. The binding constant is thermodynamically representative of the protein's affinity to the
resin.
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