Abstract
Hydrophobic interaction chromatography (HIC) is commonly used to separate protein monomer and aggregate species in the purification of protein therapeutics. Despite being used frequently, the HIC separation mechanism is quite complex and not well understood. In this paper, we examined the separation of a monomer and aggregate protein mixture using Phenyl Sepharose FF. The mechanisms of protein adsorption, desorption, and diffusion of the two species were evaluated using several experimental approaches to determine which processes controlled the separation. A chromatography model, which used homogeneous diffusion (to describe mass transfer) and a competitive Langmuir binary isotherm (to describe protein adsorption and desorption), was formulated and used to predict the separation of the monomer and aggregate species. The experimental studies showed a fraction of the aggregate species bound irreversibly to the adsorbent, which was a major factor governing the separation of the species. The model predictions showed inclusion of irreversible binding in the adsorption mechanism greatly improved the model predictions over a range of operating conditions. The model successfully predicted the separation performance of the adsorbent with the examined feed.
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Abbreviations
- c 1 :
-
monomer concentration in solution (mg/mL)
- c 2 :
-
aggregate concentration in solution (mg/mL)
- D a :
-
axial dispersion coefficient (cm2/s) (Eq. 22)
- D eff :
-
effective diffusivity (cm2/s) (Eq. 16)
- D f :
-
molecular diffusivity in aqueous solution
- k 1,ad :
-
monomer adsorption rate constant (ml/mg/s)
- k 2,ad :
-
aggregate adsorption rate constant (ml/mg/s)
- k −1,de :
-
monomer desorption rate constant (1/s)
- k −2,de :
-
monomer desorption rate constant (1/s)
- k 1,irr :
-
monomer unfolding rate constant (ml/mg/s)
- k 2,irr :
-
aggregate unfolding rate constant (ml/mg/s)
- k f :
-
external film mass transfer coefficient (cm/s)
- K 1 :
-
monomer binding constant (mL/mg) (Eq. 5)
- K 2 :
-
aggregate binding constant (mL/mg) (Eq. 5)
- K 3 :
-
aggregate irreversible binding constant (mL/mg) (Eq. 23)
- K 1,irr :
-
monomer irreversible binding constant (mL/mg) (Eq. 5)
- K 2,irr :
-
aggregate irreversible binding constant (mL/mg) (Eq. 5)
- L:
-
bed length (cm)
- Pe :
-
Peclet number (−)
- q 1 :
-
monomer concentration in particle (bound in native state) (mg/mL)
- q 2 :
-
aggregate concentration in particle (bound in native state) (mg/mL)
- q 1,irr :
-
monomer concentration in particle (irreversibly bound) (mg/mL)
- q 2,irr :
-
aggregate concentration in particle (irreversibly bound) (mg/mL)
- q m,irr :
-
irreversible aggregate static capacity (mg/mL) (Eq. 23)
- q m :
-
static capacity (mg/mL)
- q m1 :
-
monomer static capacity (mg/mL)
- q m2 :
-
aggregate static capacity (mg/mL)
- r :
-
radial coordinate (cm)
- R :
-
particle radius (cm)
- u :
-
interstitial velocity (cm/h)
- u s :
-
sperficial velocity (cm/h)
- z :
-
bed length coordinate (cm)
- β:
-
surface area ratio between reversible and irreversibly bound species (−) (Eqs. 1, 2)
- ε:
-
extraparticle void fraction (−)
- ρ:
-
liquid density (g/cm3)
- τ:
-
fluid residence time (min)
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Acknowledgments
The authors would like to thank Joshua Walker and Phil Vilmorin (Biogen Idec Corporation) for preparation of the feed solution. The authors would also like to thank Doug Cecchini (Biogen Idec Corporation) and Mina Sierou (Comsol, Inc.) for useful technical discussions.
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McCue, J.T., Engel, P., Ng, A. et al. Modeling of protein monomer/aggregate purification and separation using hydrophobic interaction chromatography. Bioprocess Biosyst Eng 31, 261–275 (2008). https://doi.org/10.1007/s00449-008-0200-1
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DOI: https://doi.org/10.1007/s00449-008-0200-1