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Bioprocess Biosyst Eng (2008) 31:261275 DOI 10.1007/s00449-008-0200-1
ORIGINAL PAPER
Modeling of protein monomer/aggregate puricationand separation using hydrophobic interaction chromatography
Justin T. McCue Philip Engel Austen Ng Rich Macniven Jrg Thmmes
Received: 19 November 2007 / Accepted: 3 January 2008 / Published online: 19 January 2008 Springer-Verlag 2008
Abstract Hydrophobic interaction chromatography (HIC) is commonly used to separate protein monomer and aggregate species in the purication 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.
Keywords Hydrophobic interaction chromatography
Preparative chromatography Diffusivity
Irreversible binding
List of symbolsc1 monomer concentration in solution (mg/mL)
c2 aggregate concentration in solution (mg/mL) Da axial dispersion coefcient (cm2/s) (Eq. 22)
Deff effective diffusivity (cm2/s) (Eq. 16)
Df molecular diffusivity in aqueous solution k1,ad monomer adsorption rate constant (ml/mg/s)
k2,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)
k1,irr monomer unfolding rate constant (ml/mg/s) k2,irr aggregate unfolding rate constant (ml/mg/s) kf external lm mass transfer coefcient (cm/s)
K1 monomer binding constant (mL/mg) (Eq. 5) K2 aggregate binding constant (mL/mg) (Eq. 5) K3 aggregate irreversible binding constant
(mL/mg) (Eq. 23)
K1,irr monomer irreversible binding constant(mL/mg) (Eq. 5)
K2,irr aggregate irreversible binding constant(mL/mg) (Eq. 5)
L bed length (cm)
Pe Peclet number (-)q1 monomer concentration in particle
(bound in native state) (mg/mL) q2 aggregate concentration in particle
(bound in native state) (mg/mL) q1,irr...