Modeling of protein monomer/aggregate purification and separation using hydrophobic interaction chromatography

  • Justin T. McCue
  • Philip Engel
  • Austen Ng
  • Rich Macniven
  • Jörg Thömmes
Original Paper

DOI: 10.1007/s00449-008-0200-1

Cite this article as:
McCue, J.T., Engel, P., Ng, A. et al. Bioprocess Biosyst Eng (2008) 31: 261. doi:10.1007/s00449-008-0200-1

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.

Keywords

Hydrophobic interaction chromatography Preparative chromatography Diffusivity Irreversible binding 

List of symbols

c1

monomer concentration in solution (mg/mL)

c2

aggregate concentration in solution (mg/mL)

Da

axial dispersion coefficient (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 film mass transfer coefficient (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

monomer concentration in particle (irreversibly bound) (mg/mL)

q2,irr

aggregate concentration in particle (irreversibly bound) (mg/mL)

qm,irr

irreversible aggregate static capacity (mg/mL) (Eq. 23)

qm

static capacity (mg/mL)

qm1

monomer static capacity (mg/mL)

qm2

aggregate static capacity (mg/mL)

r

radial coordinate (cm)

R

particle radius (cm)

u

interstitial velocity (cm/h)

us

sperficial velocity (cm/h)

z

bed length coordinate (cm)

Greek symbols

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)

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Justin T. McCue
    • 1
  • Philip Engel
    • 1
  • Austen Ng
    • 1
  • Rich Macniven
    • 1
  • Jörg Thömmes
    • 1
  1. 1.Biogen Idec CorporationBioprocess DevelopmentCambridgeUSA

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