doi:10.1016/j.chroma.2007.03.016 
Copyright © 2007 Elsevier B.V. All rights reserved.
Application of a chromatography model with linear gradient elution experimental data to the rapid scale-up in ion-exchange process chromatography of proteins
Takashi Ishiharaa, 
, 
, Toshihiko Kadoyaa and Shuichi Yamamotob
aCMC R&D Laboratories, Pharmaceutical Division, Kirin Brewery Co. Ltd., Takasaki, Gunma 370-0013, Japan
bDepartment of Chemical Engineering, Yamaguchi University, Tokiwadai, Ube 755-8611, Japan
Available online 15 March 2007.
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Abstract
We applied the model described in our previous paper to the rapid scale-up in the ion exchange chromatography of proteins, in which linear flow velocity, column length and gradient slope were changed. We carried out linear gradient elution experiments, and obtained data for the peak salt concentration and peak width. From these data, the plate height (HETP) was calculated as a function of the mobile phase velocity and iso-resolution curve (the separation time and elution volume relationship for the same resolution) was calculated. The scale-up chromatography conditions were determined by the iso-resolution curve. The scale-up of the linear gradient elution from 5 to 100 mL and 2.5 L column sizes was performed both by the separation of β-lactoglobulin A and β-lactoglobulin B with anion-exchange chromatography and by the purification of a recombinant protein with cation-exchange chromatography. Resolution, recovery and purity were examined in order to verify the proposed method.
Keywords: Proteins; Ion-exchange chromatography; Separation; Chromatography models
Fig. 1. Typical elution curves of linear gradient elution and GH–IR curves with anion-exchange gel column. (A): Elution curves of β-lactoglobulin A (LgA) and β-lactoglobulin B (LgB), u = 9.53 cm/min, GH = 0.0126, Z = 10 cm, IR: peak salt concentration. (B): GH–IR curves of LgA and LgB. Column: Q Sepharose HP (10 cm × 0.8 cm I.D., Vt = 5 mL), mobile phase pH 5.6, sample: 5 mL β-lactoglobulin (1 mg/mL) solution.
Fig. 2. Typical elution curves of linear gradient elution and GH–IR curves with cation-exchange gel column. (A): Elution curves of recombinant protein (recP) and impurities (X, Y, Z), u = 3.03 cm/min, GH = 0.00472, Z = 10 cm, IR: peak salt concentration. (B): GH–IR curves of recP and X, Y, Z. Column: SP Sepharose HP (10 cm × 0.8 cm I.D., Vt = 5 mL), mobile phase pH 8.0, sample: 5 mL SP load solution (1 mg/mL).
Fig. 3. Scale-up studies for the separation of β-lactoglobulin A (LgA) and β-lactoglobulin B (LgB) with dimensionless parameter O. (A): (HETP)LGE and u relationships of LgB. (B): Iso-resolution curves of LgB. Column: Q Sepharose HP, mobile phase pH 5.6. The open circle represents the data for the base-case condition. The closed rhombic point represents one of the scale-up chromatography conditions chosen at Z = 3.5 cm. The closed circle represents one of the scale-up chromatography conditions chosen at Z = 10 cm.
Fig. 4. Elution curves of β-lactoglobulin A (LgA) and β-lactoglobulin B (LgB) with anion-exchange chromatography in a scale-up study with dimensionless parameter O. (A) Base-case conditions: Vt = 5 mL, u = 4.85 cm/min, GH = 0.0204, Z = 10 cm, (B) 100 mL scale-up conditions: Vt = 100 mL, u = 3.07 cm/min, GH = 0.0100, Z = 3.5 cm, (C) 2.5 L scale-up conditions: Vt = 2.5 L, u = 7.24 cm/min, GH = 0.0155, Z = 10 cm. Column: Q Sepharose HP, mobile phase pH 5.6, sample load: 1 mg protein/resin mL. The O values in (A), (B) and (C) were set to be equal. Note that because HETP is not proportional to u (u·GH), its values are not equal in (A), (B) and (C).
Fig. 5. Scale-up studies for the purification of recombinant protein (recP) with dimensionless parameter O. (A): (HETP)LGE and u relationships of recP. (B): Iso-resolution curves of recP. Column: SP Sepharose HP, mobile phase pH 8.0. The open circle represents the data for the base-case conditions. The closed circle represents one of the scale-up chromatography conditions chosen.
Fig. 6. Elution curves of recombinant protein (recP) with cation-exchange chromatography in a scale-up study with dimensionless parameter O and silver-stained SDS-PAGE analysis of purified recP under non-reducing conditions. (A) Base-case conditions: Vt = 5 mL, u = 4.53 cm/min, GH = 0.00315, Z = 10 cm, (B) 2.5 L scale-up conditions: Vt = 2.5 L, u = 7.58 cm/min, GH = 0.0024, Z = 10 cm. Column: SP Sepharose HP, mobile phase pH 8.0, sample load: 1 mg protein/resin mL. X, Y, Z: impurity X, impurity Y, impurity Z. The O values in (A) and (B) were set to be equal. Note that because HETP is not proportional to u (u·GH), its values are not equal in (A) and (B). (C) Lanes 1 and 4: molecular weight standards. Lane 2: base-case (Vt = 5 mL). Lane 3: 2.5 L scale-up (Vt = 2.5 L). Note: 2 μg/lane except molecular weight standard.
Table 1.
Parameter values obtained by linear gradient elution data analysis
A: KeΛB, Ke: equilibrium association constant, Λ: total ion exchange capacity, B: number of sites (charges) involved in protein adsorption, LgA: β-lactoglobulin A, LgB: β-lactoglobulin B, recP: recombinant protein.
Table 2.
Base-case and scale-up chromatography conditions for Q Sepharose HP chromatography
LgB: β-lactoglobulin B, u: linear mobile phase velocity, Z: column length velocity.
Table 3.
Base-case and scale-up chromatography conditions for SP Sepharose HP chromatography
recP: recombinant protein, u: linear mobile phase velocity, Z: column length.
Abstract
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