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Removal of Protein A by anion exchange chromatography on Capto™ Q in the flow-through mode

IP.com Disclosure Number: IPCOM000078040D
Publication Date: 2005-Feb-25
Document File: 2 page(s) / 111K

Publishing Venue

The IP.com Prior Art Database

Abstract

The purpose of this study was to evaluate Capto™ Q as a second step in a MAb purification process in the flow-through mode. The aim was to reduce the levels of Protein A while maintaining a high yield of the MAb. Capto™ Q is a new anion-exchange medium based on a rigid agarose base matrix developed for capture and intermediate purification. More specifically, Capto™ Q comprises strong anion exchange (Q) groups coupled to a crosslinked agarose matrix via dextran An IgG4 MAb, from clarified NS0 supernatant, purified on MabSelect™ Xtra was used as sample. pl of the MAb is 6.5-7.5. Protein A content and MAb concentration was determined in the starting material as well as the collected fractions. The yield of MAb was high and the Protein A removal was complete. A Tricorn 5/50 was packed with Capto™ Q to a bed height of 50 mm and used for the purification of the MAb.

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Removal of Protein A by anion exchange chromatography on Capto™ Q in the flow-through mode

The purpose of this study was to evaluate Capto™ Q as a second step in a MAb purification process in the flow-through mode. The aim was to reduce the levels of Protein A while maintaining a high yield of the MAb. Capto™ Q is a new anion-exchange medium based on a rigid agarose base matrix developed for capture and intermediate purification. More specifically, Capto™ Q comprises strong anion exchange (Q) groups coupled to a crosslinked agarose matrix via dextran

An IgGMAb, from clarified NS0 supernatant, purified on MabSelect™ Xtra was used as sample. pl of the MAb is 6.5-7.5.  Protein A content and MAb concentration was determined in the starting material as well as the collected fractions. 

The yield of MAb was high and the Protein A removal was complete.

A Tricorn 5/50 was packed with Capto™ Q to a bed height of 50 mm and used for the purification of the MAb. Chemicals and equipment used are shown in Table 1 and the buffers used for the chromatography are shown in Table 2. Table 3 shows the chromatography method used.

Table 1. Chemicals and equipment used.

Chemicals

2 (N-morpholino) ethane sulfonic acid (MES)

Sigma

 

Ethanol, 99-100%

Kemetyl

 

Hydrocloric acid (HCl)

Merck

 

Sodium Cloride (NaCl)

Merck

 

Sodium Hydroxide (NaOH)

Merck

Equipment

ÄKTAexplorer 10

Amersham Biosciences

 

Syringe filters (Filtropur S)

Sarstedt

Table 2. Buffers used for the chromatography experiments.

Equilibration and wash

50 mM MES pH 6.8

Elution and reequilibration

50 mM MES + 1M NaCl pH 6.8

Table 3. Chromatography method used for the removal of Protein A.

Step

Amount

Equilibration

9 CV

Sample load

70 mg MAb

Wash

10 CV

Elution

7 CV

CIP

10 CV

Re-equilibration

4 CV

 

The pH of the material from MAbSelect™ Xtra was adjusted with HCl. When adjusting the pH, the pI for the MAb was passed which lead to a minor protein precipitation that was removed by passing the sample through a 0.45 µm syringe filter. After the filtration the MAb concentration was determined.

The flow was 1 mL/min during all chromatography steps except for the CIP-step, where the flow was 0.17 mL/min. The CIP was performed with 1 M NaOH using reversed flow. 70 mg of the MAb sample was applied to the column in each experiment.

The MAb and Protein A concentration was determined in the starting material, flow through, wash and eluate.

The concentration of MAb was determined spectrophotometrically, in triplicate at 280 nm, u...