Performing a separation with HiTrap Protein G HP

Extracted from Affinity Chromatography Principles and Methods, GE Healthcare, 2007

Column: HiTrap Protein G HP, 1 ml or 5 ml

Recommended flow rates: 1 ml/min (1 ml column) or 5 ml/min (5 ml column)

Binding buffer: 0.02 M sodium phosphate, pH 7.0

Elution buffer: 0.1 M glycine-HCl, pH 2.7

Neutralization buffer: 1 M Tris-HCl, pH 9.0

Centrifuge samples (10 000 g for 10 minutes) to remove cells and debris. Filter through a 0.45 µm filter. If required, adjust sample conditions to the pH and ionic strength of the binding buffer either by buffer exchange on a desalting column or by dilution and pH adjustment (see page 133, Buffer exchange and desalting for affinity chromatography).

  1. Equilibrate column with 5 column volumes of binding buffer.
  2. Apply sample.
  3. Wash with 5–10 column volumes of the binding buffer to remove impurities and unbound material. Continue until no protein is detected in the eluent (determined by UV absorbance at 280 nm).
  4. Elute with 5 column volumes of elution buffer*.
  5. Immediately re-equilibrate with 5–10 column volumes of binding buffer.

*Since elution conditions are quite harsh, it is recommended to collect fractions into neutralization buffer (60 µl – 200 µl 1 M Tris-HCl, pH 9.0 per ml fraction), so that the fnal pH of the fractions will be approximately neutral.

IgGs from most species and subclasses bind to protein G at near physiological pH and ionic strength. For the optimum binding conditions for IgG from a particular species, it is worth consulting the most recent literature. Avoid excessive washing if the interaction between the protein and the ligand is weak, since this may decrease the yield.

Most immunoglobulin species do not elute from Protein G Sepharose until pH 2.7 or less. If biological activity of the antibody or antibody fragment is lost due to the low pH required for elution, try Protein A Sepharose: the elution pH may be less harsh.

Desalt and/or transfer purifed IgG fractions to a suitable buffer using a desalting column (see page 133, Buffer exchange and desalting for affinity chromatography).

Reuse of Protein G Sepharose depends on the nature of the sample and should only be considered when processing identical samples to avoid cross-contamination.

To increase capacity, connect several HiTrap Protein G HP columns (1 ml or 5 ml) in series. HiTrap columns can be used with a syringe, a peristaltic pump or connected to a liquid chromatography system, such as ÄKTAprime plus. For greater capacity pack a larger column with Protein G Sepharose 4 Fast Flow (see Appendix 3, Column packing and preparation for affinity chromatography).

MAbTrap Kit

MAbTrap Kit, ready for use

Fig. 13. MAbTrap Kit, ready for use.

MAbTrap Kit contains a HiTrap Protein G HP 1 ml column, stock solutions of binding, elution and neutralization buffers, a syringe with fttings and an optimized purifcation protocol, as shown in Figure 13. The kit contains suffcient material for up to 20 purifcations of monoclonal or polyclonal IgG from serum, cell culture supernatant or ascitic fluid, using a syringe. The column can also be connected to a peristaltic pump, if preferred. Figure 14 shows the purifcation of mouse monoclonal IgGfrom cell culture supernatant with syringe operation and a similar purifcation with pump operation. Eluted fractions were analyzed by SDS-PAGE as shown in Figure 15.

Purifcation of mouse monoclonal IgG1 from cell culture supernatant

Fig. 14. Purifcation of mouse monoclonal IgG1 from cell culture supernatant. A. with syringe operation. B. with pump operation.

 SDS-PAGE on PhastSystem using PhastGel 10–15, non-reduced, and silver staining

Fig. 15. SDS-PAGE on PhastSystem using PhastGel 10–15, non-reduced, and silver staining.

Performing a separation with MAbTrap Kit

Column: HiTrap Protein G HP, 1 ml

Recommended flow rate: 1 ml/min

Binding buffer: Dilute buffer concentrate 10-fold

Elution buffer: Dilute buffer concentrate 10-fold

Neutralization buffer: Add 60–200 µl of neutralization buffer per ml fraction to the test tubes in which IgG will be collected

Centrifuge samples (10 000 g for 10 minutes) to remove cells and debris. Filter through a 0.45 µm flter. If required, adjust sample conditions to the pH and ionic strength of the binding buffer either by buffer exchange on a desalting column (see page 133, Buffer exchange and desalting for affinity chromatography) or by dilution and pH adjustment.

Using HiTrap Protein G HP with a syringe

Fig. 16. Using HiTrap Protein G HP with a syringe. A: Dilute buffers and prepare sample. Remove the column’s top cap and twist off the end. B: Equilibrate the column, load the sample and begin collecting fractions. C: Wash and elute, continuing to collect fractions.

  1. Allow the column and buffers to warm to room temperature.
  2. Dilute the binding and elution buffers.
  3. Connect the syringe to the column using the luer adapter supplied.
  4. Equilibrate the column with 5 ml distilled water, followed by 3 ml diluted binding buffer.
  5. Apply the sample.
  6. Wash with 5–10 ml diluted binding buffer until no material appears in the eluent.
  7. Elute with 3–5 ml diluted elution buffer. Collect fractions into tubes containing neutralization buffer.
  8. Immediately re-equilibrate the column with 5 ml diluted binding buffer.

Media Characteristics

  Ligand density Composition pH stability* Mean particle size
HiTrap Protein G HP (MAbTrap Kit) 2 mg/ml Ligand coupled to Sepharose HP by N-hydroxysuccinimide activation (gives stable attachment through alkylamine and ether links). Long term 3–9 Short term 2–9 34 µm
Protein G Sepharose 4 Fast Flow 2 mg/ml Ligand coupled to Sepharose 4 Fast Flow by cyanogen bromide activation. Long term 3–9 Short term 2–10 90 µm

* Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.

Chemical Stability

Stable in all common aqueous buffers.

Storage

Wash media and columns with 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.

Materials

     

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