Patent Application
20080029455
In each of the figures conductivity (mS/cm) and UV absorbance at 280 nm (mAu) of eluted fractions are plotted against time (min).
The following methods are for the purification of basic proteins (high pI). Resin types (charge) would be reversed to separate acidic proteins.
A running buffer (mobile phase) is selected with a pH below the pI of the protein so that the protein carries a positive charge. Purification of the protein is achieved using a negatively charged sugar polymer carboxymethyl beta-cyclodextrin and a positively charged chromatography matrix (anion exchanger). Traditionally it would not be possible to retain a positively charged protein on a positively charged matrix. However, in this method, a solution of a negatively charged sugar polymer is injected through the column prior to introduction of the protein solution. The sugar polymer binds to the chromatography matrix, temporarily derivatising the surface. This derivatised surface is capable of hydrophobic interaction with protein molecules. The protein(s) to be separated are then injected through the column. An elution is performed. The elution method can be an isocratic elution (no salt gradient) in which retention time on the column is determined by the strength of the hydrophobic protein—sugar polymer interactions. A protein separation is achieved, the most hydrophobic protein is eluted last from the column. Thus a separation based on protein hydrophobicity is achieved using an ion-exchange matrix. Alternatively a salt gradient may be used for the elution. The elution may also involve the use of one or more other compounds (e.g. urea, methanol, ethanol, isopropyl alcohol, guanidine hydrochloride or acetonitrile) to modulate the hydrophobic interaction of the sugar polymer with protein.
A 1 ml Q Sepharose anion exchanger was employed to separate the protein lysozyme from acetone in aqueous solution. The process was operated as described in method 1 using a salt gradient to elute the protein and sugar polymer.
Lysozyme has a molecular weight of 14 kDa and an isoelectric point of 11.3. The running buffer used was 100 mM Tris, 1 mM EDTA at pH 8.0 (buffer 1). Therefore, both the protein and the chromatography stationary phase were positively charged under the process conditions. Charged molecules bound to the anion exchange resin were removed by elution using a salt gradient. This was performed by mixing buffer 1 with buffer 2 (as buffer 1 plus 1 M NaCl) in gradually increasing ratios of buffer 2.
The column was loaded with 1 ml samples for each of the six runs shown. The samples contained 0.25% v/v acetone, 0.5 mg/ml lysozyme or a mixture of 0.25% v/v acetone, 0.5 mg/ml lysozyme. All samples were dissolved in buffer 1. Each sample was run on the Q Sepharose column with a flow rate of 1 mil/min. The experiments were performed by first running each of the acetone, lysozyme and mixed acetonellysozyme samples individually down the underivatised column. This process was repeated using the same column, but using a derivatisation step to derivatise the column with sulfated-beta-cyclodextrin before running each sample down the column. To derivatise the column the sulphated beta-cyclodextrin was loaded onto the column by injecting 25 ml of buffer 1 containing 4 mg/ml of the sulphated beta-cyclodextrin at a flow rate of 4 ml/min. The sugar polymer used, sulphated beta-cyclodextrin sodium salt (Aldrich, #38,915-3), is negatively charged.
Elution of acetone and lysozyme was monitored continuously by detecting UV absorbance at 280 nm (mAu) of the elute. Lysozyme and acetone absorb UV light at 280 nm. Sulphated beta-cyclodextrin does not absorb UV light at 280 nm sufficiently strongly to affect the UV signals measured. Conductivity was measured in mS/cm. The conductivity of buffer 1 was around 8 mS/cm and of buffer 2 was around 80 mS/cm.
Under normal operation, (i.e. without derivatisation of the anion exchange resin), lysozyme cannot be retained on an anion exchange column as both the column and protein have the same charge. Since the flow rate is 1 ml/mn and the column volume is 1 ml with around 50% voidage, material that cannot bind to the chromatography stationary phase begins to elute approximately 0.5 min after injection (
All three samples run on the underivatised column eluted rapidly without binding to the column (
When the lysozyme sample was injected onto the derivatised column, no increase in UV absorbance was detected in the 0 to 5 minute range, as the protein was retained on the derivatised column (
In the final run, a sample containing a mixture of acetone and lysozyme was found to be separated efficiently on the derivatised column. The acetone passed through the column, but the lysozyme protein was retained until it was eluted from the column using a salt gradient. These results demonstrate that charged amphipathic sugar polymer—protein interactions can be used in ion-exchange chromatography to achieve separation.
In this method a positively charged amphipathic sugar polymer (e.g. an amino beta-cyclodextrin or an aminoalkyl inulin) and a negatively charged chromatography matrix (cation exchanger) are used. The column is derivatised with a pulse of the positively charged sugar polymer in aqueous solution. A solution containing the positively charged weakly hydrophobic protein is then injected through the column. This method is mixed mode, that is protein—matrix interactions occur through a combination of electrostatic and hydrophobic interactions. Elution is performed using a gradually increasing salt concentration to screen out the protein—matrix electrostatic interactions. However, since the protein is bound through a combination of electrostatic and hydrophobic interactions elution of the more hydrophobic protein will occur at a higher salt concentration. This method is particularly useful for separation of two proteins with very similar charge using an ion exchange matrix.
In this method, the hydrophobic interaction between the amphipathic sugar polymer and the positively charged protein of interest is used to decrease binding to the chromatography matrix. The sugar polymer used has a weak negative charge (e.g. carboxymethyl beta-cyclodextrin). A negatively charged chromatography matrix (cation exchanger) is used to ensure that there is little or no binding of the sugar polymer to the matrix. The positively charged protein is passed through the column and binds to the negatively charged matrix. The sugar polymer is now included in the elution buffer and a salt gradient passed through the column. The sugar polymer binds to the most hydrophobic protein with greatest affinity. This reduces the protein net charge and allows the most hydrophobic protein to be eluted at a lower salt concentration than a similarly charged less hydrophobic protein.
Eluent obtained from the ion exchange column is passed through a second column, or column section, containing an immobilised enzyme. The enzyme degrades any sugar polymer that co-elutes with the protein(s) of interest. Sugar polymers are degraded to monomers, e.g. glucose, fructose and/or derivatives thereof. This releases the sugar polymers from the protein molecules and prevents any further modification of the protein properties by the sugar polymer.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0326064.3 | Nov 2003 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB04/50024 | 11/8/2004 | WO | 00 | 4/9/2007 |
