| JP2004131745 | METHOD OF SEPARATING AND RECOVERING PLATINUM GROUP ELEMENT |
| WO/2002/053256 | DEVICES AND METHODS FOR PURIFICATION |
| WO/2002/089946 | SEPARATION PROCESS BY SIMULATED MOVING BED CHROMATOGRAPHY |
JAHAGIRDHAR, Kishore (101 Surya Chandra Satya Enclave, ASR Raju NagarNear Orange Model School,Miyapur, Hyderabad 0 Andhra Pradesh, Pradesh, IN)
DR. REDDY'S LABORATORIES, INC. (200 Somerset Corporate Boulevard 7th Floor, Bridgewater, New Jersey, 08807, US)
KOTICHA, Darshan (Plot No. 27, Green AvenuesOpp Bhavyas Anandam Apts.,Nizampet Road,,Hyderaba, 0 Andhra Pradesh, Pradesh, IN)
JAHAGIRDHAR, Kishore (101 Surya Chandra Satya Enclave, ASR Raju NagarNear Orange Model School,Miyapur, Hyderabad 0 Andhra Pradesh, Pradesh, IN)
| CLAIMS: 1 . A process for isolating low pi isoforms of darbepoetin from a mixture of darbepoetin isoforms, comprising: loading the mixture onto a hydrophobic anion induction chromatography sorbent at pH values of 4 or less; and eluting low pi isoforms with an elution buffer at a pH values greater than the pH value of the loaded mixture. 2. A process according to claim 1 , wherein hydrophobic anion induction chromatography is preceded by one or more ion exchange chromatography steps. 3. A process according to claim 1 , wherein hydrophobic anion induction chromatography is preceded by an anion exchange chromatography step. 4. A process according to claim 3, wherein the anion exchange chromatography step is followed by a cation exchange chromatography step, which is followed by hydrophobic anion exchange chromatography. 5. A process according to claim 1 , wherein hydrophobic anion induction chromatography is preceded and followed by ion exchange chromatography steps. 6. A process for the isolation of low pi isoforms of darbepoetin from a mixture of darbepoetin isoforms, comprising affinity chromatography followed by hydrophobic anion induction chromatography, wherein the mixture is loaded onto a hydrophobic anion induction chromatography sorbent at pH values of 4 or less. 7. A process according to claim 6, wherein the affinity chromatography is conducted using a blue sepharose chromatography column. 8. A process according to claim 6, wherein the affinity chromatography is conducted using a phenylboranate ligand mediated chromatography column. 9. A process according to either of claims 1 or 6, wherein the mixture is loaded onto the hydrophobic anion induction chromatography sorbent at a pH about 3.5. 10. A process according to either of claims 1 or 6, wherein the mixture is loaded onto the hydrophobic anion induction chromatography sorbent at a pH about 3.5 and the low pi isoforms are eluted at a pH about 6. 1 1 . A process according to either of claims 1 or 6, wherein the mixture is loaded onto the hydrophobic anion induction chromatography sorbent at a pH value about 3.3. 12. A process according to either of claims 1 or 6, wherein the mixture is loaded onto the hydrophobic anion induction chromatography sorbent at a conductivity value of 0.3 mS/cm or less. 13. A process according to either of claims 1 or 6, wherein the low pi isoforms are eluted from the hydrophobic anion induction chromatography sorbent at a conductivity value about 15 mS/cm. 14. A process according to either of claims 1 or 6, further comprising a tangential flow filtration, concentration, diafiltration, ultrafiltration, or buffer exchange step between chromatography steps. 15. A process according to either of claims 1 or 6, further comprising viral inactivation, sterile filtration, or viral filtration steps. |
INTRODUCTION
Aspects of the present application relate to methods for purifying cytokines. Specific aspects relate to methods for purifying darbepoetin alpha. In
embodiments, the application relates to methods for separating darbepoetin alpha isoforms, comprising hydrophobic anion induction chromatography.
Erythropoiesis stimulating proteins (ESPs), such as erythropoietin and analogs of erythropoietin, are glycoprotein hormones that are the principle homeostatic regulators of red blood cell production. Though natural erythropoietin is produced by the kidney, its large scale production for therapeutic purposes is achieved by recombinant DNA methods. Purified recombinant human
erythropoietin (rHuEPO) is administered in human patients for the treatment of medical indications associated with inadequate red blood cell supply, e.g., anemia and chronic renal failure. rHuEPO and its analog darbepoetin alpha, are used for the treatment of anemia and disrupted red blood cell production associated with various conditions, such as perisurgery, chronic renal failure, as well in the treatment of side effects associated with HIV, HCV and cancer chemotherapy.
Variability in the sugar moieties that make up the glycan chains of human erythropoietin and its analogue darbepoetin alpha contribute to the
microheterogenity in the isoform compositions of these glycoproteins. This in turn has profound effects on the physico-chemical properties and biological activities of erythropoietin and darbepoetin alpha compositions.
Negatively charged sialic acid residues typically cap the ends of a glycan chain of most glycoproteins. Human erythropoietin and darbepoetin alpha expressed in Chinese hamster ovary (CHO) cells exhibit variable degrees of glycosylation and sialylation. Variability in the glycan chains, in particular in the sialic acid content, results in erythropoietin and darbepoetin alpha isoforms that differ in their overall charge and isoelectric point (R. S. Rush et al., Analytical Chemistry, Vol. 67, pages 1442-1452, 1995). Thus erythropoietin and darbepoetin alpha isoforms with higher sialic content have lower isoelectric point (pi), than those with lower sialic acid content. Variability in the extent of sialylation in turn effects the in vivo activity of erythropoietin and darbepoetin compositions. Compositions enriched in low pi isoforms exhibit higher bioactivity then those with higher content of high pi isoforms (Takeuchi et al., 1989 PNAS
86(20):7819-22, Zanette et al., 2003 Journal of Biotechnology 101(3):275-287). Thus, just as human erythropoietin, low pi isoforms of darbepoetin alpha exhibit higher specific activity than isoforms with higher pi. Hence darbepoetin alpha isoforms with lower pi are of greater therapeutic value. For example Aranesp® (Amgen) the approved and marketed form of darbepoetin alpha comprises essentially of low pi isoforms of the protein, having a pi range of 3-3.9 (Francoise Lasne et al., Analytical Biochemistry 311 (2002) 119-126). However, darbepoetin alpha is expressed in cell culture as a heterogeneous mixture of isoforms in the pi range of about 3 to 8. Hence, there is a need for efficient and effective methods for the separation of low pi isoforms from higher pi isoforms of darbepoetin alpha.
The literature discloses various methods for the purification of
erythropoietin and analogues of erythropoietin.
International Application Publication No. WO 00/27869 discloses a process for purification of erythropoietin consisting of a sequence of hydrophobic interaction, anion exchange, cation exchange and size exclusion chromatographic steps.
International Application Publication No. WO 03045996 discloses chromatographic purification of recombinant human erythropoietin by reverse phase chromatography, anion exchange and size exclusion chromatography.
International Application Publication No. WO 86/07594 discloses a method of purifying erythropoietin from a fluid comprising the steps of subjecting said fluid to reverse phase liquid chromatographic separation involving an immobilized C4 or C6 resin.
However the purification methods described in the prior art involve complex series of chromatographic steps and none of the prior art disclose methods for the purification of darbepoetin alpha by hydrophobic anion induction chromatography (HAIC). The present application provides novel and efficient methods for the separation and purification of darbepoetin alpha. SUMMARY
Aspects of the present application provide processes for separating and purifiying low pi isoforms of darbepoetin alpha from mixtures of isoforms, comprising hydrophobic anion induction chromatography.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a chromatography profile of hydrophobic anion induction chromatography as performed in Example 4.
Fig. 2 is an isoelectric focusing gel of the hydrophobic anion induction chromatography performed as described in Example 4.
Fig. 3 shows a comparison of darbepoetin alpha samples from different batches according to Example 4 with a commercially available darbepoetin alpha product, Aranesp® (pi 3-3.9, Amgen). DETAILED DESCRIPTION
Darbepoetin alpha is an erythropoietin analog with five N-linked
carbohydrate chains and up to 22 sialic acids. As a result, darbepoetin alpha exhibits a three-fold longer serum half-life and increase in vivo activity as compared to recombinant human EPO. In cell culture, darbepoetin alpha is expressed as a heterogeneous mixture of low and high pi isoforms that differ in their extent of glycosylation and sialylation. However, low pi isoforms, as a result of higher sialic acid content, exhibit much higher specific activity as compared to isoforms of higher pi having lower sialic acid content (e.g. Imai et al., European Journal of Biochemistry, 194 (1990), 457-462; European Patent Application Publication 0 428 267). Hence, isoforms with higher sialic acid content and lower pi are of greater therapeutic value.
Aspects of the present application provide efficient methods for the isolation and separation of darbepoetin alpha isoforms from a mixture comprising low and high pi isoforms, using hydrophobic anion induction chromatography.
Hydrophobic anion induction chromatography (HAIC) is a type of dual mode or mixed mode chromatography. Mixed mode chromatography exploits the unique properties of certain chromatographic ligands that show either hydrophobic or charged interactions, or both (Boschetti et al, Gen. Engineering News, 20, 2000; Ghose et al, Biotechnoogy Progress, 21, 498 - 508, 2005, for detailed discussions of mixed mode chromatography). Hydrophobic anion induction chromatography sorbents possess chromatographic ligands that are hydrophobic at one pH and positively charged at a lower pH. Hence, such chromatographic ligands contribute hydrophobic characteristics to the chromatographic resin at higher pH values and anion exchange characteristics at lower pH values. For instance the chromatographic ligand of MEP HyperCel™ (Pall Corporation, USA) is 4-mercaptoethyl pyridine (MEP). MEP has a pK a of 4.8 and the nitrogen atom in its pyridine ring acquires a positive charge at low pH. Hence at high pH, MEP HyperCel acts as a hydrophobic interaction chromatography medium, and at low pH, as an anion exchange chromatography medium. Hydrophobic anion induction chromatography has been used for the purification of monoclonal antibodies, wherein the antibodies are bound to the column at close to neutral pH. Under acidic conditions, both, the chromatographic ligand and the bound antibody take on a net positive charge. Binding is thus disrupted and elution occurs (Schwartz et al, Journal of Chromatography A, 908, 251-263 21, 2001).
In embodiments of the present application, hydrophobic anion induction chromatography is used for the separation of low pi isoforms from a mixture comprising low and high pi isoforms of darbepoetin alpha.
In embodiments, the application provides methods for the separation of low pi isoforms of darbepoetin alpha from a solution comprising low and high pi isoforms by hydrophobic anion induction chromatography, comprising:
a) loading the solution on a hydrophobic anion induction
chromatography sorbent at pH values of 4 or less, to bind the low pi isoforms to the resin,
b) eluting the low pi isoforms with an elution buffer at pH values greater than the pH of the loading solution.
In embodiments, the loading solution has pH values about 3 to about 4, In embodiments, the loading solution has pH 3.3.
In embodiments, the conductivity of the loading solution may be less than or equal to 0.3 mS/cm.
In embodiments, the elution buffer has pH values greater than about 4.5. In embodiments, the elution buffer has pH values between about 5 and about 6.
In embodiments, the elution buffer has pH about 6.
In embodiments, the conductivity of the elution buffer is about 4 mS/cm to about 30 mS/cm.
In embodiments, the conductivity of the elution buffer is about 10 mS/cm to about 20 mS/cm.
In embodiments, the conductivity of the elution buffer is about 15 mS/cm.
In embodiments, the present application provides methods for the separation of low pi isoforms of darbepoetin alpha from a solution comprising low and high pi isoforms by hydrophobic anion induction chromatography, comprising loading the solution onto the hydrophobic anion induction chromatography resin at pH about 3.3 and conductivity about 0.3 mS/cm, and eluting the low pi isoforms at pH about 6 and conductivity about 15 mS/cm.
In embodiments, the hydrophobic anion induction chromatography may be preceded by ion exchange chromatography.
In embodiments, the hydrophobic anion induction chromatography may be preceded by two ion exchange chromatography.
In embodiments, the hydrophobic anion induction chromatography may be preceded and followed by ion exchange chromatography.
In embodiments, the hydrophobic anion induction chromatography may be preceded by an anion exchange chromatography.
In embodiments, the hydrophobic anion induction chromatography may be preceded by an anion exchange chromatography and a cation exchange chromatography.
In embodiments, the hydrophobic anion induction chromatography may be preceded by an affinity chromatography.
The embodiments mentioned here may include one or more of viral inactivation, sterile filtration, and viral filtration steps.
In embodiments, a mixed-mode chromatography sorbent such as MEP
HyperCel™ (Pall Corporation, USA) is used. MEP HyperCel sorbent is a 80-100 μΐη particle size high porosity cross-linked cellulose matrix having 80-125 μΐηοΙ/nnL of a 4-mercaptoethyl pyridine (4-MEP) ligand. In embodiments of the application, affinity chromatography using materials such as Blue Sepharose™ 6 Fast Flow (GE Healthcare Life Sciences) or a Prosep®-PB (Millipore) is used. Blue Sepharose 6 Fast Flow resin is made using a Cibacron Blue 3G coupled to Sepharose 6 Fast Flow, which is a highly cross- linked 6% agarose matrix. ProSep-PB media is composed of a synthetic m- aminophenyl ligand (phenyl boronate ligand) immobilized on controlled pore size glass beads.
Anion exchange chromatography in embodiments may be carried out using any weak or strong anion exchange chromatographic resin, or a membrane which can function as a weak or a strong anion exchanger. Commercially available anion exchange resins include, but are not limited to, DEAE cellulose, Poros PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Applied Biosystems, MonoQ, MiniQ, Source 15Q and 3OQ, Q, DEAE and ANX Sepharose Fast Flow, Q Sepharose high Performance, QAE SEPHADEX and FAST Q SEPHAROSE from GE
Healthcare, Macro-Prep DEAE and Macro-Prep High Q from Biorad, Q-Ceramic Hyper D, DEAE-Ceramic Hyper D, from Pall Corporation. In embodiments of the application, a strong anion exchange resin, such as Q-Sepharose Fast Flow™ (GE Healthcare Life Sciences) is used. This resin is made using a highly cross- linked 6% agarose matrix attached to a -O-CH2CHOHCH2OCH2CHOHCH2- N + (CH 3 )3 functional group.
Cation exchange chromatographic steps in embodiments may be carried out using any weak or strong cation exchange chromatographic resin, or a membrane which could function as a weak or a strong cation exchanger.
Commercially available cation exchange resins include, but are not limited to, those having a sulfonate based group, e.g., MonoS, MiniS, Source 15S and 30S, SP Sepharose Fast Flow, SP Sepharose High Performance from GE Healthcare, Toyopearl SP-650S and SP-650M from Tosoh, S-Ceramic Hyper D, from Pall Corporation or a carboxymethyl based group e.g., CM Sepharose Fast Flow from GE Healthcare, Macro-Prep CM from BioRad, CM-Ceramic Hyper D, from Pall Corporation, Toyopearl CM-650S, CM-650M and CM-650C from Tosoh. In embodiments of the application a weak cation exchange column, such as CM - Sepharose™ (GE Healthcare Life Sciences) is used. This resin is made using a highly cross-linked 6% agarose matrix attached to a carboxymethyl group. The buffering agents used for making buffer solutions may comprise acetate or phosphate buffers, including any of their salts or derivatives.
The term "isoform", as used herein, refers to proteins with identical amino acid sequences, but differing with respect to charge and therefore isoelectric points, as a result of differences in glycosylation, acylation, deamidation, or sulfation.
The "isoelectric point" or "pi" is the pH at which a particular molecule or surface carries no net electrical charge. The "pi" of a polypeptide refers to the pH at which the polypeptide's positive charge balances its negative charge. The pi can be estimated by various methods known in the art, e.g., from the net charge of the amino acid and/or sialic acid residues on the polypeptide, or by using isoelectric focusing, chromatofocussing, etc.
The low pi isoforms refer to isoforms with pi of 4 or less.
Certain specific aspects and embodiments are more fully described in the following examples. These examples should not, however, be construed to limit the scope of the application as defined by the appended claims.
Expression and Harvest of Protein
Chinese hamster ovary (CHO) production cell lines are made by
transduction of the CHO-S parental cell line with retrovector from the darbepoetin alpha expression vector. The pooled population of cells is diluted to very low cell density (1 -3 viable cells/200 μΙ_ media) and plated in 96 well microtiter plates to establish clonal cell lines that originate from single cells. Clones are screened for darbepoetin alpha production and clones with high productivity are selected for expression.
The cells expressing darbepoetin alpha are expanded from the master cell bank a stages of spinners and one stage of seed reactor before being inoculated into the production reactor.
PF-CHO medium is used for culturing the cells in spinners in order to obtain good cell growth and high viability. The PF-CHO medium contains, per liter of medium: PF-CHO main powder 6.0 g, PF-CHO base powder 10.4 g, L- Glutamine 0.58 g, Pluronic F-68 1 .0 g, sodium bicarbonate 2.0 g. The pH of the medium is adjusted to 7 before inoculation. Cells from the master cell bank are inoculated in a spinner bottle containing PF-CHO medium at an initial cell count of 0.2 million cells/mL. The spinner bottles are incubated in a 5% CO2 incubator maintained at 37°C. After 72 hours of incubation, cells are inoculated in a 6 L seed reactor containing 4 L SFM-6(1 ) medium. The SFM-6(1 ) medium contains, "DMEM/F-12" basal media, amino acids, insulin, vitamins, trace elements, plant peptone, bicarbonate, and fructose sugar. In the seed reactor, the pH is
maintained at 7.0 and temperature of culture is controlled at 37.0°C. Dissolved oxygen is maintained at 40% by controlling agitation and aeration. After 72 hours, the culture is aseptically harvested and cells are transferred to a 10 L production reactor containing 9 L of SFM-6(2) medium at an initial cell density of 0.2 million cells/mL. The culture is harvested after 12 days to collect the supernatant liquid containing the desired product.
EXAMPLE 1
Anion Exchange Chromatography
After clarification, the cell culture broth from the Expression and Harvest of Protein procedure described above is concentrated and the conductivity is reduced by diafiltration (using tangential flow filtration (TFF) with a molecular weight cut off of 30 kDa) using 25 mM Tris, 60 mM NaCI buffer having pH 7.1 . The concentrated cell culture broth is then loaded onto a Q-Sepharose column (300 ml_, XK 50/20) that was pre-equilibrated with 5 column volumes (CV) of 25 mM Tris, 60 mM NaCI, pH 7.1 buffer. The column is then washed with 5 CV of the equilibration buffer (25 mM Tris, 60 mM NaCI, pH 7.1 ). This is followed by a low pH wash with 80 mM sodium acetate, 50 mM NaCI buffer of pH 4.0. The desired protein loaded onto the column is eluted using 25 mM Tris buffer, 250 mM pH 7.1 . Impurities bound to the column were subsequently eluted with 25 mM Tris, 500 mM NaCI buffer of pH 7.1 .
EXAMPLE 2
Hydrophobic anion induction chromatography
Eluate from Example 1 , containing the desired protein, is concentrated and exchanged with buffer containing 83.4 mM sodium acetate (or 80-85 mM sodium acetate) pH 3.3 (or pH 3-3.5) using TFF. Sample from TFF is loaded onto a MEP chromatographic column (4 ml_, Tricorn 5/20) pre-equilibrated with 5 CV of 83.4 mM sodium acetate buffer of pH 3.3. After loading, the column is washed with 83.4 mM sodium acetate buffer of pH 3.3 (3 CV) followed by a wash of 73 mM acetate buffer, pH 4.8, conductivity 4 mS/cm. To elute the desired protein, the column is treated with 10 CV of 20 mM phosphate, 140 mM NaCI, pH 6.0, conductivity 15 mS/cm. The column is further washed with 3 CV of 40 mM phosphate, 280 mM NaCI, pH 6.0 buffer, at a conductivity of 30 mS/cm. Impurities bound to the column were subsequently washed with 3 CV washing with 25 mM Tris, 500 mM NaCI buffer of pH 7.1 at a conductivity 48 mS/cm.
EXAMPLE 3
Cation Exchange Chromatography
As an alternative to Example 2, the eluate from Example 1 may be subjected to cation exchange chromatography.
Eluate from Example 1 (anion exchange chromatography) is concentrated and the conductivity and pH are reduced by diafiltration by a TFF step using 73 mM sodium acetate buffer, pH 4.8. This step acts as a buffer exchanging step wherein the pooled eluate of Q-Sepharose column is brought into 73 mM sodium acetate buffer of pH 4.8 conductivity 4 mS/cm. The buffer exchanged sample is then loaded onto a CM-Sepharose column (300 ml_, XK 50/20) that was pre- equilibrated with 73 mM sodium acetate buffer, pH 4.8. This is a flow through step wherein the desired product does not bind to the column but impurities bind, thus resulting in purification. As the column loading is initiated, the flow through containing the desired protein is collected. For a more complete recovery of the product, the column is further washed with 73 mM sodium acetate buffer, pH 4.8 until the absorbance at 280 nm returns to baseline (approximately 5 CV) and is pooled with the initial flow through fractions. Impurities bound to the column are subsequently eluted with 25 mM Tris, 500 mM NaCI buffer of pH 7.1 . EXAMPLE 4
Hydrophobic anion induction chromatography
The flow through fraction from Example 3 containing the desired protein is concentrated and exchanged with buffer containing 83.4 mM sodium acetate (or 80-85 mM sodium acetate) pH 3.3 (or pH 3-3.5) using TFF. Sample from the TFF is loaded onto a MEP chromatographic column (4 ml_, Tricorn 5/20) pre- equilibrated with 5 CV of 83.4 mM sodium acetate buffer of pH 3.3. After loading, the column is washed with 83.4 mM sodium acetate buffer of pH 3.3 (3 CV) followed by a wash of 73 mM acetate buffer, pH 4.8, conductivity 4 mS/cm. To elute the desired protein, the column is treated with 10 CV of 20 mM phosphate, 140 mM NaCI, pH 6, conductivity 15 mS/cm. The column is further washed with 3 CV of 40 mM phosphate, 280 mM NaCI, pH 6, at a conductivity of 30 mS/cm. Impurities bound to the column are subsequently washed with 3 CV wash with 25 mM Tris, 500 mM NaCI buffer of pH 7.1 at a conductivity of 48 mS/cm.
The eluate fractions from hydrophobic anion induction chromatography of Example 4 are analyzed by isoelectric focusing (IEF). The IEF gel is prepared using water, urea, 30% acrylamide, and Ampholyte (pH range 2-4 and 3-10). The above components are mixed gently and 10% w/v ammonium persulfate and TEMED are added to the mixture and the mixture is cast in a gel sandwich apparatus (BIORAD Mini Protean Cell) and fitted with a comb. The gel is allowed to polymerize for 45 minutes at room temperature. A small amount of protein solution (sample) is mixed with an equal volume of sample buffer (glycerol, Ampholyte and Milli-Q water) and protein samples are loaded into the gel. The gel is then placed in a BIORAD Mini Protean Cell assembly and filled with a cathode buffer (25 mM sodium hydroxide) and anode buffer (25 mM orthophosphoric acid) in separate compartments. The flow through fractions from Example 5 are run at 200 V constant voltage for 1 .5 hours for pre-focusing of ampholytes at room temperature and then the voltage is increased to 400 V and run for the next 1 .5 hours at room temperature. After the run, the gel is carefully removed and stained by silver staining.
The isoelectric focusing procedure can also be used with eluate fractions from hydrophobic anion induction chromatography of Examples 2 and 6.
Fig. 1 is an illustration of a chromatogram from the procedure as described in this example. The line marked "Cond" shows the increase in conductivity in mS/cm. Peaks A and B represent the eluate obtained at conductivities 15 mS/cm and 30 mS/cm, respectively. Fig. 2 is an isoelectric focusing gel of the hydrophobic anion induction chromatography performed as described in this example. Lane 1 is the internal reference standard. Lane 2 corresponds to fraction obtained by elution at pH 6 and conductivity 15 mS/cm. Lane 3 corresponds to fractions obtained by elution at pH 6 and conductivities 30 mS/cm.
Fig. 3 shows a comparison of darbepoetin alpha samples from different batches. Lanes 1 -9 corresponds to darbepoetin alpha samples from different batches. Lane 10 corresponds to a commercially available darbepoetin alpha product, Aranesp® (pi 3-3.9). Lane 3 is used as an internal reference standard.
EXAMPLE 5
Affinity Chromatography
As an alternative to Examples 1 , 2, 3, or 4, cell culture broth from the Expression and Harvest of Protein procedure above may be subjected to an affinity chromatography procedure.
After clarification, the cell culture broth is loaded onto a Blue-Sepharose FF (20 mL, VL 1 1 /21 ) column that is pre-equilibrated with 5 CV of 50 mM phosphate, 100 mM NaCI, pH 7.5 buffer at a conductivity of 8 mS/cm. The desired protein bound onto the column is eluted using 250 mM Tris buffer of pH 7.5.
Alternatively, a phenylboronate column (20 mL, VL 1 1/21 ) may be used as an affinity chromatography step wherein the crude extract of the clarified cell culture broth is loaded onto the phenylboronate column. The column is pre- equilibrated with 5 CV of 50 mM phosphate, 100 mM NaCI, pH 7.5 buffer at a conductivity of 8 mS/cm. The desired protein bound onto the column is eluted using 50 mM phosphate, 100 mM NaCI, 100 mM sorbitol buffer of pH 7.5.
EXAMPLE 6
Hydrophobic anion induction chromatography
Eluate from Example 5, containing the desired protein, is concentrated and exchanged with buffer containing 83.4 mM sodium acetate (or 80-85 mM sodium acetate) pH 3.3 (or pH 3-3.5) using TFF. Sample from TFF is loaded onto a MEP chromatographic column (4 mL, Tricorn 5/20) pre-equilibrated with 5 CV of 83.4 mM sodium acetate buffer of pH 3.3. After loading, the column is washed with 83.4 mM sodium acetate buffer of pH 3.3 ( 3 CV) followed by a wash of 73 mM acetate buffer, pH 4.8, conductivity 4 mS/cm. To elute the desired protein, the column is treated with 10 CV of 20 mM phosphate, 140 mM NaCI, pH 6, conductivity 15 mS/cm. The column is further washed with 3 CV of 40 mM phosphate, 280 mM NaCI, pH 6, conductivity 30 mS/cm. To remove bound impurities, the column is subsequently washed with 3 CV of 25 mM Tris, 500 mM NaCI buffer of pH 7.1 and conductivity 48 mS/cm.
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