WO2005070513A1

WO2005070513A1 - Improved recovery of analytes for process chromatography

Info

Publication number: WO2005070513A1
Application number: PCT/US2005/000461
Authority: WO
Inventors: Derek Wai-Ho Chan; Kervin Hershal Harrison; Reno Thanh-Chuong Nguyen
Original assignee: Alltech Associates Inc.
Priority date: 2004-01-09
Filing date: 2005-01-07
Publication date: 2005-08-04

Abstract

The present invention provides for a process scale or preparative scale chromatographic method of purifying biological material comprising the steps of providing chromatographic media including inorganic material in the form of porous particles; applying a solution comprising the biological material to the media, wherein the biological material is reversibly bonded to the media; and eluting the biological material from the media with a solvent, wherein the porous particles are subjected to a pretreatment in order to reduce BET surface area.

Description

Improved Recovery of Analytes for Process Chromatography

BACKGROUND OF THE INVENTION

[0001] The present invention relates to the separation of biological compounds utilizing reverse-phase chromatographic media. [0002] Biological compounds, such as proteins, have become commercially important as drug candidates in other therapeutic applications. One of the greatest challenges lies in the development of cost effective and efficient processes for purification of proteins on a commercial scale. While many methods are now available for large-scale preparation of proteins, crude products contain not only the desired product but also closely related impurities that are difficult to separate from the desired product. Moreover, biological sources of proteins usually produce complex mixtures of materials. [0003] Generally, proteins are produced by cell culture, using either mammalian or bacterial strains engineered to produce the protein of the interest by insertion of a recombinant plasmid containing the gene for that protein. Since the strains used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids and growth factors that are usually supplied from preparations of animal serum. Separation of the desired protein from the mixture of compounds fed to the cells and from the byproducts from the cells themselves to a purity sufficient for use as a human therapeutic poses a formidable challenge. Usually, the separation procedure is multi-step requiring expensive apparatus and chromatographic media.

[0004] Procedures for purification of proteins from cell debris initially depend on the sight of expression of the protein. Some proteins may be secreted directly from the cell into the surrounding growth media, while others are made intra-cellularly. For the latter proteins, the first step of a purification process involves lysing or destruction of the cell, which can be done by a variety of methods, including mechanical shear, osmotic shock, or enzymatic treatments. Such destruction releases the entire contents of the cell into the homogenate, and in addition produces sub-cellular fragments that are difficult to remove due to their small size. Usually, these are removed by differential centrifugation or by filtration. [0005] After a clarified solution containing the protein of interest has been obtained, its separation from the other proteins produced by the cell is usually attempted using a combination of different chromatography techniques. These techniques separate mixtures of proteins on the basis of their charge, degree of hydrophobicity, or size. Several different chromatography media are available for each of these techniques, allowing accurate tailoring of the purification scheme to the particular protein involved. Affinity chromatography, which exploits a specific interaction between the proteins to be purified in a second protein, such as a specific antibody, may also be a separation technique for some proteins.

[0006] The essence of each of the separation methods is that proteins can be caused either to move at different rates through chromatographic media, achieving a physical separation that increases as they pass further through the media, or to adhere selectively to the separation media, being then differentially eluted by different solvents. In some cases, the desired protein is separated from impurities when the impurities specifically adhere to the media, and the protein of interest does not.

[0007] The major performance measures of chromatography techniques are productivity and peak resolution. Productivity refers to specific throughput. It is a measure of the mass of solute that can be processed per unit volume of chromatography matrix. Generally, productivity improves with increases in the surface area per unit volume of the matrix, the rate of solute mass transfer to the sorbent surface, the rate of adsorption and desorption, and the fluid flow velocity through the matrix. Resolution is a measure of the degree of purification that a system can achieve. It is specified by the difference in affinity among solutes in the mixture to be separated and by the systems inherent tendency towards dispersion or bandspreading. Affinity of the solutes is controlled by the nature in the process liquid and the chemical properties of the chromatography media. Bandspreading is controlled primarily by the geometry of the chromatographic media- (e.g. the surface area, particle size, etc.) and the mass transfer rates from the solute to the media surface during the chromatographic procedure. [0008] The application of modern liquid chromatographic techniques by high performance liquid chromatographic (HPLC) has led to an improvement of separation, characterization, and purification of proteins. Liquid chromatographic using a reverse phase packing has been found to be an effective tool in both qualitative and quantitative analysis for drug substances in blood, serum, or plasma. Typically, the reverse phase packing material is made up of bonding alkyl groups to porous inorganic oxides, e.g., silica, and most typically the packing is a pore silica having octadecylsilane bonded to it. The porous silica particles are available in a variety of forms, with different sizes of particle and pore size within the particle. The size of particle chiefly determines the packing properties of the material, which determine the rate of flow and the backpressure when the material is used as a column. The pore size, however, determines the size of protein that has access to the interior of the pore. Typically, pore sizes vary from 100 to 500 Angstroms in size. The size of the particle typically varies from 1 micron to 100 microns, depending upon the nature of the separation process. A mixture of water and organic solvent are used to elute molecules of interest. Biomolecules are often loaded under highly aqueous conditions to maximize their binding to the reversed-phase material. These molecules will elute at the threshold organic solvent concentration. One means for their elution involves a gradient separation whereby the organic solvent content is increased per unit time. [0009] In analytical chromatography peak resolution is of paramount importance, whereas the amount of recovered or purified material (i.e., productivity) is not. Thus, chromatographic media utilized in analytical applications possesses smaller particle size, which minimizes bandspreading and maximizes peak resolution. In contrast, preparative and process chromatography requires high throughput and recovery of the target material, and thus, requires large particle size and large surface area to volume ratio of the media, respectively. Larger particle size allows increased fluid flow velocity through the media as does a larger surface area to volume ratio. However, this combination results in significantly lower peak resolution. [0010] There have been numerous efforts of utilizing inorganic porous media to separate biological materials using high pressure liquid chromatography (HPLC). See for example US Patent Nos. 4,289,690 and 4,959,340. Additionally, US Patent No. 5,451 ,660 describes the use of reverse phase high-pressure liquid chromatography for the purification of polypetides. In such a process, the different components of the mixture introduced into the column possess different respective degrees of solubility in the stationary phase (chromatographic media) and in the mobile phase (the mixture passing through the column). As the mobile phase flows over the stationary phase, there is an equilibrium in which the sample components are partitioned between the stationary phase and the mobile phase. As the mobile phase passes through the column, the equilibrium is constantly shifted in favor of the mobile phase. This occurs because the equilibrium mixture, at any time, is exposed to fresh mobile phase and partitions into the fresh mobile phase. As the mobile phase is carried down the column, the mobile phase is exposed to fresh stationary phase and partitions into the stationary phase. A separation of a mixture of components occurs because the mixture of components has slightly differently affinities for the stationary phase and/or solubilities in the mobile phase, and therefore, have different partition equilibrium values. Thus, the mixture of components passed down the column at different rates.

[0011] In US Patent No. 5,585,236, ion pair reverse-phase high-pressure liquid chromatography is described as a process for separation DNA using a non-poly separation media, wherein the process utilizes a counter-ion agent and an organic solvent to release the DNA from the separation media. More recently, analysis and separation of RNA molecules has been performed using matched ion polynucleotide chromatography. See US Patent No. 6,475,388 B1.

[0012] Profusive chromatography has been utilized to increase the efficiency in chromatographic separations by increasing the surface area of the chromatographic media and the fluid flow velocity through the media in order to increase the liquid through the column. See US Patent No. 5,833,861. This is accomplished using chromatographic media possessing a first pore set having a great mean diameter than the members of a second pore set.

[0013] Heretofore, there has been no high-pressure chromatographic process that provides for efficient separation of biological substances combined with the advantages of high throughput. Therefore, there is a need for a separation process for such biological materials that allows for high throughput as well as increased recovery of the target biological material that may be utilized in preparative and process chromatographic applications.

SUMMARY OF THE INVENTION

[0014] The present invention provides for a process scale or preparative scale chromatographic method of purifying biological compounds comprising the steps of providing chromatographic media including inorganic material in the form of porous particles applying a solution comprising said biological compounds to the media, wherein the biological compound is reversibly bonded to the media; and eluting the biological material from the media with a buffer wherein said porous inorganic particles are treated to reduce BET surface area.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention relates to improved processes for purifying biological compounds.

[0016] By the term "biological compound" as used herein refers to amino acid containing compounds, such as proteins and peptides and also includes non-peptidyl compounds as used herein "peptide" refers to a molecule of up to about 30 covalently bonded amino acids including natural and unnatural amino acids of the L- isomeric form of D-isomeric, as well as derivatives and/or analogues thereof. Examples of peptides include compounds, such as enkephalin, somatostatin, somatopin and alpha-MSH. Other peptides that may be purified according to a method of the subject invention include but are not limited to, a list of peptides set forth in PCT International Publication number WO 02/074791 A1 , the entire subject matter of which is incorporated herein by reference.

[0017] Proteins typically have a molecular weight of 10,000 or greater and peptides possess a molecular weight of less than 10,000 and they are also known as polypeptides. These compounds may be obtained naturally or synthetically.

[0018] As used herein, "protein" or "polypeptide" refers to peptides having more than 30 amino acids covalently bonded together. The polypeptides or proteins may be homologous to the whole cell or may be exogenous, meaning that they are heterologous, i.e., foreign to the whole cell, being utilized, such as a human protein produced by a Chinese Hamster ovary cell or by a bacterial cell, or yeast polypeptide produced by different yeast or a bacterial or mammalian cell such as those set forth in US Patent No. 5,451 ,660, the entire subject matter of which is incorporated herein by reference. Examples of bacterial polypeptides or proteins include alkaline phosphatase and β- lactamase. Examples of mammalian polypeptides or proteins include compounds such as Renin a growth hormone, including human growth hormone bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1- antitrypsin; insulin A-chain, insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VI IIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natmietic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-l-alpha); a serum albumin such as human serum albumin; mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, such as beta-lactamase; DNase; inihibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; integrin; protein A or D, rheumatoid factors; a neurotropbic factor such as bone-derived neurotrophic factor (BDNF), neurotro-phin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-P; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF), transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-β1 , TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factor-l and -II (IGF-I and IGF-II); des (I-3) -IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins such as CD-3, CD-4, CD-8 and CD-19; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic. protein (BMP); an interferon such as interferonalpha, -beta, and -gamma; colony stimulating factors (CSFs), e. g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g, IL-I to IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral antigen such as; for example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; antibodies, and fragments of any of the above-listed polypeptides.

[0019] Polypeptides of interest are those that are easily produced in cells with a minimum of proteolysis and need not be glycosylated for their intended utility. Examples of such mammalian polypeptides include IGF-I, IGF-II, brain IGF-I, growth hormone, relaxin chains, growth hormone releasing factor, insulin chains or pro-insulin, urokinase, immunotoxins, NGF, NT-5, RANTES, MIP-l-alpha, vascular endothelial growth factor, an IGF-I binding protein, a GH binding protein, and antigens. Particularly preferred mammalian polypeptides include IGF-I, brain IGF-I, growth hormone, a neurotrophin such as NGF, NT- 3, NT-4, m-5, and NT-6, including NT-5, an IGF-I binding protein, vascular endothelial growth factor, or RANTES. The most preferred mammalian polypeptide is IGF-I, including full-length IGF-I and brain IGF-I. As used herein, "IGF-1" refers to insulin-like growth factor-1 from any species, including bovine, ovine, porcine, equine, and preferably human, in native sequence or in variant form (such as des-1-3-IGF-l, or brain IGF-I) and recombinantly produced. One method for IGF-I is described in EP 128,733 published December 19, 1984.

[0020] Synthetic peptides may also be separated utilizing the subject invention. Such peptides include, but are not limited to, short synthetic peptides, e.g., growth hormone releasing peptides (GHRPs), etc. [0021] As used herein, "non-peptidal compound" refers to an organic or inorganic compound that is not composed of amino acids and possesses a molecular weight of between about 100 and 1000 Daltons. The compound is preferably an organic compound and includes antibiotics, such as vancomycins, cephalosporins, penicillins, and the like; other organic molecules that may be purified according to the invention include, but are not limited to, polyene macrolides, teypenes, alkaloids, carbohydrates, polypetides, and the like.

[0022] As used herein, "buffer" refers to a solution containing the buffer that resists changes in pH through acid-base congregate components. Examples of materials utilized to prepare buffers include free acids such as citric, phosphoric, maleic, malonic, phthalic, salicylic, fumaric, dimethyl malonic, mandelic, malic, formic, tartaric, itaconic, lactic, barbituric, butyric, ascorbic, succinic, benzoic, propionic, acetic, such as trifluoroacetic, etc. Suitable free bases for forming buffers include triethylamine, imidazole, brucine, tricine, glycinamide, listidine, ethanolamine, glycine, ethylamine, dimethylamine, and the like. Those of ordinary skill in the art will readily recognize that many other acids and basis may be used to prepare such buffers. Typically, buffers possess a pH between about 2.0 and about 12.0, and preferably between about 2.0 and about 10.0, and more preferably between about 2.0 and about 7.0 is typically used for reversed-phase silica resin.

[0023] As used herein, "solvent" refers to alcohols and aprotic solvents, as set forth in US Patent No. 6,475,388 B1 , the entire subject matter of which is incorporated herein by reference. Alcohols include those with 1 to 10 carbon atoms, such as methanol, ethanol, iso-propanyl, n-propanol, butanol, ethylene glycol, polyethylene glycol etc. Aprotic solvents include such compounds as dimethyl sulfoxide (DMSO) dimethyl formamide (DMF), N-methyl pyrrolidone (NMP), tetrahydrofuran (THF), dioxanes, nitriles, esters, ethers, etc. These are product solvents which may be utilized alone or in conjunction with alcohols as defined herein. Preferred solvents include methanol, ethanol, iso- propanol, n-propanol, acetonitrile and mixture thereof. Typically, the buffer is prepared in water and is commonly mixed with organic solvents for reversed- phase separations.

[0024] In the process according to this invention for separating a biological compound from other components in a mixture, there are at least two steps. In the first, the mixture is loaded into a reverse-phase liquid chromatography column. The mixture may contain a number of biological components, such as carbohydrates, lipids, proteins, peptides, polypeptides, nucleic acids, and the like. The mixture may also contain closely related isomers of the target biological compound to be separated, such as regioisomers, geometric isomers, stereoisomers, and the like.

[0025] As mentioned herein, the biological compound to be separated may be carbohydrate, lipid, protein, peptide, polypeptide, or nucleic acid. Preferably, the biological compound is protein or peptide. [0026] The chromatographic column utilized in the process of the present invention may be a preparative or process column. As used herein, "preparative" column refers to columns that are used to purify 1 to 200 mg and "process" column refers to columns that are used to purify 200 mg and above. Typically, the diameter of a preparative column ranges between about 10 mm and about 22 mm. The diameter of a process column typically ranges from about 50 mm to about 1000 mm. The column diameter utilized in the present invention may be between about 2 mm and 2 m, preferably between about 10 mm and about 200 mm, and more preferably, between about 20 mm and about 150 mm and even more preferably, between about 22 and about 100 mm.

[0027] The chromatographic media or stationary phase utilized in the process of the present invention is a porous inorganic oxide in the form of particles or beads. The particles may have a diameter of between about 1 micron to about 300 microns, preferably between about 15 microns to about 100 microns, and more preferably, between about 10 microns to about 50 microns, and even more preferably, between about 15 and about 20 microns. The particles may have a pore size between about 50 Angstroms to about 1000 Angstroms, preferably between about 100 Angstroms to about 800 Angstroms, and more preferably, from about 150 Angstroms to about 700 Angstroms. The BET surface area of the particles utilized according to the present invention may be about 50 to about 500 m²/g, and preferably, about 100 to about 125 m²/g. The surface areas set forth herein are measured by the nitrogen BET method. The particles may be spherical or irregular. The inorganic oxide particles are preferably porous silica prepared from silica gel. The surface of the inorganic oxide particles is treated in such a fashion to reduce the BET surface area. Such methods are described in US Patent Nos. 6,074,983, 5,998,329, 4,868,147, 4,434,280 and 4,131 ,542. The surface area of the particles may be reduced by more than 1 %, preferably by more than 5%, and more preferably by more than 10%. For example, the reduction in surface area may range from 1% to 50%.

[0028] The surface of the inorganic oxide particles may be treated in a fashion to form hydrocarbon entities bonded thereto. This renders the surface hydrophobic and allows the biological substance alkyls to be reversibly attached to the particles. The length of the hydrocarbon entities may comprise alkyls having a length of from Ci to C-ι₈ groups, or the entities may include phenyl derivatives, aminoalkyl or aminophenyl derivatives, silanes, diols, etc. Such treatment is described in US Patent Nos. 3,917,527; 6,045,697; 4,773,994; 4,477,492; 4,959,340 and 4,415,631. [0029] A preferred media (238HR) is available from The Separations Group, 17434 Mojave Street, Hesparia, California, 92345, USA. [0030] The separation process of the present invention may be conducted under low pressure, medium pressure or high pressure. Preferably, the process of the present invention is conducted at pressures above atmospheric pressure.

[0031] The column length must be scaled properly to the column diameter. In general, this is an empirical determination that may be readily made by those of ordinary skill in the art. The factors that may be important in determining the relationship between column diameter and column length include particle size, type of elution (e.g., isocratic as compared to gradient solution).

[0032] The mobile phase, which includes a mixture solvent and buffer, is mixture introduced into the column includes the target biological compound as well as other components, such as impurities, as mentioned herein, and also includes solvent and buffer in an amount of from about 0.05 to about 0.15 percent by volume, and more preferably about 0.08 to about 0.12 percent by volume of the mixture. The solvent may be an aqueous buffered solution, such as a solution of trifluoroacetic acid, heptafluorobutyric acid or phosphoric acid. Preferably, the solvent is trifluoroacetic acid. The pH of the mobile phase is preferably within the range of about pH2 to about pH9, more preferably about pH 2 to about pH 7.

[0033] The amount of biological compound loaded onto the column is generally between about 0.01 g molecule/liter bed volume to about 70.0g molecule/liter bed volume, preferably between about 0.02g molecule/liter bed volume to about 40.0g molecule/liter bed volume, more preferably, between about 0.05g molecule/liter bed volume to about 30. Og molecule/liter bed volume. [0034] The linear velocity of the column may vary between about 30 cm/hour to about 300 cm/hr, and preferably, between about 40 cm/hour to about 250 cm/hour and even more preferably between about 50 to about 200 cm/hour when loading the biological compound onto the column media. During the elution process, i.e., removing the biological compound from the chromatographic media, the elution flow rate into the column may range between about 60 cm/hour to about 160 cm/hour, and preferably from about 100 cm/hour to about 140 cm/hour. It should be noted that the flow rate depends on the molecule phase viscosity, media head or particle size, biological compound binding efficiency to the media, operating pressure of the process, etc. Preferably, the process is conducted under pressure in a range from about 50 to about 5000 psi.

[0035] In the next step of the present invention, the biological compound is eluted from the column with a buffer and/or solvent. The eluant may be in the form of an aqueous solution containing a solvent as defined herein in an amount of about 5 to about 50% (v/v), preferably about 10 to about 40% (v/v) of the solution and may have a pH of from about 2.0 to about 12.0, preferably between about 2.0 and about 10.0, and more preferably, between about 2.0 to about 7.0. The organic solvent may be acetonitile, ethanol, isao-propanol, or n-propanol or combinations thereof. The temperature for elution may be about room temperature or about 25°C, although higher or lower temperatures may be employed.

[0036] The eluted biological compound may be further processed depending on the desired final product.

[0037] In some instances, the biological compounds may need only buffer exchange or concentration prior to their intended use. In other instances, the biological compounds may need further purification after the separation process of the current invention prior to their intended use. For example, any purification method known to one of ordinary skill in the art may be utilized for further processing. In general, techniques including, but not limited to, ammonium sulfate precipitation, centrifugation, ion exchange chromatography, affinity chromatography, gel filtration, reverse phase chromatography, recrystalization and adsorption chromatography, ultra filtration, etc. May be used to further purify the biological compounds. EXAMPLES

[0038] The following Examples are given as specific illustrations of the claimed invention. It should be understood, however, that the invention is not limited to the specific details set forth in the Examples. All parts and percentages in the Examples, as well as in the remainder of the specification, are by weight unless otherwise specified.

[0039] Furthermore, any range of numbers recited in the specification or claims, such as that representing a particular set of properties, conditions, physical states or percentages, is intended to literally incorporate expressly herein any number and/or fraction of any number flowing within such range, including any subset of numbers with any range so recited. Any modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. All publications cited herein are incorporated by reference in their entirety.

EXAMPLE I

[0040] In this comparative Example, high performance chromatopgraphic silica comprised of spheroidal porous (i.e., pores having diameters of 30θA) particles of silica is tested in a chromatographic column to determine its ability to recover various biological substances, such as proteins. The silica includes a surface treatment that yields a layer of C₄or C-ι₈ silane polymers covalently bonded to the silica surface, which renders the particles hydrophobic. This silica is available from The Separations Group as VYDAC^® 238TP in two forms, C₄ or C-ie surface modified. Various properties of this silica are set forth in Table I.

[0041] Preparative or process scale reversed-phase chromatography is utilized as the separation technique with the C₁₈238TP media. Various proteins, listed in Table II, are injected into VYDAC^® 238TP54 columns (4.6 mm x 150 mm) under the following conditions: a mobile phase including solvent A comprising 0.1% v/v TFA in water with 1% protein; and solvent B comprising 0.085% v/v TFA in acetonitile. A gradient recovery process is used wherein the column is equilibrated at 20% solvent B for 20 minutes; followed by increasing from 20% up to 80% solvent B in 15 minutes; holding the flow of solvent B at 80% for 5 minutes; and then reducing the flow of solvent B from 80 to 20% in 2 minutes. The flow rate is ml/minute. The detection is performed using a UVD 1705 detector (available from Dionex Corp., Sunnyvale, CA) at 280 nm. A Dionex HPLC system (P580A LPG available from Dionex Corp.), ASI-100 autosampler (available from Dionex Corp.), and CHROMELEON^®data system (available from Dionex Corp.), are also utilized in the analyses. The results are shown in Table III.

EXAMPLE II

[0042] The identical testing method as set forth in EXAMPLE I is used with the same chromatographic media of Example I except that the surface area of the inorganic oxide is reduced as shown in Table I. The silica is available from The Separations Group as C-ι₈ reversed-phase VYDAC^® 238 HR media. The column utilized is VYDAC^® 238 HR54 and is 4.6 mm x 150 mm. The results, are shown in TABLE III, clearly demonstrate that the protein recovery for EXAMPLE II according to the present invention is significantly higher than the protein recovery for comparative EXAMPLE I.

EXAMPLE III

[0043] In this comparative Example, a C reversed-phase VYDAC^® 238TP media is utilized in a reversed-phase chromatographic process similar to EXAMPLE I separation technique with the C-ι₈238TP media. Various proteins, listed in Table IV, are injected into VYDAC^® 238TP54 columns (4.6 mm x 150 mm) under the following conditions: a mobile phase including solvent A comprising 0.01 %v/v TFA in water with 1% protein; and solvent B comprising 0.085% v/v TFA in acetonitile. A gradient recovery process is used wherein the column is equilibrated at 20% of solvent B for 20 minutes; followed by increasing from 20% up to 80% solvent B in 15 minutes; holding the flow of solvent B at 80% for 5 minutes; and then reducing the flow of solvent B from 80 to 20% in 2 minutes. The flow rate is 1 ml/minute. The detection is performed using a UVD 1705 detector (available from Dionex Corp., Sunnyvale, CA) at 280 nm. A Dionex HPLC system (P580A LPG available from Dionex Corp.), ASI-100 autosampler (available from Dionex Corp.), and CH ROM ELEON^® data system (available from Dionex Corp.) are also utilized in the analyses. The proteins recovered are listed in TABLE IV. The chromatographic column is identical to that utilized in EXAMPLE I except for the media. The results are set forth in TABLE IV.

EXAMPLE IV

[0044] The identical testing method as set forth in EXAMPLE I is used with the same chromatographic media of Example I except that the surface area of the inorganic oxide is reduced as shown in Table I. The silica is available from The Separations Group as C-ι₈ reversed-phase VYDAC^® 238 HR media. The column utilized is VYDAC^® 238 HR54 and is 4.6 mm x 150 mm. The results, are shown in TABLE III, clearly demonstrate that the protein recovery for EXAMPLE II according to the present invention is significantly higher than the protein recovery for comparative EXAMPLE I.

TABLE I Inorganic Oxides

*The surface area is measured with the use of nitrogen by the method of Brunauer, Emmett, and Teller, Journal of American Chemical Society, Vol. 60, pp309 (1938)

j Area (mAU*mϊn.) % Higher HR t _Λ y " ■. ⁱ , Area (mAU* icytochrome e Amount (ug) Example I Example II Recovery trnyoglo ij __ _L, Amount (ug) Example I 20 48 ^" 64 ~ 33 20 21 20 41 62 53 20 22 50 104 146 41 50 57 50 104 144 38 50 58 100 208 286 37 100 1 12 100_ 211 281 33 100 113

Average Ret. Time 8.0 7.6. f vørage RgtL TirrjΘ, ^*10i49 Area (mAU*min.) % Higher HR $> -_t ^" Area (mAU* rtbonuclease A Amount (ug) Example I Example ll Recovery_! ¹ insulin_* Amount (ug) Example I 20 8 1 1 38 20 13 20 20 13 9 1 1 33 50 35 50 22 29 29 50 37 50 22 29 28 100 71 10 44 57 30 100 70

Average Ret. Tun

Area (mAU^*min.) % Higher HR lysozyme Amount (ug) ExampleX Example H Recovery , • Recoveries for 238HR are typically 20 36 40 10 than 238TP at low sample load; 4-3 20 37 40 8 sample load. 50 95 103 9 50 94 104 10 100 189 206 9 100 204 _ 8 j Average Ret TTiimmee 8.62 7.37 - ^"1

TABLE IV c₄ Test Proteins

Protein

cytochrome c bovine heart 11,572 9.52 -087 Hydrophilic ribonuclease A bovine pancreas 13,690 8.64 -0.66 carbonic anhy rase II bovine erthyrocytes 28,981 7.93 -0.56 bovine serum albumin bovine serum 66,433 560 -0.48 conalbumin (ovotransferrin) chicken egg 75,828 6.69 -0.44 ovalbumin chicken egg 42,750 5.19 -0.006 V insulin * bovine pancreas 5,722 5.39 0.31 Hydrophobic

All proteins prepared at 10 mg/mL concentration.

* dissolved 10 mg in 200 uL n-propanol, 200 uL 50% acetic acid, 600 uL water.

Molecular mass, isolelectric point, and hydrophobicity were determined using the program ProtParam (Swiss Prot

Databank).

Hydrophobicity based on: Kyte, J. and Doolittle, R. F. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157: 105-132.

Recovery Data for C₄ Reversed-Phase Columns

Area (mAU*min.) % Higher H Area (rnAJf min. "

carbonic anhydrase Amount (ug) Example III Example IV- RecoveryExample III Example IV Recovery 20 21 22 8 8 8 impurity peak issue 20 21 23 11 9 8 50 59 59 21 19 50 60 60 22 19 100 119 118 42 39 100 118 42 39

[Average Ret Time (mm ) 10.93 J0.18 *2,6'l 11 53

Area (mAU*rrnn.) ϊ ϊ⁴ t ^•*- % Higher 1 Area (mAU*mιn.) % Higher HR

BSA_^ Amount Example lit Example IV Rβ e v 'eyfpchrome c Amount ug Example III Example IV Recovery 20 10 12 17 2θ^" 36 58 62 20 11 12 14 20 36 55 51 50 50 89 126 41 28 32 13 50 50 90 109 21 28 32 12 100 180 194 8 100 57 63 12 100 180 188 4 100 57 63 11 Average Het Time ( iη.) 7.7& ^"7.22

Average Ret Time -& _ .a-a_

Area (mAU*m,n.) o_{/o H}j_gh^ A ,_. -* 7 1 , Area (mAU*πιin.) % Higher HR

Conalbumin Amount Example ill Example IV Recovery ribonuclease *- A ~* Amount (ugλ Example III Example IV Recovery 20 13 14 8 20 10 9 20 13 15 13 20 10 9 50 25 24 50 34 37 7 50 24 28 15 50 35 37 6 100 48 58 19 100 69 74 6 100 70 75 7

Average Ret Time

Claims

1. A process scale or preparative scale chromatographic method of purifying biological material comprising the steps of: providing chromatographic media including inorganic material in the form of porous particles; applying a solvent comprising said biological material to the media, wherein the biological material is reversibly bonded to the media; and eluting the biological material from the media with a solvent, wherein said porous particles are subjected to a pretreatment in order to reduce BET surface area.

2. A method according to claim 1 , wherein said biological material comprises proteins, peptides.

3. A method according to claim 1, wherein said biological material comprises proteins.

4. A method according to claim 3, wherein said proteins comprise cytochrome c, ribonuclease, carbonic anhydrase II, bovine serum albumin, conalbumin, ovalbumin, myoglobin, lysozyme, and insulin, or mixtures thereof.

5. A method according to claim 3, wherein said proteins comprise 1% of the solution.

6. A method according to claim 1 , wherein said particles comprise silica having a hydrocarbon material bonded thereto.

7. A method according to claim 6, wherein said hydrocarbon material comprises alkyl groups having a chain length of Ci to Cι₈.

8. A method according to claim 6, wherein said silica particles comprise a median particle size ranging from about 4.0 μm to about 5.3 μm.

9. A method according to claim 1 , wherein said pretreatment reduces BET surface area of the particles by more than 1%.

10. A method according to claim 1 , wherein said solvent comprises water, acetonitrile, and trifluoroacetic acid.

11. A method according to claim 1 , wherein said solvent comprises 0.1 % v/v trifluoroacetic acid in pure water and acetonitrile.

12. A process scale or preparative scale chromatographic method of purifying biological material comprising the steps of: providing chromatographic media including inorganic material in the form of porous particles; applying a solvent comprising said biological material to said media, wherein said biological material is reversibly bonded to said media; and eluting said biological material from the media with a solvent, wherein said chromatographic media is subjected to a pretreatment that reduces BET surface area and recovers increased amounts of said biological material compared to chromatographic media that is not subjected to said pretreatment.

13. A method according to claim 12, wherein said biological material comprises proteins, peptides.

14. A method according to claim 12, wherein said biological material comprises proteins.

15. A method according to claim 14, wherein said proteins comprise cytochrome c, ribonuclease, carbonic anhydrase II, bovine serum albumin, conalbumin, ovalbumin, myoglobin, lysozyme, and insulin, or mixtures thereof.

16. A method according to claim 14, wherein said proteins comprise 1% of the solution.

17. A method according to claim 12, wherein said particles comprise silica having a hydrocarbon material bonded thereto.

18. A method according to claim 17, wherein said hydrocarbon material comprises alkyl groups having a chain length of Cι to Cιβ.

19. A method according to claim 12, wherein said silica particles comprise a median particle size ranging from about 4.0 μm to about 5.3 μm.

20. A method according to claim 12, wherein said pretreatment reduces BET surface area of the particles by more than 1%.

21. A method according to claim 12, wherein said solvent comprises water, acetonitrile, and trifluoroacetic acid.