US20150275195A1 - Elution of biomolecules from multi-modal resins using mes and mops as mobile phase modifiers - Google Patents

Elution of biomolecules from multi-modal resins using mes and mops as mobile phase modifiers Download PDF

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US20150275195A1
US20150275195A1 US14/438,401 US201314438401A US2015275195A1 US 20150275195 A1 US20150275195 A1 US 20150275195A1 US 201314438401 A US201314438401 A US 201314438401A US 2015275195 A1 US2015275195 A1 US 2015275195A1
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chloride
phosphate
potassium
good
biomolecule
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Rahul Godawat
Daniel Cummings
Veena Warikoo
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Genzyme Corp
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Genzyme Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
    • A61B19/02—
    • A61B19/026—
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B50/00—Containers, covers, furniture or holders specially adapted for surgical or diagnostic appliances or instruments, e.g. sterile covers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B50/00—Containers, covers, furniture or holders specially adapted for surgical or diagnostic appliances or instruments, e.g. sterile covers
    • A61B50/30—Containers specially adapted for packaging, protecting, dispensing, collecting or disposing of surgical or diagnostic appliances or instruments
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61J—CONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE
    • A61J1/00—Containers specially adapted for medical or pharmaceutical purposes
    • A61J1/14—Details; Accessories therefor
    • A61J1/16—Holders for containers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3847—Multimodal interactions
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/165—Extraction; Separation; Purification by chromatography mixed-mode chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
    • B01D15/424—Elution mode
    • B01D15/426—Specific type of solvent
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01022—Alpha-galactosidase (3.2.1.22)

Definitions

  • the present invention relates to methods for purifying or enriching a biomolecule using multimodal resins and an elution buffer containing a Good's buffer.
  • Multimodal resins are chromatography media that contain at least two different groups that interact with the target molecule by at least two different mechanisms.
  • multimodal ion exchangers carry a charged group (e.g., a positively charged amino group, a positively charged quarternary ammonium group; a negatively charged carboxylic acid group, a negatively charged sulfonic acid group) which may interact by charge-charge interactions with the target molecule.
  • These multimodal ion exchangers additionally carry another group that may interact with the target molecule by different modes of action, such as hydrophobic interactions, van der Waals interactions, dipole interactions, cation-pi interactions, or hydrogen bonding.
  • EP 2 167 526 B1 describes the purification of coagulation Factor VIII using a multimodal resin containing ligands which comprise a hydrophobic part and a negatively charged part.
  • the elution is carried out with an elution buffer containing a high concentration of salt (>1.5 M), at least 40% (w/v) ethylene glycol, propylene glycol, or a mixture thereof, and calcium ions.
  • US 2011/0160435 A1 describes another process for the purification of coagulation factor VIII using a multimodal resin.
  • the elution buffer comprises at least one amino acid that is positively charged at pH 6 to 8.
  • the amino acid is selected from lysine, arginine and histidine and is used in concentrations above 0.4 M, especially above 0.5 M.
  • US 2011/0166332 A1 describes a method for separating at least one non-aggregated protein from a liquid protein preparation by contacting said preparation with a multimodal anion exchanger.
  • the experimental section describes the separation of non-aggregated IgG using an elution buffer containing 1 M glycine in 20 mM Tris, 20 mM Hepes, 20 mM MES, 50 mM NaCl with a linear gradient from pH 9.0 to pH 4.5.
  • the present invention relates to a method for purifying or enriching a biomolecule, said method comprising the steps:
  • aqueous buffer solution comprises, essentially consists of or consists of: at least 15 mM at least one Good's buffer; between 0 and 400 mM in total concentration of one or more of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; between 0 and 400 mM in total concentration of one or more of urea, guanidinium chloride, guanidin
  • the aqueous buffer solution comprises 15 mM to 2.5 mM Good's buffer, such as at least 20 mM, 50 mM, 100 mM, 150 mM, 200 mM or more of a Good's buffer.
  • the aqueous buffer comprises between 10 mM and 400 mM of one or more of alanine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; between 10 mM and 400 mM of one or more of guanidinium thiocyanate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium acetate, sodium chloride, sodium citrate, sodium phosphate, sodium iodide, potassium chloride, potassium citrate, potassium sulfate, potassium phosphate, potassium iodide, magnesium chloride, magnesium sulfate, magnesium phosphate, calcium chloride, calcium sulfate, calcium phosphate, and taurine; and between 1% and 60%
  • the Good's buffer in the aqueous buffer is MES, MOPS or Tris.
  • the invention relates to a purified biomolecule, such as a protein, obtainable by the method according to the first aspect.
  • the present invention relates to a preparation comprising:
  • aqueous buffer solution comprising, essentially consisting of or consisting of at least 15 mM of at least one Good's buffer; between 0 and 400 mM in total concentration of one or more of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; between 0 and 400 mM in total concentration of one or more of urea, guanidinium chloride, guanidinium thiocyanate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium acetate, sodium chloride, sodium citrate, sodium caprylate, sodium phosphate, sodium iodide, potassium chloride, potassium
  • the aqueous buffer solution comprises 15 mM to 2.5 mM of a Good's buffer, such as at least 20 mM, 50 mM, 100 mM, 150 mM, 200 mM or more of a Good's buffer.
  • the aqueous buffer comprises between 10 mM and 400 mM of one or more of alanine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; between 10 mM and 400 mM of one or more of guanidinium thiocyanate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium acetate, sodium chloride, sodium citrate, sodium phosphate, sodium iodide, potassium chloride, potassium citrate, potassium sulfate, potassium phosphate, potassium iodide, magnesium chloride, magnesium sulfate, magnesium phosphate, calcium chloride, calcium sulfate, calcium phosphate, and taurine; and between 1% and 60%
  • the Good's buffer in the aqueous buffer is MES, MOPS or Tris.
  • the invention relates to a use of an aqueous buffer solution for desorbing a biomolecule from a multimodal resin, wherein said aqueous buffer solution comprises, essentially consists of or consists of at least 15 mM of at least one Good's buffer; between 0 and 400 mM in total concentration of one or more of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; between 0 and 400 mM in total concentration of one or more of urea, guanidinium chloride, guanidinium thiocyanate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium acetate, sodium chloride,
  • the aqueous buffer solution comprises 15 mM to 2.5 mM of a Good's buffer, such as at least 20 mM, 50 mM, 100 mM, 150 mM, 200 mM or more of a Good's buffer.
  • the aqueous buffer comprises between 10 mM and 400 mM of one or more of alanine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; between 10 mM and 400 mM of one or more of guanidinium thiocyanate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium acetate, sodium chloride, sodium citrate, sodium phosphate, sodium iodide, potassium chloride, potassium citrate, potassium sulfate, potassium phosphate, potassium iodide, magnesium chloride, magnesium sulfate, magnesium phosphate, calcium chloride, calcium sulfate, calcium phosphate, and taurine; and between 1% and 60%
  • the Good's buffer in the aqueous buffer is MES, MOPS or Tris.
  • the present invention relates to use of an aqueous buffer solution for increasing the recovery rate of a biomolecule adsorbed on a multimodal resin by contacting (a) the multimodal resin onto which said biomolecule has been adsorbed with (b) the aqueous buffer solution, wherein the aqueous buffer solution comprises, essentially consists of or consists of at least 15 mM of at least one Good's buffer; between 0 and 400 mM in total concentration of one or more of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; between 0 and 400 mM in total concentration of one or more of urea, guanidinium chloride, guanidinium thiocyan
  • the aqueous buffer solution for increasing the recovery rate of a biomolecule adsorbed on a multimodal resin can include at least 15 mM, at least 100 mM, or at least 200 mM of a Good's buffer; between 10 mM and 400 mM of one or more of alanine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; between 10 mM and 400 mM of one or more of guanidinium thiocyanate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium acetate, sodium chloride, sodium citrate, sodium phosphate, sodium iodide, potassium chloride, potassium citrate, potassium sulfate, potassium phosphate,
  • the solution comprising the biomolecule recovered from the multimodal resin is applied to one or more of ultrafiltration/diafiltration (UF/DF), tangential flow filtration (TFF), microfiltration (MF) and hydrophobic interaction chromatography (HIC). In one embodiment, the solution comprising the biomolecule recovered from the multimodal resin is applied to HIC.
  • UF/DF ultrafiltration/diafiltration
  • TFF tangential flow filtration
  • MF microfiltration
  • HIC hydrophobic interaction chromatography
  • the present invention relates to an article of manufacture comprising:
  • the aqueous buffer solution in the article of manufacture includes at least 15 mM, at least 100 mM, or at least 200 mM of a Good's buffer; between 10 mM and 400 mM of one or more of alanine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; between 10 mM and 400 mM of one or more of guanidinium thiocyanate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium acetate, sodium chloride, sodium citrate, sodium phosphate, sodium iodide, potassium chloride, potassium citrate, potassium sulfate, potassium phosphate, potassium iodide, magnesium chloride, magnesium s
  • FIG. 1 is a contour plot for yield for an experiment in which agalsidase beta is desorbed from the multimodal chromatographic resin CaptoTM Adhere (GE Healthcare) by an elution buffer containing MES and arginine.
  • the contour plot is calculated for a pH value of 6.25.
  • FIG. 2 is a contour plot for yield for an experiment in which agalsidase beta is desorbed from the multimodal chromatographic resin CaptoTM Adhere (GE Healthcare) by an elution buffer containing MOPS and arginine.
  • the contour plot is calculated for a pH value of 7.0.
  • FIG. 3 is a contour plot for yield for an experiment in which agalsidase beta is desorbed from the multimodal chromatographic resin CaptoTM Adhere (GE Healthcare) by an elution buffer containing MES and glycol.
  • FIG. 4 is the multimodal ligand of CaptoTM Adhere. The leftmost part of the molecule forms the linkage to the support matrix.
  • FIG. 5 is the multimodal ligand of CaptoTM MMC.
  • FIG. 6 is the multimodal ligands of Pall PPA HyperCelTM, Pall HEA HyperCelTM, and Pall MEP HyperCelTM.
  • FIG. 6A shows the phenylpropyl substituent of Pall PPA HyperCelTM, which is bound to an amine group (not shown).
  • FIG. 6B shows the n-hexyl substituent of Pall HEA HyperCelTM, which is bound to an amine group (not shown).
  • FIG. 6C shows the 4-mercapto-ethyl-pyridine ligand of Pall MEP HyperCelTM.
  • FIG. 7 is the multimodal ligand of Eshmuno® HCX resin obtainable from Merck Millipore, Germany.
  • the invention is based on the discovery that both MES and MOPS, which are typically used as buffering agents, are surprisingly effective in disrupting multimodal interactions.
  • MES, MOPS or other Good's buffers it is possible to lower the concentration of arginine or related mobile phase modifiers with high conductivity or to use elution buffers free, or substantially free, from arginine and related mobile phase modifiers when desorbing biomolecules from multimodal chromatographic resins.
  • an increased concentration of MES, MOPS or other Good's buffers in accordance with the present invention it is possible to significantly reduce or even completely avoid the presence of chaotropic agents in the elution buffer.
  • Solutions of the above-mentioned mobile phase modifiers typically exhibit a high conductivity, which is disadvantageous for subsequent purification steps (e.g., ion exchange steps) that are often carried out after the multimodal resin chromatography. More specifically, a high conductivity may require large dilutions or extended times of dialysis.
  • several of the commonly used mobile phase modifiers can denature the quaternary structure or even the tertiary structure of biomolecules (e.g., proteins), if such mobile phase modifiers are present in high concentration. The risk of denaturation is particularly high if the mobile phase modifier is a chaotropic agent, such as urea or a guanidinium salt.
  • arginine or other additives with high conductivity and to identify mobile phase modifiers that exhibit low conductivity. It is further desirable to reduce the concentration of chaotropic agents (such as urea, gunadinium chloride, guanidinium thiocyanate) in the elution buffer and to identify mobile phase modifiers that exert a weak effect, or have no denaturing effect, on biomolecules.
  • chaotropic agents such as urea, gunadinium chloride, guanidinium thiocyanate
  • the term “Good's buffer” originally referred to one of twelve buffering agents that were selected and described by Norman Good and colleagues in 1966 as particularly suitable for biological research. In the context of the present specification, the “Good's buffer” does not only encompass the originally described twelve buffering agents but further encompasses structurally related buffering agents. Accordingly, as used herein, the term “Good's buffer” comprises the following chemical compounds: ADA, ACES, BES, Bicine, CAPS, CAPSO, CHES, HEPES, MES, MOPS, PIPES, TAPS, TES, and Tris.
  • ADA is an abbreviation for N-(2-Acetamido)iminodiacetic acid.
  • ACES is an abbreviation for N-(2-Acetamido)-2-aminoethanesulfonic acid.
  • BES is an abbreviation for N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic.
  • Bicine is an abbreviation for N,N-Bis(2-hydroxyethyl)glycine
  • CAPS is an abbreviation for 3-(Cyclohexylamino)-1-propanesulfonic acid.
  • CAPSO is an abbreviation for 3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid.
  • CHES is an abbreviation for 2-(Cyclohexylamino)ethanesulfonic acid.
  • HEPES is an abbreviation for 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid.
  • MES is an abbreviation for 2-(N-Morpholino)ethanesulfonic acid.
  • MOPS is an abbreviation for 3-(N-Morpholino)propanesulfonic acid.
  • PIPES is an abbreviation for 1,4-Piperazinediethanesulfonic acid.
  • TAPS is an abbreviation for N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid.
  • TES is an abbreviation for 2-[(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethane-sulfonic acid.
  • Tris is an abbreviation for Tris(hydroxymethyl)aminomethane.
  • Pall MEP HyperCelTM is a chromatography sorbent composed of a rigid cellulose matrix to which 4-Mercapto-Ethyl-Pyridine (4-MEP) is linked (see FIG. 6C ).
  • the cellulose bead confers high porosity, chemical stability and low non-specific interaction.
  • the average bead diameter is 80 ⁇ m to 100 ⁇ m.
  • Pall MEP HyperCelTM operates by a mixed-mode or multi-mode mechanism also described as Hydrophobic Charge Induction Chromatography (HCIC).
  • HCIC Hydrophobic Charge Induction Chromatography
  • Pall PPA HyperCelTM is a chromatography mixed-mode sorbent.
  • the sorbent material is based on high porosity crossed-linked cellulose and contains phenylpropylamine groups as ligands (see FIG. 6A ).
  • Pall HEA HyperCelTM is a chromatography mixed-mode sorbent.
  • the sorbent material is based on high porosity crossed-linked cellulose and contains hexylamine groups as ligands (see FIG. 6B ).
  • CaptoTM Adhere is a chromatography sorbent employing the ligand N-benzyl-N-methyl-ethanolamine immobilized on porous agarose particles. It is obtainable from GE Healthcare Life Sciences. The detailed chemical structure of this ligand is shown in FIG. 4 .
  • CaptoTM MMC is a chromatography sorbent employing a ligand derived from N-benzoyl-methionine immobilized on porous agarose particles. It is obtainable from GE Healthcare Life Sciences. The detailed chemical structure of this ligand is shown in FIG. 5 .
  • Eshmuno® HCX media is a mixed-mode resin that couples a tentacle structure (of the ligands) with a hydrophilic polyvinyl ether base matrix. More specifically, the base matrix is a surface grafted rigid polyvinyl ether hydrophilic polymer. The mean particle size (d 50 ) is 75 ⁇ m to 95 ⁇ m.
  • the detailed chemical structure of the ligand employed in Eshmuno® HCX media is shown in FIG. 7 .
  • the Eshmuno® HCX media is obtainable from Merck Millipore, Germany, or EMD Millipore, USA.
  • Alpha-galactosidase is a glycoside hydrolase enzyme that hydrolyses the terminal alpha-galactosyl moieties from glycolipids and glycoproteins. It is encoded by the GLA gene. This enzyme is a homodimeric glycoprotein that hydrolyses the terminal alpha-galactosyl moieties from glycolipids and glycoproteins. It predominantly hydrolyzes ceramide trihexoside, and it can catalyze the hydrolysis of melibiose into galactose and glucose.
  • Agalsidase alpha and beta are both recombinant forms of the human ⁇ -galactosidase A enzyme and both have the same amino acid sequence as the native enzyme.
  • Agalsidase alpha and beta differ in the structures of their oligosaccharide side chains.
  • the pharmaceutical company Shire manufactures agalsidase alpha under the brand name Replagal® as a treatment for Fabry's disease.
  • the pharmaceutical company Genzyme produces synthetic agalsidase beta under the brand name Fabrazyme® for treatment of Fabry's disease.
  • the present invention is directed to a method for purifying or enriching a biomolecule, said method comprising the steps:
  • aqueous buffer solution comprises, essentially consists of or consists of:
  • the method according to the first aspect provides several advantages as compared to previous methods for purifying or enriching biomolecules.
  • Previous methods of purifying or enriching biomolecules using multimodal chromatography typically employ elution buffers containing either high concentrations of arginine, glycine, or other amino acids (e.g., 1 M glycine, cf. example 1 of US 2011/0166332 A1; or 0.8 mol/kg L-arginine, cf. example 10 of US 2011/0160435 A1) or high concentrations of salt (e.g., up to 2.5 M of NH 4 Cl or NaCl, cf. example 6 of EP 2 167 526 B1).
  • high concentrations of arginine, glycine, or other amino acids e.g., 1 M glycine, cf. example 1 of US 2011/0166332 A1; or 0.8 mol/kg L-arginine, cf. example 10 of US 2011/0160435 A1
  • arginine and other additives exhibit a high conductivity which is disadvantageous in subsequent purification steps (e.g., ion exchange steps) that are often carried out after the multimodal resin chromatography.
  • a high conductivity may require large dilutions or extended times of dialysis.
  • high concentrations of salts can denature the quaternary structure or even the tertiary structure of biomolecules (e.g., proteins). The risk of denaturation is particularly high if the salt is a chaotropic agent (e.g., guanidinium salts or lithium perchlorate).
  • the method according to the first aspect provides the advantage that the desorption of the biomolecule is effected by an elution buffer containing only low concentrations of amino acids (such as between 0 and 400 mM, such as between 10 mM and 300 mM) and only low concentrations of salts or chaotropic agents (such as between 0 and 400 mM, such as between 10 mM and 300 mM), thereby reducing the risk of denaturing the biomolecule and facilitating subsequent purification steps.
  • This advantage is achieved by increasing the concentration of the Good's buffer in the elution buffer to values of at least 15 mM, at least 50 mM, at least 100 mM or at least 200 mM.
  • Good's buffers are well known for their use in biological research and typically have mild to no effects on biomolecules. Accordingly, the purification or enrichment method of the present invention is gentler than methods traditionally employed.
  • desorption of a biomolecule is effected in accordance with the invention either by a Good's buffer in a concentration of 15 mM or more (such as 50 mM, 100 mM or 200 mM or more) in combination with an amino acid in a concentration below 400 mM or by a Good's buffer in a concentration of 15 mM or more, such as 50 mM, 100 mM or 200 mM, in combination with ethylene glycol and/or propylene glycol in a total concentration of not more than 60% (v/v).
  • a Good's buffer in a concentration of 15 mM or more (such as 50 mM, 100 mM or 200 mM or more) in combination with an amino acid in a concentration below 400 mM or by a Good's buffer in a concentration of 15 mM or more, such as 50 mM, 100 mM or 200 mM, in combination with ethylene glycol and/or propylene glycol in a total concentration of not more than 60%
  • the aqueous buffer solution contains at least one amino acid (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), or ethylene glycol or propylene glycol or a mixture of ethylene glycol and propylene glycol.
  • the concentrations of ethylene glycol and propylene glycol are each 0%, if the aqueous buffer solution contains one or more amino acids.
  • the total concentration of said one or more amino acids is 0 mM, if the aqueous buffer solution contains ethylene glycol or propylene glycol or both.
  • the aqueous buffer solution comprises between 0% and 60% (v/v) (such as between 1% and 50% (v/v), between 5% and 40% (v/v), between 8% and 30% (v/v), between 10% and 25% (v/v), or between 15% and 20% (v/v)) of ethylene glycol.
  • the at least one amino acid is typically selected from the group consisting of alanine, asparagine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and valine.
  • aspartic acid, glutamic acid, tryptophan and tyrosine are absent.
  • the at least one amino acid is selected from the group consisting of alanine, cysteine, glycine, histidine, lysine, proline, serine, threonine, and valine.
  • asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine are typically absent.
  • the at least one amino acid is typically selected from the group consisting of alanine, glycine, histidine, and lysine.
  • asparagine, aspartic acid, cysteine, glutamic acid, glutamine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine are typically absent.
  • the at least one Good's buffer is selected from the group consisting of MES, MOPS, Tris, ADA, ACES, BES, Bicine, CAPS, CAPSO, CHES, HEPES, PIPES, TAPS, and TES.
  • the at least one Good's buffer is MES, and in another embodiment, the at least one Good's buffer is MOPS.
  • the biomolecule is a protein (such as an enzyme, a structural protein, a transport protein, a signaling protein, a transmembrane protein, a peptide hormone or an antibody) or a fragment thereof, or a nucleic acid (e.g., a single-stranded oligonucleotide, a double-stranded oligonucleotide, a single-stranded polynucleotide, or a double-stranded polynucleotide).
  • the nucleic acid can be, for example, DNA, RNA or a hybrid double-strand formed from DNA and RNA.
  • the biomolecule is an enzyme (e.g., an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, or a ligase).
  • the enzyme is a hydrolase, such as a glycosidase (often also termed glycoside hydrolase).
  • the glycosidase is an alpha-galactosidase, such as an alpha-galactosidase selected from the group consisting of agalsidase alpha (e.g., Replagal® manufactured by Shire, United Kingdom) and agalsidase beta (e.g., Fabrazyme® manufactured by Genzyme, Cambridge, Mass.).
  • the alpha-galactosidase is agalsidase beta.
  • the biomolecule is a peptide hormone, such as thyroid-stimulating hormone (also known as TSH or thyrotropin).
  • TSH is a glycoprotein consisting of two subunits: the alpha subunit and the beta subunit.
  • the biomolecule is an antibody, such as a monoclonal antibody.
  • the biomolecule is a glycosylated protein.
  • the multimodal resin is selected from the group consisting of CaptoTM Adhere, CaptoTM MMC, Pall PPA HyperCelTM, Pall HEA HyperCelTM, Pall MEP HyperCelTM, and Eshmuno® HCX media.
  • the ligands used in these multimodal resins on the filing day of the present application are shown in FIGS. 4 , 5 , 6 A, 6 B, 6 C, and 7 .
  • the multimodal resin is CaptoTM Adhere.
  • the present invention is directed to a purified biomolecule obtainable or obtained by the method according to the first aspect.
  • a biomolecule can be purified or enriched by a method that is gentler than the purification or enrichment methods typically known in the art. Accordingly, it is expected that the purified biomolecule is in a state that is more native than that of a purified biomolecule obtainable by methods of the prior art.
  • the aqueous buffer solution contains either at least one amino acid (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), or ethylene glycol or propylene glycol or a mixture of ethylene glycol and propylene glycol.
  • the concentrations of ethylene glycol and propylene glycol are each 0%, if the aqueous buffer solution contains one or more amino acids.
  • the total concentration of said one or more amino acids is 0 mM, if the aqueous buffer solution contains ethylene glycol or propylene glycol or both.
  • the aqueous buffer solution comprises between 0% and 60% (v/v) (such as between 1% and 50% (v/v), between 5% and 40% (v/v), between 8% and 30% (v/v), between 10% and 25% (v/v), or between 15% and 20% (v/v)) of ethylene glycol.
  • the at least one amino acid is typically selected from the group consisting of alanine, asparagine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and valine.
  • aspartic acid, glutamic acid, tryptophan and tyrosine are typically absent.
  • the at least one amino acid is typically selected from the group consisting of alanine, cysteine, glycine, histidine, lysine, proline, serine, threonine, and valine.
  • asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine are typically absent.
  • the at least one amino acid is typically selected from the group consisting of alanine, glycine, histidine, and lysine.
  • asparagine, aspartic acid, cysteine, glutamic acid, glutamine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine are typically absent.
  • the at least one Good's buffer is selected from the group consisting of MES, MOPS, Tris, ADA, ACES, BES, Bicine, CAPS, CAPSO, CHES, HEPES, PIPES, TAPS, and TES.
  • the at least one Good's buffer is MES, and in another embodiment, the at least one Good's buffer is MOPS.
  • the biomolecule is a protein (e.g., an enzyme, a structural protein, a transport protein, a signaling protein, a transmembrane protein, a peptide hormone or an antibody) or a fragment thereof, or a nucleic acid (e.g., a single-stranded oligonucleotide, a double-stranded oligonucleotide, a single-stranded polynucleotide, or a double-stranded polynucleotide).
  • the nucleic acid can be, for example, DNA, RNA or a hybrid double-strand formed from DNA and RNA.
  • the biomolecule is an enzyme (e.g., an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, or a ligase).
  • the enzyme can be a hydrolase, such as a glycosidase (often also termed glycoside hydrolase).
  • the glycosidase is an alpha-galactosidase, such as an alpha-galactosidase selected from the group consisting of agalsidase alpha (e.g., Replagal® manufactured by Shire, United Kingdom) and agalsidase beta (e.g., Fabrazyme® manufactured by Genzyme, Cambridge, Mass.).
  • the alpha-galactosidase is agalsidase beta.
  • the biomolecule is a peptide hormone, such as thyroid-stimulating hormone (also known as TSH or thyrotropin).
  • TSH is a glycoprotein consisting of two subunits: the alpha subunit and the beta subunit.
  • a recombinant form of human TSH-alpha is obtainable from Genzyme Corp. under the tradename “Thyrogen.”
  • the biomolecule is an antibody, such as a monoclonal antibody.
  • the biomolecule is a glycosylated protein.
  • the multimodal resin is selected from the group consisting of CaptoTM Adhere, CaptoTM MMC, Pall PPA HyperCelTM, Pall HEA HyperCelTM, Pall MEP HyperCelTM, and Eshmuno® HCX media.
  • the ligands used in these multimodal resins on the filing day of the present application are shown in FIGS. 4 , 5 , 6 A, 6 B, 6 C, and 7 .
  • the multimodal resin is CaptoTM Adhere.
  • the present invention is directed to a preparation comprising:
  • the aqueous buffer solution described herein involves a reduced risk of denaturing a biomolecule as compared to elution buffer solutions described in the art. Therefore, it is expected that the preparation according to the third aspect contains the biomolecule in a state that is more native than that of biomolecules in preparations described in the art.
  • the aqueous buffer solution contains either at least one amino acid (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), or ethylene glycol or propylene glycol or a mixture of ethylene glycol and propylene glycol.
  • the concentrations of ethylene glycol and propylene glycol are each 0%, if the aqueous buffer solution contains one or more amino acids.
  • the total concentration of said one or more amino acids is 0 mM, if the aqueous buffer solution contains ethylene glycol or propylene glycol or both.
  • the aqueous buffer solution comprises between 0% and 60% (v/v) (such as between 1% and 50% (v/v), between 5% and 40% (v/v), between 8% and 30% (v/v), between 10% and 25% (v/v), or between 15% and 20% (v/v)) of ethylene glycol.
  • the at least one amino acid is typically selected from the group consisting of alanine, asparagine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and valine.
  • aspartic acid, glutamic acid, tryptophan and tyrosine are typically absent.
  • the at least one amino acid is typically selected from the group consisting of alanine, cysteine, glycine, histidine, lysine, proline, serine, threonine, and valine.
  • asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine are typically absent.
  • the at least one amino acid is selected from the group consisting of alanine, glycine, histidine, and lysine.
  • asparagine, aspartic acid, cysteine, glutamic acid, glutamine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine are typically absent.
  • the at least one Good's buffer is selected from the group consisting of MES, MOPS, Tris, ADA, ACES, BES, Bicine, CAPS, CAPSO, CHES, HEPES, PIPES, TAPS, and TES.
  • the at least one Good's buffer is MES, and in another embodiment, the at least one Good's buffer is MOPS.
  • the biomolecule is a protein (e.g., an enzyme, a structural protein, a transport protein, a signaling protein, a transmembrane protein, a peptide hormone or an antibody) or a fragment thereof, or a nucleic acid (e.g., a single-stranded oligonucleotide, a double-stranded oligonucleotide, a single-stranded polynucleotide, or a double-stranded polynucleotide).
  • the nucleic acid can be, for example, DNA, RNA or a hybrid double-strand formed from DNA and RNA.
  • the biomolecule is an enzyme (e.g., an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, or a ligase).
  • the enzyme is a hydrolase, such as a glycosidase (often also termed glycoside hydrolase).
  • the glycosidase is an alpha-galactosidase, such as an alpha-galactosidase selected from the group consisting of agalsidase alpha (e.g., Replagal® manufactured by Shire, United Kingdom) and agalsidase beta (e.g., Fabrazyme® manufactured by Genzyme, Cambridge, Mass.).
  • the alpha-galactosidase is agalsidase beta
  • the biomolecule is a peptide hormone, such as thyroid-stimulating hormone (also known as TSH or thyrotropin).
  • TSH is a glycoprotein consisting of two subunits: the alpha subunit and the beta subunit.
  • a recombinant form of human TSH-alpha is obtainable from Genzyme Corp. under the tradename “Thyrogen”.
  • the biomolecule is an antibody, such as a monoclonal antibody.
  • the biomolecule is a glycosylated protein.
  • the present invention is directed to a use of an aqueous buffer solution for desorbing a biomolecule from a multimodal resin, wherein said aqueous buffer solution comprises, essentially consists of or consists of:
  • the aqueous buffer solution described herein is particularly well suited for desorbing biomolecules from a multimodal resin. It was especially unexpected that Good's buffers could be effective in desorbing biomolecules from a multimodal resin to an extent that is comparable to that of arginine.
  • the aqueous buffer solution contains either at least one amino acid (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), or ethylene glycol or propylene glycol or a mixture of ethylene glycol and propylene glycol.
  • the concentrations of ethylene glycol and propylene glycol are each 0%, if the aqueous buffer solution contains one or more amino acids.
  • the total concentration of said one or more amino acids is 0 mM, if the aqueous buffer solution contains ethylene glycol or propylene glycol or both.
  • the aqueous buffer solution comprises between 0% and 60% (v/v) (such as between 1% and 50% (v/v), between 5% and 40% (v/v), between 8% and 30% (v/v), between 10% and 25% (v/v), or between 15% and 20% (v/v)) of ethylene glycol.
  • the at least one amino acid is typically selected from the group consisting of alanine, asparagine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and valine.
  • aspartic acid, glutamic acid, tryptophan and tyrosine are typically absent.
  • the at least one amino acid is typically selected from the group consisting of alanine, cysteine, glycine, histidine, lysine, proline, serine, threonine, and valine.
  • asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine are typically absent.
  • the at least one amino acid is typically selected from the group consisting of alanine, glycine, histidine, and lysine.
  • asparagine, aspartic acid, cysteine, glutamic acid, glutamine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine are typically absent.
  • the at least one Good's buffer is selected from the group consisting of MES, MOPS, Tris, ADA, ACES, BES, Bicine, CAPS, CAPSO, CHES, HEPES, PIPES, TAPS, and TES.
  • the at least one Good's buffer is MES, and in another embodiment, the at least one Good's buffer is MOPS.
  • the biomolecule is a protein (e.g., an enzyme, a structural protein, a transport protein, a signaling protein, a transmembrane protein, a peptide hormone or an antibody) or a fragment thereof, or a nucleic acid (e.g., a single-stranded oligonucleotide, a double-stranded oligonucleotide, a single-stranded polynucleotide, or a double-stranded polynucleotide).
  • the nucleic acid can be, for example, DNA, RNA or a hybrid double-strand formed from DNA and RNA.
  • the biomolecule is an enzyme (e.g., an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, or a ligase).
  • the enzyme is a hydrolase, such as a glycosidase (often also termed glycoside hydrolase).
  • the glycosidase is an alpha-galactosidase, such as agalsidase alpha (e.g., Replagal® manufactured by Shire, United Kingdom) or agalsidase beta (e.g., Fabrazyme® manufactured by Genzyme, Cambridge, Mass.).
  • the alpha-galactosidase is agalsidase beta.
  • the biomolecule is a peptide hormone, such as thyroid-stimulating hormone (also known as TSH or thyrotropin).
  • TSH is a glycoprotein consisting of two subunits: the alpha subunit and the beta subunit.
  • a recombinant form of human TSH-alpha is obtainable from Genzyme Corp. under the tradename “Thyrogen”.
  • the biomolecule is an antibody, such as a monoclonal antibody.
  • the biomolecule is a glycosylated protein.
  • the multimodal resin is selected from the group consisting of CaptoTM Adhere, CaptoTM MMC, Pall PPA HyperCelTM, Pall HEA HyperCelTM, Pall MEP HyperCelTM, and Eshmuno® HCX media.
  • the ligands used in these multimodal resins on the filing day of the present application are shown in FIGS. 4 , 5 , 6 A, 6 B, 6 C, and 7 .
  • the multimodal resin is CaptoTM Adhere.
  • the present invention is directed to a use of an aqueous buffer solution for increasing the recovery rate of a biomolecule adsorbed on a multimodal resin by contacting (a) said multimodal resin onto which said biomolecule has been adsorbed with (b) said aqueous buffer solution, wherein said aqueous buffer solution comprises, essentially consists of or consists of:
  • the aqueous buffer solution described herein is particularly well-suited for desorbing biomolecules from a multimodal chromatography resin at a high recovery rate.
  • the inventors achieved recovery rates of up to 99% using aqueous buffer solutions featured in the invention (see Table 1).
  • the aqueous buffer solution contains either at least one amino acid (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), or ethylene glycol or propylene glycol or a mixture of ethylene glycol and propylene glycol.
  • the concentrations of ethylene glycol and propylene glycol are each 0%, if the aqueous buffer solution contains one or more amino acids.
  • the total concentration of said one or more amino acids is 0 mM, if the aqueous buffer solution contains ethylene glycol or propylene glycol or both.
  • the aqueous buffer solution comprises between 0% and 60% (v/v) (such as between 1% and 50% (v/v), between 5% and 40% (v/v), between 8% and 30% (v/v), between 10% and 25% (v/v), or between 15% and 20% (v/v)) of ethylene glycol.
  • the at least one amino acid is typically selected from the group consisting of alanine, asparagine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and valine.
  • aspartic acid, glutamic acid, tryptophan and tyrosine are typically absent.
  • the at least one amino acid is typically selected from the group consisting of alanine, cysteine, glycine, histidine, lysine, proline, serine, threonine, and valine.
  • asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine are typically absent.
  • the at least one amino acid is typically selected from the group consisting of alanine, glycine, histidine, and lysine.
  • asparagine, aspartic acid, cysteine, glutamic acid, glutamine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine are typically absent.
  • the at least one Good's buffer is selected from the group consisting of MES, MOPS, Tris, ADA, ACES, BES, Bicine, CAPS, CAPSO, CHES, HEPES, PIPES, TAPS, and TES.
  • the at least one Good's buffer is MES, and in another embodiment, the at least one Good's buffer is MOPS.
  • the biomolecule is a protein (e.g., an enzyme, a structural protein, a transport protein, a signaling protein, a transmembrane protein, a peptide hormone or an antibody) or a fragment thereof, or a nucleic acid (e.g., a single-stranded oligonucleotide, a double-stranded oligonucleotide, a single-stranded polynucleotide, or a double-stranded polynucleotide).
  • the nucleic acid can be, for example, DNA, RNA or a hybrid double-strand formed from DNA and RNA.
  • the biomolecule is an enzyme (e.g., an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, or a ligase).
  • the enzyme is a hydrolase, such as a glycosidase (often also termed glycoside hydrolase).
  • the glycosidase is an alpha-galactosidase, such as an alpha-galactosidase selected from the group consisting of agalsidase alpha (e.g., Replagal® manufactured by Shire, United Kingdom) and agalsidase beta (e.g., Fabrazyme® manufactured by Genzyme, Cambridge, Mass.).
  • the alpha-galactosidase is agalsidase beta.
  • the biomolecule is a peptide hormone, such as thyroid-stimulating hormone (also known as TSH or thyrotropin).
  • TSH is a glycoprotein consisting of two subunits: the alpha subunit and the beta subunit.
  • a recombinant form of human TSH-alpha is obtainable from Genzyme Corp. under the tradename “Thyrogen”.
  • the biomolecule is an antibody, such as a monoclonal antibody.
  • the biomolecule is a glycosylated protein.
  • the multimodal resin is selected from the group consisting of CaptoTM Adhere, CaptoTM MMC, Pall PPA HyperCelTM, Pall HEA HyperCelTM, Pall MEP HyperCelTM, and Eshmuno® HCX media.
  • the ligands used in these multimodal resins on the filing day of the present application are shown in FIGS. 4 , 5 , 6 A, 6 B, 6 C, and 7 .
  • the multimodal resin is CaptoTM Adhere.
  • the present invention is directed to an article of manufacture comprising:
  • the aqueous buffer solution featured in the invention involves a reduced risk of denaturing a biomolecule as compared to elution buffer solutions described in the prior art.
  • the article of manufacture according to the sixth aspect provides this advantageous aqueous buffer solution in combination with a container or packaging material and with a data carrier containing instructions for carrying out a method that makes use of this advantageous aqueous buffer solution.
  • the preparation according to the present aspect contains the biomolecule in a state that is more native than that of biomolecules in preparations described in the prior art.
  • the data carrier is selected from the group consisting of a label present on the packaging material or container (e.g., in the form of an adhesive label, a tag, a chip, or an RFID tag) and a packaging insert (e.g., a leaflet; a booklet; a chip; or a computer-readable storage medium, such as a floppy disk, a CD-ROM, a CD-R, a DVD, or a blue-ray disk).
  • the data carrier comprises written information, visual information (e.g., pictures), or computer-readable information (e.g., a program or a bar code).
  • the data carrier further comprises one or more of the following information:
  • the article of manufacture further comprises one or more of the following components:
  • a chromatography medium (d) a chromatography medium; (e) a chromatography column; (f) a reference biomolecule usable for calibrating a method for purifying or enriching a biomolecule, such as for calibrating a method according to the first aspect.
  • the chromatography column is prepacked with the chromatography medium.
  • the chromatography medium is a multimodal chromatography resin.
  • the multimodal chromatography resin is typically selected from the group consisting of CaptoTM Adhere, CaptoTM MMC, Pall PPA HyperCelTM, Pall HEA HyperCelTM, Pall MEP HyperCelTM, and Eshmuno® HCX media.
  • the ligands used in these multimodal resins are shown in FIGS. 4 , 5 , 6 A, 6 B, 6 C, and 7 .
  • the multimodal resin is CaptoTM Adhere.
  • the reference biomolecule is selected from the group consisting of a protein (e.g., an enzyme, a structural protein, a transport protein, a signaling protein, a transmembrane protein, a peptide hormone or an antibody) and a nucleic acid (e.g., a single-stranded oligonucleotide, a double-stranded oligonucleotide, a single-stranded polynucleotide, or a double-stranded polynucleotide).
  • the nucleic acid can be DNA, RNA or a hybrid double-strand formed from DNA and RNA.
  • the reference biomolecule is an enzyme (e.g., an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, or a ligase).
  • the reference enzyme can be, for example, a hydrolase, such as a glycosidase (often also termed glycoside hydrolase).
  • the reference glycosidase is an alpha-galactosidase, such as an agalsidase alpha (e.g., Replagal® manufactured by Shire, United Kingdom) or an agalsidase beta (e.g., Fabrazyme® manufactured by Genzyme, Cambridge, Mass.).
  • the reference alpha-galactosidase is agalsidase beta.
  • the reference biomolecule is a peptide hormone, such as thyroid-stimulating hormone (also known as TSH or thyrotropin).
  • TSH is a glycoprotein consisting of two subunits: the alpha subunit and the beta subunit.
  • a recombinant form of human TSH-alpha is obtainable from Genzyme Corp. under the tradename “Thyrogen”.
  • the reference biomolecule is an antibody, such as a monoclonal antibody.
  • the reference biomolecule is a glycosylated protein.
  • the aqueous buffer solution contains either at least one amino acid (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), or ethylene glycol or propylene glycol or a mixture of ethylene glycol and propylene glycol.
  • the concentrations of ethylene glycol and propylene glycol are each 0%, if the aqueous buffer solution contains one or more amino acids.
  • the total concentration of said one or more amino acids is 0 mM, if the aqueous buffer solution contains ethylene glycol or propylene glycol or both.
  • the aqueous buffer solution comprises between 0% and 60% (v/v) (such as between 1% and 50% (v/v), between 5% and 40% (v/v), between 8% and 30% (v/v), between 10% and 25% (v/v), between 15% and 20% (v/v)) of ethylene glycol.
  • the at least one amino acid is typically selected from the group consisting of alanine, asparagine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and valine.
  • aspartic acid, glutamic acid, tryptophan and tyrosine are absent.
  • the at least one amino acid is selected from the group consisting of alanine, cysteine, glycine, histidine, lysine, proline, serine, threonine, and valine.
  • asparagine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, methionine, phenylalanine, tryptophan and tyrosine are absent.
  • the at least one amino acid is typically selected from the group consisting of alanine, glycine, histidine, and lysine.
  • asparagine, aspartic acid, cysteine, glutamic acid, glutamine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine are typically absent.
  • the at least one Good's buffer is selected from the group consisting of MES, MOPS, Tris, ADA, ACES, BES, Bicine, CAPS, CAPSO, CHES, HEPES, PIPES, TAPS, and TES.
  • the at least one Good's buffer is MES.
  • the at least one Good's buffer is MOPS.
  • a biomolecule agalsidase beta (Fabrazyme® manufactured by Genzyme, Cambridge, Mass.) was bound on multimodal chromatographic resin CaptoTM Adhere (GE Healthcare) and desorbed using MES and MOPS (see Table 1 and FIGS. 1 and 2 ).
  • the table shows the detailed experimental design, where a range of MES concentrations from 50 mM to 200 mM and MOPS concentrations from 20 mM to 200 mM was tested in the presence of 150 mM to 400 mM arginine within a pH range of 5.5 to 7.5. Yield was monitored in these experiments as an indicator of desorption from the chromatographic resin. Under a given buffer condition, 0% yield means no desorption and 100% means complete desorption of the biomolecule. The experimental design and analysis was done using Design-Expert®.
  • FIG. 1 and FIG. 2 show contour plots for yield with MES at pH 6.25 and MOPS at pH 7.0, respectively.
  • yield increases monotonically with increasing Arginine concentration.
  • yield increases monotonically with MES and MOPS concentration as well.
  • MES was found to be at least as effective as arginine in desorption, whereas, MOPS was slightly less effective than arginine.
  • both MES and MOPS act similar to additives like arginine in desorbing the biomolecule from the multimodal resin.
  • MES and MOPS are considered to be particularly suitable additives for elution of biomolecules in downstream processing. This is because (i) MES and MOPS solutions are of lower conductivity than arginine and guanidine, allowing easier processing on a following ion exchange step; and (ii) solutions containing MES and MOPS are expected to be less disruptive for biomolecules than other additives, because biomolecular solutions of MES and MOPS are regularly used in biochemistry as part of Good's buffer systems.
  • MES and MOPS can be used in desorption of biomolecules from other multimodal chromatographic resins available, such as Pall PPA HyperCelTM, Pall HEA HyperCelTM, Pall MEP HyperCelTM, CaptoTM MMC, and potentially various others multimodal resins which combine hydrophobic, ionic and/or hydrogen bonding interactions.
  • Example 2 The experiment from Example 1 was modified in that arginine was replaced by ethylene glycol.
  • the contour plot of this experiment is shown in FIG. 3 .
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US11203747B2 (en) 2021-12-21
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CA2888824C (fr) 2021-02-02
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HK1222868A1 (zh) 2017-07-14
SG11201502918RA (en) 2015-05-28
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