WO1998013119A1 - Cristaux proteiniques reticules utilises comme supports de separation universels - Google Patents

Cristaux proteiniques reticules utilises comme supports de separation universels Download PDF

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Publication number
WO1998013119A1
WO1998013119A1 PCT/US1997/017167 US9717167W WO9813119A1 WO 1998013119 A1 WO1998013119 A1 WO 1998013119A1 US 9717167 W US9717167 W US 9717167W WO 9813119 A1 WO9813119 A1 WO 9813119A1
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crystals
crosslinked
chromatography
separation media
protein crystals
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PCT/US1997/017167
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English (en)
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Alexey L. Margolin
Lev Z. Vilenchik
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Altus Biologics Inc.
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Priority to AU47381/97A priority Critical patent/AU4738197A/en
Publication of WO1998013119A1 publication Critical patent/WO1998013119A1/fr

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    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/06—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising oxides or hydroxides of metals not provided for in group B01J20/04
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/06—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising oxides or hydroxides of metals not provided for in group B01J20/04
    • B01J20/08—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising oxides or hydroxides of metals not provided for in group B01J20/04 comprising aluminium oxide or hydroxide; comprising bauxite
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
    • B01J20/103—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate comprising silica
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
    • B01J20/16—Alumino-silicates
    • B01J20/165—Natural alumino-silicates, e.g. zeolites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
    • B01J20/267—Cross-linked polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/283—Porous sorbents based on silica
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/284—Porous sorbents based on alumina
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/285—Porous sorbents based on polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/29—Chiral phases
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3204—Inorganic carriers, supports or substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens

Definitions

  • the present invention relates to the use of crosslinked protein crystals in methods, apparatus and systems for separating a substance or molecule of interest from a sample.
  • crosslinked protein crystals are used in chromatographic methods, apparatus and systems in which separation is based on a physical or chemical property of that substance or molecule of interest.
  • the crosslinked protein crystals which characterize the methods, apparatus and systems of this invention possess excellent mechanical strength and well developed porous structure, demonstrate significant affinity and chiral selectivity and are extremely stable in organic solvents and aqueous-organic solvent mixtures. These properties render the crystals particularly useful as sorbents for separations, including size exclusion, affinity and chiral chromatography.
  • Chromatography is a variation of a dynamic adsorption process in a two-phase system, in which a mixture of substances or a crude substance migrates through a porous medium with a gas or solvent flow. Individual components of the mixture or, contaminants from the crude substance, are separated according to their respective sorption activities [B.G. Belenkii and L.Z. Vilenchik, Modern Liquid Chromatography of Macromolecules . Elsevier Press, Amsterdam-Oxford-New York-Tokyo (1983); J.C. Giddings, Dynamics of Chromatography. Marcel Dekker, New York (1965); L.R. Snyder and J.J. Kirkland, Introduction to Modern Liquid Chromatography. Wiley-Interscience, New York (1979)].
  • chromatography is divided into gas and liquid chromatography. It is also divided according to the type of sorbent used as the stationary phase.
  • chromatography types include size exclusion [J. Porath and P. Flodin, Nature. 183, p. 1651 (1959); J.C. Moore, J. Polvm. Sci.. A, p. 835 (1964)], affinity [J.A. Jonsson, Ed., Chromatographic Theory and Basic Principles. Marcel Dekker, New York (1987) ] and chiral chromatography [J. Hermansson et al., in M. Ziefand and L. Crane, Eds., Chromatographic Chiral Separations. 40, pp. 245-81, Marcel Dekker, New York (1987)].
  • Macroporous sorbents which are adsorptionally inert with respect to the molecules of the substances undergoing chromatography, are used in size exclusion chromatography to separate molecules according to their hydrodynamic size.
  • the method is applied to macromolecules ranging from oligomers (with molecular weights of a few 100 to 10,000 daltons) to polymers (with molecular weights of 10,000 to a few million daltons).
  • macromolecules ranging from oligomers (with molecular weights of a few 100 to 10,000 daltons) to polymers (with molecular weights of 10,000 to a few million daltons).
  • stationary phases are employed in size exclusion chromatography.
  • Macromolecular crosslinked swelling polymer gels, macroporous polymer sorbents and macroporous silicate sorbents are those primarily used fBeschii and Vilenchik. supra! .
  • Ion exchange chromatography separates molecules, such as proteins, that have an electrical charge. This type of chromatography is widely used for preparative scale separations. Different types of adsorption chromatography are employed to separate and analyze low molecular weight substances. Adsorption chromatography is divided into three major categories, according to the interaction between the stationary phase and molecules of the substances to be separated: reverse-phase chromatography (hydrophobic interaction with bonded non-polar groups of the stationary phase) ; affinity chromatography (biospecific sorption) and chiral chromatography (steric interactions that can involve hydrogen bonding, dipole-dipole, ⁇ - ⁇ , electrostatic and hydrophobic interaction) . Affinity chromatography sorbents are typically porous materials [P.
  • Chiral chromatography is of special interest to the industrial-scale synthesis of specialty chemicals, pharmaceuticals and agrochemicals .
  • Many drugs and pesticides contain at least one chiral center. Enantiomeric purity is an important factor in the biological activity and safety of these materials.
  • the FDA has called for complete pharmacodynamics and pharmacokinetics on the individual isomers of proposed pharmaceutical agents. As a result, most new chiral drugs will be developed as a single enantiomer.
  • a third limitation is narrow operational conditions. Protein chiral stationary phases are effective only in a narrow range of conditions.
  • an Ultron ES-OVM column can function in the range of pH 3-7.5 (the same limitation exists for Pirkle phases) , at temperatures lower than 40°C and in eluents containing less than 50% organic solvent.
  • a fourth limitation relates to high cost.
  • the price of the majority of small analytical chiral chromatography columns is between $1,000 - $2,000.
  • the price of bulk chiral stationary phase is around $8,000 per kg, making it the greatest single element of the cost of chiral chromatographic separations.
  • PCT patent application WO95/09907 relates to methods using crystals of biological macromolecules to separate compounds from mixtures, based on selective binding affinity.
  • the chromatography features of protein crystals have not been commercially exploited, due to widely held misconceptions. These include perceived difficulties of protein crystal preparation on large scales; perceived general instability of protein crystals and perceived mechanical fragility of protein crystals [M.A. Navia et al., Stability and Stabilization of Enzvmes. W.J.J. van den Tweel et al. Eds., Elsevier, .Amsterdam, pp. 63- 73 (1993) ⁇ .
  • Crosslinked enzyme crystal (“CLEC®”) technology put an end to misconceptions surrounding protein crystal preparation [N.L. St. Clair and M.A. Navia, "Cross-Linked Enzyme Crystals as Robust Biocatalysts", J. Am. Chem. Soc. 114, pp. 7314-16 (1992)].
  • Crosslinked enzyme crystals retain their activity in environments that are normally incompatible with enzyme (soluble or immobilized) function. Such environments include prolonged exposure to high temperature and extreme pH. Additionally, in organic solvents and aqueous-organic solvent mixtures, crosslinked enzyme crystals exhibit both stability and activity far beyond that of their soluble or conventionally-immobilized counterparts.
  • Crosslinked enzyme crystals represent an important advance in the area of biocatalysis, as attractive and broadly applicable catalysts for organic synthesis reactions
  • CLEC® technology represents a general stabilization technology, providing proteins characterized by increased stability against heat, organic solvents and exogenous proteolysis [C.
  • crosslinked protein crystals also represent a means for satisfying the present need for commercially and technically feasible separation media for techniques, devices and systems, including those based on chromatography.
  • the present invention provides methods, apparatus and systems using crosslinked protein crystals for separating a substance or molecule of interest from a sample, based on a physical or chemical property of the substance or molecule of interest.
  • Such separation methods are characterized by the step of contacting the crosslinked protein crystals with the substance or molecule of interest by any means, for a sufficient time and under conditions which permit said protein to retain said substance or molecule of interest, thus separating it from said sample.
  • crosslinked protein crystals may be used in place of or in combination with conventional sorbents in methods, devices and systems for separating molecules of interest from mixtures or crude preparations thereof. Separation methods, apparatus and systems using crosslinked protein crystals according to this invention are advantageously characterized by higher degrees of sensitivity, volumeric productivity and throughput than those of methods, apparatus and systems based on conventional sorbents .
  • Chromatographic techniques and systems which may be carried out using crosslinked protein crystals include size exclusion chromatography, adsorption chromatography (for example, reverse phase chromatography) , affinity chromatography and chiral chromatography. Such types of chromatography may be advantageously carried out in the presence of an aqueous solvent, organic solvent or an aqueous-organic solvent mixture.
  • FIG. 1 is a graph representing the pore size distribution of crosslinked thermolysin crystals (TR-3) useful in this invention.
  • Figure 2 is a graph representing the distribution coefficient as a function of the root mean square radius of polyethylene glycol ("PEG") macromolecules run on a chromatographic column packed with crosslinked thermolysin crystals (TR-3) .
  • Figure 3 is a graph representing the relationship between the root mean square radius of PEG macromolecules and retention times on a chromatographic column packed with crosslinked thermolysin crystals
  • Figure 4 is a graph representing the relationship between the molecular weight of PEG macromolecules and retention times on a chromatographic column packed with crosslinked thermolysin crystals
  • Figures 5 and 6 are chromatograms illustrating the separation of PEG samples by size exclusion chromatography on a chromatographic column packed with crosslinked thermolysin crystals (TR-3) .
  • Figure 5 is a chromatogram of three PEG standards with average molecular weights of 62, 600 and 10,000 daltons.
  • Figure 6 illustrates the separation of six PEG standards with average molecular weights of 62,
  • Figure 7 is a chromatogram illustrating the separation of phenylacetic acid, hydroxyphenylglycine and R-phenylglycine by affinity chromatography on a chromatographic column packed with crosslinked thermolysin crystals (TR-3) .
  • Figure 8 is a chromatogram illustrating the separation of phenylacetic acid, hydroxyphenylglycine, R-phenylglycine and L-phenylalaninamide by affinity chromatography on a chromatographic column packed with crosslinked thermolysin crystals (TR-3) .
  • Figure 9 is a chromatogram illustrating the separation of a mixture of D-isomer of phenyllactic acid and ibuprofen by affinity chromatography on a chromatographic column packed with crosslinked thermolysin crystals (TR-3) .
  • Figure 10 is a chromatogram of flurbiprofen on a chromatographic column packed with crosslinked thermolysin crystals (TR-3) .
  • Figure 11 is a chromatogram illustrating the separation of a mixture of flurbiprofen and ibuprofen by affinity chromatography on a chromatographic column packed with crosslinked thermolysin crystals (TR-3) .
  • Figure 12 is a chromatogram illustrating the separation of a mixture of the D and L optical isomers of phenyllactic acid by chiral chromatography on chromatographic columns packed with crosslinked thermolysin crystals (TR-2) and (TR-3) in series.
  • Figure 13 is a chromatogram illustrating the separation of a mixture of the D and L optical isomers of phenyllactic acid by chiral chromatography on a chromatographic column packed with crosslinked thermolysin crystals (TR-2) .
  • Figure 14 is a chromatogram illustrating the separation of a mixture of the R and S optical isomers of phenylglycine by chiral chromatography on chromatographic columns packed with crosslinked thermolysin crystals (TR-2) and (TR-3) (injection volume 10 ⁇ l) .
  • Figure 15 is a chromatogram illustrating the separation of a mixture of the R and S optical isomers of phenylglycine by chiral chromatography on chromatographic columns packed with crosslinked thermolysin crystals (TR-2) and (TR-3) (injection volume 1.5 ⁇ l).
  • Figure 16 is a graph representing the pore size distribution of crosslinked Candida rugosa lipase crystals useful in this invention.
  • Figure 17 is a graph representing the distribution coefficient as a function of the root mean square radius of PEG macromolecules run on a chromatographic column packed with crosslinked Candida rugosa lipase crystals.
  • Figure 18 is a graph representing the relationship between the root mean square radius of PEG macromolecules and retention times on a chromatographic column packed with crosslinked Candida ru ⁇ osa lipase crystals.
  • Figure 19 is a graph representing the relationship between the molecular weight of PEG macromolecules and retention times on a chromatographic column packed with crosslinked Candida ru ⁇ osa lipase crystals.
  • Figure 20 is a chromatogram illustrating the separation of PEG samples by size exclusion chromatography on a chromatographic column packed with crosslinked Candida rugosa lipase crystals.
  • the figure includes chromatograms of four PEG standards with average molecular weights of 62, 400, 1,500 and 10,000 daltons .
  • Figure 21 is a chromatogram illustrating the separation of PEG samples by size exclusion chromatography on a chromatographic column packed with crosslinked Candida rugosa lipase crystals.
  • the figure includes chromatograms of six PEG standards with average molecular weights of 62, 106, 194, 400, 1,500 and 10,000 daltons separated in 50% acetonitrile.
  • Figure 22 is a chromatogram illustrating the separation of PEG samples by size exclusion chromatography on a chromatographic column packed with crosslinked Candida rugosa lipase crystals.
  • the figure includes chromatograms of four PEG standards with average molecular weights of 194, 900, 1,500 and 5,000 daltons .
  • Figure 23 is a graph representing the pore size distribution of crosslinked Pseudomonas cepacia lipase crystals useful in this invention.
  • Figure 24 is a graph representing the distribution coefficient as a function of the root mean square radius of PEG macromolecules run on a chromatographic column packed with crosslinked
  • Figure 25 is a graph representing the relationship between the root mean square radius of PEG macromolecules and retention times on a chromatographic column packed with crosslinked Pseudomonas cepacia lipase crystals.
  • Figure 26 is a graph representing the relationship between the molecular weight of PEG macromolecules and retention times on a chromatographic column packed with crosslinked Pseudomonas cepacia lipase crystals.
  • Figure 27 is a chromatogram illustrating the separation of a mixture of phenylacetic acid, D-4- hydroxyphenylglycine and R-phenylglycine by affinity chromatography on a chromatographic column packed with crosslinked Pseudomonas cepacia lipase crystals.
  • Figure 28 is a chromatogram illustrating the separation of a mixture of ibuprofen and L-phenyllactic acid by affinity chromatography on a chromatographic column packed with crosslinked Pseudomonas cepacia lipase crystals.
  • Figure 29 is a chromatogram illustrating the separation of a mixture of ibuprofen and flurbiprofen by affinity chromatography on a chromatographic column packed with crosslinked Pseudomonas cepacia lipase crystals.
  • Figure 30 is a chromatogram illustrating the separation of a mixture of ketoprofen, suprofen and flurbiprofen by affinity chromatography on a chromatographic column packed with crosslinked Pseudomonas cepacia lipase crystals.
  • Figure 31 is a chromatogram illustrating the separation of a mixture of the R and S optical isomers of methylmandelate by chiral chromatography on a chromatographic column packed with crosslinked Pseudomonas cepacia lipase crystals.
  • Figure 32 is a graph representing the pore size distribution of crosslinked penicillin acylase crystals useful in this invention.
  • Figure 33 is a graph representing the distribution coefficient as a function of the root mean square radius of PEG macromolecules run on a chromatographic column packed with crosslinked penicillin acylase crystals.
  • Figure 34 is a graph representing the relationship between the root mean square radius of PEG macromolecules and retention times on a chromatographic column packed with crosslinked penicillin acylase crystals.
  • Figure 35 is a graph representing the relationship between the molecular weight of PEG macromolecules and retention times on a chromatographic column packed with crosslinked penicillin acylase crystals.
  • Figure 36 is a graph representing the pore size distribution of crosslinked bovine serum albumin crystals useful in this invention.
  • Figure 37 is a graph representing the distribution coefficient as a function of the root mean square radius of PEG macromolecules run on a chromatographic column packed with crosslinked bovine serum albumin crystals.
  • Figure 38 is a graph representing the relationship between the root mean square radius of PEG macromolecules and retention times on a chromatographic column packed with crosslinked bovine serum albumin crystals.
  • Figure 39 is a graph representing the relationship between the molecular weight of PEG macromolecules and retention times on a chromatographic column packed with crosslinked bovine serum albumin crystals.
  • Figure 40 is a chromatogram illustrating the separation of PEG samples by size exclusion chromatography on a chromatographic column packed with crosslinked bovine serum albumin crystals.
  • the figure includes chromatograms of four PEG standards with average molecular weights of 62, 400, 1,500 and 10,000 daltons .
  • Figure 41 is a chromatogram illustrating the separation of a mixture of ketoprofen, suprofen and naproxen by affinity chromatography on a chromatographic column packed with crosslinke ⁇ bovine serum albumin crystals.
  • Figure 42 is a chromatogram illustrating the separation of a mixture of the D and L optical isomers of phenyllactic acid by chiral chromatography on a chromatographic column packed with crosslinked bovine serum albumin crystals.
  • Figure 43 is a chromatogram illustrating the separation of PEG samples by size exclusion chromatography on a chromatographic column packed with crosslinked human serum albumin crystals.
  • the figure includes chromatograms of two PEG standards with average molecular weights of 400 and 5,000 daltons.
  • Figure 44 is a chromatogram illustrating the separation of a mixture of S ibuprofen and R phenyllactic acid by adsorption chromatography on a chromatographic column packed with crosslinked thermolysin crystals (TR-2) .
  • Figure 45 is a chromatogram illustrating the separation of a mixture of the R and S optical isomers of phenylglycine by chiral chromatography on a chromatographic column packed with crosslinked thermolysin crystals (TR-2) .
  • Figure 46 is a chromatogram illustrating the separation of the chiral compound folinic acid on a chromatographic column packed with crosslinked human serum albumin crystals (particle size 10 ⁇ ) .
  • Figure 47 is a chromatogram illustrating the separation of the chiral compound 2-phenylprop ⁇ on ⁇ c acid on a chromatographic column packed with crosslinked human serum albumin crystals.
  • Figure 48 is a chromatogram illustrating the separation of the chiral compound N-2, -DNP-DL- -amino- n-butyric acid on a chromatographic column packed with crosslinked human serum albumin crystals.
  • Figure 49 is a chromatogram illustrating the separation of the chiral compound N-2, 4-DNP-DL-glutamic acid on a chromatographic column packed with crosslinked human serum albumin crystals.
  • Figure 50 is a chromatogram illustrating the separation of the chiral compound phenylglycine on a chromatographic column packed with crosslinked human serum albumin crystals.
  • Figure 51 is a chromatogram illustrating the separation of the chiral compound N-2, 4-DNP-citrulline on a chromatographic column packed with crosslinked human serum albumin crystals.
  • Figure 52 is a chromatogram illustrating the separation of the chiral compound folinic acid on a chromatographic column packed with crosslinked human serum albumin crystals (particle size 25 ⁇ ) .
  • Figure 53 is a chromatogram illustrating the separation of the chiral compound folinic acid on a chromatographic column packed with crosslinked human serum albumin crystals (particle size 3-5 ⁇ ) .
  • Separation Based on Physical or Chemical Properties separation by means other than specific binding affinity between the active binding site of the protein component of the crosslinked protein crystals and the substance to be isolated, i.e., the substance is not a substrate, substrate analog, ligand or inhibitor of the protein.
  • separation or purification based on physical or chemical properties include those based on molecular weight, molecular size, solubility, charge, hydrophobicity, hydrophilicity, polarity and chirality.
  • Separation Separation of a substance from a mixture of two or more different substances, i.e., substances having different physical or chemical properties, or two or more forms of the same substance.
  • separation is defined as purification of a substance from a crude form thereof. Separation may be carried out by any means including, for example, chromatography, membrane separation, filtration, electrophoresis and simulated moving bed technology.
  • Sample a mixture of two or more different substances or two or more forms of the same substance.
  • a sample may be a crude form of a substance.
  • Organic Solvent any solvent of non-aqueous origin.
  • a ⁇ ueous-Or ⁇ anic Solvent Mixture a mixture comprising n% organic solvent, where n is between 1 and 99 and m% aqueous, where m is 100 - n.
  • Crosslinked protein crystals such as crosslinked enzyme crystals, grown from aqueous solution and crosslinked with a bifunctional agent, such as glutaraldehyde, exhibit remarkable characteristics, that are superior to both soluble and conventionally immobilized enzymes [St. Clair and Navia, supra1.
  • crosslinked protein crystals produced using CLEC® technology are also mechanically stable under extreme conditions of temperature and pH, making them excellent candidates for chromatography media.
  • protein crystals are macroporous materials.
  • solvent constitutes from 30% to 65% of crystal weight [B.W. Matthews, J. Mol. Biol.. 33, pp. 491-97 (1968)].
  • the uniform solvent-filled channels traverse the body of a crystal and thus, facilitate the transport of substances in and out of the crystal.
  • the diameter of the channels depends on the nature of the protein and its crystal form, and ranges from 20 to 100 A, that allows for macromolecules with molecular weight up to 100,000 daltons to penetrate inside the crystals with different probability, and therefore to be separated according to their size.
  • protein crystals are asymmetric molecules made of L-amino acids and can, in principle, provide stereoselective sorption of chiral ligands. Given the fact that proteins are weak ion-exchangers with isoelectric points from 2 to 12, one can easily manipulate binding of small molecules by changing pH and buffer content of the eluent.
  • Crosslinked protein crystals are useful in all types of chromatography that use a solid as either a stationary phase or support thereof. Given their high mechanical stability, affinity and enantioselectivity, crosslinked protein crystals are advantageously useful in separation of molecules via at least three different mechanisms: size exclusion, affinity and chiral. By virtue of these characteristics, crosslinked protein crystals constitute universal separation media, enabling use of columns packed with the same type crystals to perform size exclusion, affinity and chiral chromatography.
  • chromatography may be carried out using crosslinked protein crystals.
  • Such techniques include, but are not limited to, reverse phase chromatography, high pressure liquid chromatography, low pressure liquid chromatography, gel filtration chromatography, gel permeation chromatography, batch chromatography, ion exchange chromatography, elution chromatography, electrochro atography, flat-bed chromatography, thin layer chromatography, paper chromatography, simulated moving bed chromatography, column gel electrophoresis and capillary gel electrophoresis. It will be appreciated by those of skill in the art that, the precise parameters for these various techniques may be determined without undue experimentation.
  • crosslinked protein crystals achieve uniformity across the entire crosslinked crystal volume. This uniformity is maintained by the intermolecular contacts and chemical crosslinks between the protein molecules constituting the crystal lattice, even when exposed to organic or mixed aqueous-organic solvents. In such solvents, the protein molecules maintain a uniform distance from each other, forming well-defined stable pores within the crosslinked protein crystals that facilitate access of substrate to the catalyst, as well as removal of product. In these crosslinked protein crystals, the lattice interactions, when fixed by chemical crosslinks, are particularly important in preventing denaturation, especially in organic solvents or mixed aqueous-organic solvents.
  • crosslinked protein crystals and the constituent proteins within the crystal lattice remain monodisperse in organic solvents, thus avoiding the problem of aggregation.
  • These features of crosslinked protein crystals contribute to their utility in separations involving harsh solvent environments that may be components of many preparative or analytical scale preparations.
  • crosslinked protein crystals are particularly resistant tc proteolysis, as well as temperature and pH extremes.
  • crosslinked protein crystals are characterized by stability and integrity under elution conditions used in separations, particularly chromatography elution conditions, as compared with the soluble uncrosslinked form of the protein that is crystallized to form the protein crystals that are crosslinked.
  • crosslinked protein crystals are characterized by stability and integrity in the presence of a solvent contained in the sample to be separated, as compared with the soluble uncrosslinked form of the protein that is crystallized to form the protein crystals that are crosslinked.
  • the crosslinked protein crystals may be used for separations involved in any number of chemical processes. Such processes include industrial and research-scale processes, such as organic syn-hesis of specialty chemicals and pharmaceuticals, synthesis of intermediates for the production of such products, chiral synthesis and resolution for optically pure pharmaceutical and specialty chemicals.
  • Products which may be separated include macromolecules, such as oligomers, polymers and copolymers, small organic molecules, such as chiral organic molecules, peptides, proteins, carbohydrates, nucleic acids, lipids and other chemical species. Examples of macromolecules include, flexible chain macromolecules having a molecular weight of about 10,000 daltons or less and globular proteins having a molecular weight of about 20,000 daltons or less.
  • Organic solvents may be selected from the group consisting of hydrophobic solvents, hydrophilic solvents and mixtures thereof.
  • hydrophobic solvents include ethers, polyethers, ethers of poly (ethylene glycol), toluene, octane, isooctane, hexane and cyclohexane.
  • hydrophilic solvents examples include alcohols, diols, polyols, methanol, ethanol, isopropanol, tetrahydrofuran, acetonitrile, acetone, pyridine, diethylene glycol, 2-methyl-2, 4- pentanediol, poly (ethylene glycol), triethylene glycol, 1, 4-butanediol, 1, 2-butanediol, 2, 3, -dimethyl-2, 3- butanediol, acetonitrile, and polyvinylpyrrolidone, or mixtures thereof.
  • Crosslinked protein crystals may also be used for air purification in conjunction with air filtration. For example, air may be passed through a column packed with crosslinked protein crystals to filter out any unwanted contaminants.
  • crosslinked protein crystals are useful as separation media in devices such as, for example, sensing devices.
  • Crosslinked protein crystals useful in the methods, devices and systems of this invention may be prepared by the steps of crystallizing and crosslinking the protein, which may be carried out as described in PCT patent application WO92/02617, which is incorporated herein by reference.
  • crosslinked protein crystals may be prepared as illustrated below.
  • Protein crystals are grown by the controlled precipitation of protein out of aqueous solution or aqueous solution-containing organic solvents. Conditions to be controlled include, for example, the rate of evaporation of solvent, the presence of appropriate co-solutes and buffers, pH and temperature.
  • Conditions to be controlled include, for example, the rate of evaporation of solvent, the presence of appropriate co-solutes and buffers, pH and temperature.
  • an intelligent trial and error search strategy can, in most instances, produce suitable crystallization conditions for many proteins, provided that an acceptable level of purity can been achieved for them [see e.g., C.W. Carter, Jr. and C.W. Carter, J. Biol. Chem.. 254, pp. 12219-23 (1979)].
  • the protein constituent of the crosslinked protein crystals may be any protein including, for example, an enzyme, antibody or receptor.
  • Proteins which may be crystallized to form crosslinked protein crystals useful in this invention include, for example, bovine serum albumin, human serum albumin, hormones, such as insulin, and immunoglobulins and their Fab fragments.
  • Enzymes which may be crystallized to form crosslinked enzyme crystals useful in this invention include hydrolases, isomerases, lyases, ligases, transferases and oxidoreductases.
  • hydrolases examples include thermolysin, elastase, esterase, lipase, nitrilase, hydantoinase, asparaginase, urease, subtilisin and other proteases and lysozyme.
  • lyases examples include aldolases and hydroxynitril lyase.
  • oxidoreductases include glucose oxidase, alcohol dehydrogenase and other dehydrogenases.
  • Microcrystals are defined as crystals which are lOO ⁇ m or less in their largest dimension.
  • crystals are produced by combining the protein to be crystallized with an appropriate aqueous solvent or aqueous solvent containing appropriate precipitating agents, such as salts or organics.
  • the solvent is combined with the protein at a temperature determined experimentally to be appropriate for the induction of crystallization and acceptable for the maintenance of protein activity and stability.
  • the solvent can optionally include co- solutes, such as divalent cations, co-factors or chaotropes, as well as buffer species to control pH.
  • the need for co-solutes and their concentrations are determined experimentally to facilitate crystallization.
  • the controlled precipitation leading to crystallization can best be carried out by the simple combination of protein, precipitant, co-solutes and, optionally, buffers in a batch process.
  • Crosslinking results in stabilization of the crystal lattice by introducing covalent links between the constituent protein molecules of the crystal. This makes possible the transfer of protein into an alternate reaction environment that might otherwise be incompatible with the existence of the crystal lattice or even with the existence of intact protein.
  • Crosslinking can be achieved by a wide variety of multifunctional reagents, including bifunctional reagents.
  • the crosslinking agent is glutaraldehyde.
  • Crosslinking with glutaraldehyde forms strong covalent bonds primarily between lysine amino acid residues within and between the protein molecules in the crystal lattice.
  • the crosslinking interactions prevent the constituent protein molecules in the crystal from going back into solution, effectively insolubilizing or immobilizing the protein molecules into microcrystalline particles.
  • the crosslinked protein crystals are typically of a shape selected from the group consisting of plates, ellipsoids, needles, polyhedrons or rods. In addition, they typically have pores with cross- sections between about 15A and about lOOA in length.
  • Pore volume per crystal volume typically ranges between about 25% and about 80%.
  • the length of the crosslinked protein crystals typically ranges between about 1 ⁇ m and 200 ⁇ m. As used herein with respect to crosslinked protein crystals, the term "length" refers to the longest dimension. In some embodiments of this invention, the crosslinked protein crystals are between about 1 ⁇ m and about 50 ⁇ m in length. The thickness of the crosslinked protein crystals typically ranges between about 1 ⁇ m and 10 ⁇ m and, in some embodiments, between about 1 ⁇ m and about 5 ⁇ m. Crosslinked protein crystals may comprise between about 20% and 80% solvent by weight.
  • crosslinked proteins crystals have the following characteristics:
  • Pore size range: between about 2 ⁇ A and about lOOA in diameter
  • Porosity range between about 0.5 and about 0.8
  • Pore volume between about 0.9 ml/g and about 4 ml/g 2
  • Pore surface areas between about 800 m /g and about 2000 m /g
  • Crosslinked protein crystals (in slurry or dried form) , prepared as described above, or commercially obtained, may be used to prepare stationary phases using conventional supports and packing techniques, including slurry packing.
  • the crosslinked protein crystals may be bound or linked to a solid support, included in a solid support, packed into a housing, such as a column or a capillary tube, layered onto beads or layered in a plate.
  • the crosslinked protein crystals may be packed into a column for size exclusion chromatography or gel permeation chromatography.
  • the crosslinked protein crystals may be incorporated into a flat-bed, batch or column chromatography system.
  • crosslinked protein crystals may be incorporated into or deposited on membranes, for example batch membranes, for use in membrane-based separations.
  • Crosslinked protein crystals may also be included in other types of filtration devices.
  • the crosslinked protein crystals may be packed into a standard chromatography housing, such as a column, having an inlet port at the top and, at the bottom, means (such as a frit, filter or disk) for retaining the crystals and an outlet tube. Packing may be at pressures between atmospheric pressure and about 7,000 psi. The top of the column is then covered and connected to an inlet tube. Equilibration solution may then be run through the column and the pH and conductivity of the flowthrough monitored, to ensure that the media is properly equilibrated. Subsequently, a sample containing the substance to be purified or isolated is loaded onto the column. The substance to be separated is retained in the crosslinked protein crystal stationary phase, with the remaining components of the mixture being recovered in the flowthrough.
  • a standard chromatography housing such as a column, having an inlet port at the top and, at the bottom, means (such as a frit, filter or disk) for retaining the crystals and an outlet tube. Packing may be at pressures between atmospheric pressure and about
  • the column is then subjected to washing or elution to recover the substance of interest.
  • the substance of interest may be recovered from the stationary phase by washing or elution with a gas, supercritical fluid or a solvent selected from the group consisting of aqueous solvents, organic solvents and aqueous-organic solvent mixtures.
  • a gas, supercritical fluid or a solvent selected from the group consisting of aqueous solvents, organic solvents and aqueous-organic solvent mixtures.
  • the particular eluant or washing agent will depend on the particular separation technique and substance involved.
  • Useful analytical chromatography columns include, for example, those having dimensions of between about 5 cm and about 25 cm in length and between about 20 mm and about 50 mm in diameter.
  • Useful preparative chromatography columns include, for example, those having dimensions of between about 50 cm and about 150 cm in length and about 5 cm and 25 cm in diameter.
  • the separation media may be packed into a capillary tube of about 50 ⁇ m in diameter for example.
  • the separation media may be layered onto a membrane of between about 100 ⁇ m and about 500 ⁇ m in thickness, or layered onto a plate of between about 100 um and about 300 urn in thickness, for example.
  • the chromatography columns may be made of any conventional material, preferably metal or glass.
  • One example of an apparatus useful in this invention comprises a housing, a stationary phase comprising crosslinked protein crystals contained in the housing, means (for example, a tube) for contacting a sample with the stationary phase and means (for example, a tube) for collecting the sample after it has been contacted with the stationary phase.
  • Conventional materials may also be used for the plate or membrane.
  • the crosslinked protein crystal separation media further comprises or supports a separation media selected from the group consisting of solid sorbents or soft sorbents.
  • Such sorbents include, for example, polysaccharides, including cellulose, derivatives of cellulose, starch, dextran, dextranes, derivatives of dextranes, agar or agarose; natural polymers, synthetic polymers, such as substituted or unsubstituted polyacrylamides, polyvinyl hydrophilic polymers or polystyrene.
  • support materials include silica, alumina, zirconia oxide, aluminosilicates, zeolites, ceramic structures, cellulose, hydrated crosslinked polymers, silica gels, SephadexTM and derivatives of Sephadex 15*1 .
  • the versatility of stationary phases based on crosslinked protein crystals permits the use of chromatography arrangements using a plurality of columns, each packed with the same or different crosslinked protein crystals in tandem, to separate substances by different types of chromatography at each stage.
  • a first column may trap contaminants by ion exchange chromatography and a second column may isolate the target substance by size exclusion chromatography.
  • the outlet of the first column is connected to the inlet of the second column and eluant from the first column runs directly into the second column.
  • one or more columns packed with crosslinked protein crystals may be used in tandem with one or more conventional chromatography columns.
  • crosslinked protein crystals Based on the high mechanical stability, affinity and enantioselectivity of crosslinked protein crystals, we packed chromatographic columns with various types of crosslinked protein crystals. More particularly, we employed crosslinked protein crystals of bovine serum albumin, human serum albumin, subtilisin, thermolysin, penicillin acylase and lipases of Candida rugosa and Pseudomonas c paci as stationary phases for liquid chromatography.
  • crosslinked protein crystals can separate polyethylene glycol (PEG) molecules, according to size, by a simple isocratic mode of chromatography in aqueous and organic solutions and racemic mixtures according to their stereosymmetry.
  • PEG polyethylene glycol
  • crosslinked protein crystals can separate a variety of compounds according to their physical properties. Accordingly, chromatographic columns packed with the same types of crosslinked protein crystals may be used to carry out size exclusion, affinity and chiral chromatography. The examples were carried out using the following reagents, processes and conditions.
  • Crosslinked Pseudomonas cepacia lipase crystals sold under the name ChiroCLEC-PCTM, available from Altus Biologies, Inc (Cambridge, Massachusetts) .
  • Crosslinked Candida rugosa lipase crystals sold under the name ChiroCLEC-CRTM, available from Altus Biologies, Inc. (Cambridge, Massachusetts) .
  • Crosslinked thermolysin crystals sold under the name PeptiCLEC-TRTM, available from Altus Biologies, Inc. (Cambridge, Massachusetts) . Starting from a batch of PeptiCLEC-TRTM, we separated crystals by size. "TR- 2 " crystals were those having an average length of 7 ⁇ m. "TR-3" crystals were those having an average length of 30 ⁇ m.
  • Crosslinked penicillin acylase crystals sold under the name ChiroCLEC-ECTM, available from Altus Biologies, Inc. (Cambridge, Massachusetts) .
  • Seed crystals were prepared by washing a sample of crystals free of precipitate with a solution of 50% saturated ammonium sulfate and 100 mg/ml soluble BSA in 100 mM phosphate buffer pH 5.3. The seeded crystallization solution was incubated at 4°C overnight on a rotating platform. Crystal plates (20-100 ⁇ ) appeared in the solution overnight (16 hr) .
  • Seed crystals were prepared by washing a sample of crystals free of precipitate with a solution of 50% saturated ammonium sulfate in 100 mM phosphate buffer pH 5.5. The seeded crystallization solution was incubated at 4°C overnight on a vigorously rotating platform. Crystals rods (20 ⁇ ) appeared in the solution overnight (16 hr) .
  • the crosslinking procedure used involved conditions designed not to disrupt crystal structure. The conditions were those which allowed crosslinking of the crystals, to prevent their dissolution in harsh conditions and render them sufficiently rigid for the purpose of chromatography.
  • Crosslinking was performed in an identical manner for the crystal solutions of each of bovine and human serum albumin. Crosslinking was performed at 4°C in a stirred solution of crystals and mother liquor containing 50% saturated ammonium sulfate as described above. The crystals were not washed prior to crosslinking with borate-pretreated glutaraldehyde.
  • Pretreated glutaraldehyde was prepared by adding one volume of 50% glutaraldehyde ("GA") to an equal volume of 300 mM sodium borate (pH 9) . The glutaraldehyde solution was incubated at 60°C for 1 hour. The pH of the solution was then adjusted to 5.5 with concentrated HCI and the solution was rapidly cooled to 4°C (on ice) .
  • the pretreated glutaraldehyde (25%) was added to the crystallization solution stepwise, in 0.05% increments (total concentration) at 15 minute intervals to a concentration of 2%. Aliquots of crystallization solution used ranged between 1 ml and 500 ml volume. The crystals were then brought to 5% GA and incubated at 4°C for 4 hours to allow crosslinking. Albumin crosslinked crystals were collected by low speed centrifugation and washed repeatedly with pH 7.5, 100 mM Tris HCI. Washing was stopped when the crosslinked crystals could be centrifuged at high speed without aggregation.
  • the crosslinked crystals described above were used to pack standard chromatographic columns having the following dimensions: 25 cm x 4.6 mm, 10 cm x 4.6 mm, and 15 cm x 2.1 mm (Alltech, Waters or Hewlett Packard) . Packing was carried out by means of a slurry packing method, using deionized water as packing fluid, at a pressures up to 7000 psi and a slurry packer machine (Alltech, Catalog No. 1666). The loaded columns remained useful for 4 months (up to 1000 injections) .
  • the solute concentration was about 5 mg/ml.
  • Affinity chromatography was performed under the same conditions, but using a flow rate of 0.5 ml/min. The solute concentration was about 0.2 mg/ml and injection volume was 20 ⁇ l .
  • Size exclusion chromatography of various PEG samples was performed, first, in the same sodium phosphate buffer with pH 8.3, second, in deionized water, and third, in 15% and 50% of aqueous acetonitrile solution, with a flow rate of 0.5 ml/min and detection by means of Rl and UV at 192 nm.
  • the concentration of the PEG solution was about 3 mg/ml.
  • BSA Bovine serum albumin
  • Pore size, pore size distribution and porosity of the crosslinked protein crystals was determined by macromolecular porosimetry [L.Z..
  • crosslinked protein crystals meet all the major requirements for chromatographic stationary phases.
  • their pore size is comparable to that of the molecules being analyzed.
  • the property permits analysis of flexible chain macromolecules in the range of molecular weight up to 10,000 daltons and globular molecules up to 20,000 daltons.
  • porosity of the crosslinked protein crystals i.e., pore volume per crystal volume is close to 50%, a level attractive for chromatographic stationary phases.
  • Such properties establish crosslinked protein crystals as a new generation of nanoporous materials that can perform as a separation media suitable for different types of chromatography and simulating moving bed technology [M.J. Gattuso et al ., "Simulated Moving Bed Technology — The Preparation of Single Enantiomer Drugs", Pharmaceutical Technology Europe, pp. 20-25 (June 1996) .
  • Example 1 A chromatographic column was packed with TR-3 crosslinked thermolysin crystals, prepared as described above, by means of a slurry packing method using deionized water as packing fluid at a pressure of 1500 psi. An aliquot of about 1 g of the crystals was packed into the column, which was 25 cm x 4.6 mm in size. The PEG standards were dissolved in water. Concentration of the solutions was 3 mg/ml. The temperature was ambient. Injection volume was 25 ⁇ l, flow rate 0.5 ml/min.
  • Figure 1 shows pore size distribution in the TR-3 crystals.
  • Figure 2 shows the distribution coefficient for PEG macromolecules inside the TR-3- packed column with as a function of root mean square radius of the macromolecules.
  • Figures 3 and 4 show raw chromatographic data for the PEG macromolecules run on the chromatographic column. The data were used to calculate the distribution coefficient shown in Figure 2 and that coefficient was used to calculate the pore size distribution in Figure 1 using the macromolecular porosimetry technique.
  • Figures 5 and 6 show separation of PEG samples according to size of their macromolecules by means of size exclusion chromatography on the TR-3- crystal packed column described in Example 1.
  • Figure 5 shows separation of a mixture of three PEG standards in the chromatography run (Rl detection) . They were: PEG with average molecular weights of 62, 600 and 10,000 daltons. Tris buffer 10 mM (pH 7.4) was used as a solvent. Flow rate was 0.5 ml/min. Injection volume was 20 ⁇ l. The pressure drop inside the column during the chromatographic run was 220 psi. Concentrations of the PEG samples used to prepare the mixture were 3 mg/ml.
  • Figure 6 shows the overlaid chromatograms (Rl detection) of six PEG standards with molecular weights of 62, 106, 194, 400, 1,500 and 10,000 daltons. They were run on the same column that was used to generate Figure 5, except that the solvent employed was 50% of CH 3 CN + 50% of H 2 0. That solvent was used in order to demonstrate the ability of the column to perform properly in the presence of organic solvent. pH value of the solvent was 7.0, flow rate was 0.5 ml/min.
  • Figures 7-10 show application of TR-3 crosslinked thermolysin crystals for affinity chromatography.
  • the chromatography was carried out in Tris buffer 10 mM (pH 8.4) at a flow rate of 0.5 ml/min.
  • Figure 9 shows separation according to chemical structure of a mixture of D-isomer of phenyllactic acid and ibuprofen on the TR-3-packed column.
  • the solvent was Tris buffer 10 mM (pH 8.15). Flow rate was 0.5 ml/mm.
  • Figure 10 is a chromatogram (Rl detection) of flurbiprofen on the TR-3-packed column.
  • Figure 11 demonstrates separation according to chemical structure of a mixture of ibuprofen (two peaks) and flurbiprofen (Rl detection) .
  • the solvent was Tris buffer 10 mM (pH 7.5) and the flow rate was 0.5 ml/mm.
  • Example 4
  • Example 4 shows the application of TR-2 and TR-3 crosslinked thermolysin crystals, prepared as described above, for chiral chromatography.
  • Figure 12 demonstrates chiral separation of two optical isomers of phenyllactic acid R and S on two columns: one packed with TR-3 and the other packed with TR-2.
  • the pressure drop inside the columns at the chromatographic run was 285 psi.
  • Flow rate was 0.3 ml/min.
  • the pressure drop inside the column at the chromatographic run was 500 psi.
  • Figures 14 and 15 show a chiral separation of two optical isomers of phenylglycine (R and S) in a chromatographic system using two columns: TR-3-packed and TR-2-packed, with an injection volume of 10 ⁇ l
  • a chromatographic column was packed with ChiroCLEC-CR m crystals by means of a slurry packing method using deionized water as packing fluid at a pressure of 2000 psi. An aliquot of about 1 g of the crystals was packed into the column, which was 25 cm x 4.6 mm in size. The PEG standards were dissolved in water. Concentration of the solutions was 3 mg/ml. The temperature was ambient. Injection volume was 25 ⁇ l, flow rate 0.5 ml.
  • Figure 16 shows pore size distribution in the ChiroCLEC-CRTM crystals.
  • Figure 17 shows the distribution coefficient for PEG macromolecules inside the ChiroCLEC-CRTM-packed column, as a function of root mean square radius of the macromolecules.
  • Figures 18 and 19 show raw chromatographic data for the PEG macromolecules run on the chromatographic column. The data were used to calculate the distribution coefficient shown in Figure 17 and that coefficient was used to calculate the pore size distribution in Figure 16 by means of macromolecular porosimetry.
  • Figures 20, 21 and 22 show separation of PEG samples according to size of their macromolecules by means of size exclusion chromatography on the column packed with ChiroCLEC-CRTM crystals, as described in Example 5.
  • Figure 20 shows the overlaid chromatograms
  • FIG. 21 shows PEG separation on the same
  • Figure 22 shows PEG separation on the same ChiroCLEC-CR ⁇ -packed column using 15% of CH 3 CN + 50% of H 0 as solvent.
  • the pH value of the solvent was 7.0, flow rate was 0.5 ml/min and the pressure drop inside the column at the chromatographic run was 296 psi.
  • a chromatographic column was packed with ChiroCLEC-PCTM crystals by means of a slurry packing method using deionized water as packing fluid at a pressure of 2500 psi. An aliquot of about 0.5 g of the crystals was packed into the column, which was
  • FIG. 24 shows the distribution coefficient for PEG macromolecules inside the column with ChiroCLEC-PCTM crystals as a function of root mean square radius of the macromolecules.
  • Figures 25 and 26 show raw chromatographic data for the PEG macromolecules run on the chromatographic column. The data were used to calculate the distribution coefficient shown in Figure 24 and that coefficient was used to calculate the pore size distribution in Figure 23 by macromolecular porosimetry.
  • FIGS. 27-30 show application of ChiroCLEC-CR 1M crystals for affinity chromatography to separate substances according to their chemical nature using the column described in
  • Example 7 The substances were: phenyllactic acid, R- phenylglycine and D-4-hydroxyphenylglycine in Figure 27; ibuprofen and phenylacetic acid-dl in Figure 28; ibuprofen and flurbiprofen in Figure 29; suprofen, ketoprofen and flurbiprofen in Figure 30.
  • Tris buffer 10 mM pH 7.4 was used as solvent and flow rate was 0.5 ml/min.
  • Figure 31 demonstrates a chiral separation of two optical isomers of methylmandelate, R and S on the ChiroCLEC-PCTM crystal-packed column (Rl detection) .
  • a chromatographic column was packed with ChiroCLEC-EC IM crystals by means of a slurry packing method using deionized water as packing fluid at a pressure of 2500 psi. .An aliquot of about 0.5 g of the crystals was packed into the column, which was 10 cm x 4.6 mm in size. The PEG standards were dissolved in water. Concentration of the solutions was 3 mg/ml. The temperature was ambient. Injection volume was 25 ⁇ l and flow rate 0.5 ml.
  • Figure 32 shows pore size distribution in the penicillin acylase crystals.
  • Figure 33 shows the distribution coefficient for PEG macromolecules inside the column with ChiroCLEC-EC IM crystals as a function of root mean square radius of the macromolecules.
  • Figures 34 and 35 show raw chromatographic data for the PEG macromolecules run on the chromatographic column. The data were used to calculate the distribution coefficient shown in Figure 33 and that coefficient was used to calculate the pore size distribution in Figure 32 by means of macromolecular porosimetry.
  • a chromatographic column was packed with bovine serum albumin crosslinked protein crystals, prepared as described above, by means of a slurry packing method using deionized water as packing fluid at a pressure of 7000 psi. An aliquot of about 1 g of the crystals was packed into the column, which was 25 cm x .6 mm in size. The PEG standards were dissolved in water. Concentration of the solutions was 3 mg/ml.
  • the temperature was ambient. Injection volume was 25 ⁇ l, flow rate 0.5 ml.
  • Figure 36 shows pore size distribution in the
  • Figure 37 shows the distribution coefficient for PEG macromolecules inside the column with BSA crystals as a function of root mean square radius of the macromolecules.
  • Figures 38 and 39 show raw chromatographic data for the PEG macromolecules run on the chromatographic column. The data were used to calculate the distribution coefficient shown in Figure 37 and that coefficient was used to calculate the pore size distribution in Figure 36 by means of macromolecular porosimetry.
  • Example 12 shows separation of PEG samples according to size of their macromolecules by means of size exclusion chromatography on the column packed with bovine serum albumin crosslinked protein crystals, as described in Example 11.
  • Figure 40 shows the overlaid chromatograms
  • Figure 41 demonstrates the overlaid Chromatograms of ketoprofen, suprofen and naproxen.
  • the solvent used was Tris buffer (pH 8.2), flow rate was 0.5 ml/min and the pressure drop inside the column at the chromatographic run was 280 psi.
  • FIG. 42 shows the application of bovine serum albumin crosslinked protein crystals for chiral chromatography .
  • Figure 42 demonstrates a chiral separation of two optical isomers of phenyllactic acid, D and L, on a BSA-packed column. The column and the chromatographic conditions were the same as in Example 13 (Rl detection) .
  • a chromatographic column was packed with human serum albumin crosslinked protein crystals by means of a slurry packing method using deionized water as packing fluid at a pressure of 4000 psi. An aliquot of about 0.2 g of the crystals was packed into the column, which was 15 cm x 2.1 mm in size. The PEG standards were dissolved in water. Concentration of the solutions was 3 mg/ml. The temperature was ambient. Injection volume was 25 ⁇ l, flow rate 0.15 ml/mm.
  • the solvent used was Tris buffer 10 mM (pH 7.5), flow rate was 0.15 ml/min and the pressure drop inside the column at the chromatographic run was 790 psi (Rl detection) .
  • FIG. 44 demonstrates separation of S ibuprofen and R phenyllactic acid on a column packed with TR-2.
  • FIG. 45 demonstrates chiral separation of two optical isomers of phenylglycine R and S on a column packed with TR-2.
  • Human serum albumin crosslinked crystals prepared as described above, were used to carry out chiral chromatography of various chiral compounds.
  • Crosslinked human serum albumin crystals were mixed with silica in the proportion specified below for each chromatography. The mixture was then filtered through a 1 ⁇ filter to remove fines.
  • aqueous slurry containing 50 mg pure silica was packed into each column, followed by the silica/crosslinked crystal mixture using a slurry packing machine (Alltech) under packing pressure below 2,000 psi. Packing time was 30 to 40 minutes. The details of each separation are listed below:
  • Folinic Acid Column 16% crosslinked HSA crystals and 84% silica gel in a 50 x 4 mm column.
  • Injection volume 0.5 ⁇ l.
  • N-2.4-DNP-DL- ⁇ -amino-n-butvric acid Column : 16% crosslinked HSA crystals and 84% silica gel in a 50 x 4.6 mm column.
  • Injection volume 0.5 ⁇ l.
  • Injection volume 0.5 ⁇ l.
  • Figures 46-51 depict these chiral separations .
  • This example shows the correlation between separation efficiency and particle size of crosslinked human serum albumin crystals, prepared as described above, in the chiral chromatography of the chiral compound folinic acid.
  • Injection volume 0.5 ⁇ l.
  • Example 20 shows the stability of crosslinked human serum albumin crystals, prepared as described above, toward organic solvents.
  • Example 21 This example shows the high loading capacity of crosslinked human serum albumin crystals as prepared above m the resolution of folinic acid.
  • a standard column (100 x 4.6 mm) was packed with a slurry of 14% crosslinked human serum albumin crystals and 86% silica.
  • the mobil phase was 4% 2-propanol in 0.1 M phosphate buffer (pH 7) .
  • the flow rate was 0.5 ml/min.
  • selectivity of the separation remained good.
  • HSA Folinic acid HSA
  • Capacity Capacity Selectivity Loading (mg) Factor k, Factor k 2 a mg/g HSA

Abstract

La présente invention porte sur l'utilisation de cristaux protéiniques réticulés dans des procédés, systèmes et appareil de séparation d'une substance ou molécule d'un échantillon. Selon une réalisation préférée, les cristaux protéiniques réticulés sont utilisés dans des procédés, systèmes et appareil de chromatographie dans lesquels la séparation est basée sur une propriété physique ou chimique de ladite substance ou molécule. De manière avantageuse, les cristaux protéiniques réticulés, caractérisant les procédés, systèmes et appareil de cette invention, possèdent une excellente résistance mécanique ainsi qu'une structure poreuse bien développée, une affinité et sélectivité chirale importantes, et sont extrêmement stables dans des solvants aqueux et organiques. Ces propriétés permettent d'utiliser ces cristaux notamment comme sorbants dans des séparations telles que les chromatographies chirale, d'exclusion stérique sur gel et d'affinité.
PCT/US1997/017167 1996-09-24 1997-09-24 Cristaux proteiniques reticules utilises comme supports de separation universels WO1998013119A1 (fr)

Priority Applications (1)

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US7300775B2 (en) 1999-12-29 2007-11-27 Verenium Corporation Methods for producing α-substituted carboxylic acids using nitrilases and strecker reagents
US7521216B2 (en) 1999-12-29 2009-04-21 Verenium Corporation Nitrilases and methods for making and using them
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WO1998046732A1 (fr) * 1997-04-11 1998-10-22 Altus Biologics Inc. Dissolution commandee de cristaux reticules de proteine
EP2267122A1 (fr) * 1997-04-11 2010-12-29 Altus Pharmaceuticals Inc. Cristaux de protéine réticulés par dissolution contrôlée
US6359118B2 (en) 1997-09-05 2002-03-19 Altus Biologies, Inc. Carbohydrate crosslinked glycoprotein crystals
US6500933B1 (en) 1997-09-05 2002-12-31 Altus Biologics Inc. Methods of preparing carbohydrate crosslinked glycoprotein crystals
US7087728B2 (en) 1997-09-05 2006-08-08 Altus Pharmaceuticals Inc. Carbohydrate crosslinked glycoprotein crystals
US8034595B2 (en) 1999-12-29 2011-10-11 Verenium Corporation Nitrilases and methods for making and using them
US9315792B2 (en) 1999-12-29 2016-04-19 Basf Enzymes Llc Nitrilases, nucleic acids encoding them and methods for making and using them
US7608445B1 (en) 1999-12-29 2009-10-27 Verenium Corporation Nitrilases, nucleic acids encoding them and methods for making and using them
US7651849B2 (en) 1999-12-29 2010-01-26 Darcy Madden, legal representative Nitrilases
US7811804B2 (en) 1999-12-29 2010-10-12 Verenium Corporation Nitrilases, nucleic acids encoding them and methods for making and using them
US7300775B2 (en) 1999-12-29 2007-11-27 Verenium Corporation Methods for producing α-substituted carboxylic acids using nitrilases and strecker reagents
US7993901B2 (en) 1999-12-29 2011-08-09 Verenium Corporation Nitrilases and methods for making and using them
US9828594B2 (en) 1999-12-29 2017-11-28 Basf Enzymes Llc Nitrilases, nucleic acids encoding them and methods for making and using them
US8088613B2 (en) 1999-12-29 2012-01-03 Verenium Corporation Nitrilases, nucleic acids encoding them and methods for making and using them
US8318471B2 (en) 1999-12-29 2012-11-27 Verenium Corporation Nitrilases, nucleic acids encoding them and methods for making and using them
US8334125B2 (en) 1999-12-29 2012-12-18 Verenium Corporation Nucleic acids encoding nitrilases
US7521216B2 (en) 1999-12-29 2009-04-21 Verenium Corporation Nitrilases and methods for making and using them
US8501451B2 (en) 1999-12-29 2013-08-06 Verenium Corporation Nitrilases and methods for making and using them
US8778651B2 (en) 1999-12-29 2014-07-15 Verenium Corporation Nitrilases, nucleic acids encoding them and methods for making and using them
US8906663B2 (en) 1999-12-29 2014-12-09 Verenium Corporation Nitrilases
WO2003099410A1 (fr) * 2002-05-29 2003-12-04 Teknillinen Korkeakoulu Procede de separation chromatographique de nucleosides
US8372608B2 (en) 2002-06-13 2013-02-12 Verenium Corporation Processes for making [R]-ethyl 4-cyano-3 hydroxybutyric acid
US9217164B2 (en) 2007-03-01 2015-12-22 Basf Enzymes Llc Nitrilases, nucleic acids encoding them and methods for making and using them
CN109781921A (zh) * 2019-03-22 2019-05-21 上海海洋大学 一种利用反相高效液相色谱法快速检测苯乳酸含量的方法
CN112986569A (zh) * 2019-12-02 2021-06-18 中国科学院大连化学物理研究所 一种单端交联肽去除方法及其在蛋白质复合物交联位点分析中的应用
CN112986569B (zh) * 2019-12-02 2022-05-06 中国科学院大连化学物理研究所 一种单端交联肽去除方法及其在蛋白质复合物交联位点分析中的应用

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