WO2012116048A1 - N-désacétylation et dépolymérisation d'héparosane en une seule étape pour préparer de l'héparine biologiquement modifiée - Google Patents

N-désacétylation et dépolymérisation d'héparosane en une seule étape pour préparer de l'héparine biologiquement modifiée Download PDF

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WO2012116048A1
WO2012116048A1 PCT/US2012/026081 US2012026081W WO2012116048A1 WO 2012116048 A1 WO2012116048 A1 WO 2012116048A1 US 2012026081 W US2012026081 W US 2012026081W WO 2012116048 A1 WO2012116048 A1 WO 2012116048A1
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heparin
heparosan
bioengineered
reaction
molecular weight
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PCT/US2012/026081
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Jonathan Dordick
Zhenyu Wang
Robert J. Linhardt
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Rensselaer Polytechnic Institute
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
    • C08B37/0078—Degradation products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/727—Heparin; Heparan

Definitions

  • Heparin and heparan sulfate are biologically important molecules involved in blood anticoagulation, viral and bacterial infection, angiogenesis, inflammation, cancer and development. Heparin is currently prepared from animal tissues in amounts of approximately 1 00 metric tons/year, but such heparin may be contaminated with other biological products. Porcine intestinal heparin typically contains 10 - 1 5 trisulfated disaccharides in a single heparin chain, and 1 - 2 disulfated disaccharides per chain (Loganathan et al. (1990), ''Structural variation in the antithrombin III binding site region and its occurrence in heparin from different sources.” Biochemistry 29:4362-80). While porcine intestinal heparin is widely used, problems with contamination and supply persist.
  • Heparosan is a polysaccharide with the repeating disaccharide unit [GlcAoc-(l -4)GlcNAcR(l - 4)] n that is naturally produced by E. coli K5.
  • Laboratory-scale studies have shown that heparosan with a weight average molecular weight (M w ) > 10,000, obtained from E. coli K5 strain can be enzymatically converted to an anticoagulant polysaccharide similar to heparin.
  • M w weight average molecular weight
  • the inventors have identified a single step reaction between heparosan and NaOH that gives optimal levels of ⁇ -deacetylation and depolymerization to obtain a product that may be processed ( Figure 1 ) to bioengineered heparin that is substantially identical to pharmaceutical heparin.
  • the inventors have also identified relevant factors and algorithms for adjusting reaction conditions to obtain the desired N-deacetylated N-sulfonated product for further processing into bioengineered heparin from variable heparosan sources.
  • bioengineered heparin that is chemically identical to pharmaceutical heparin remains elusive. It has now been shown that heparosan can be treated in a single step base-catalysed reaction that gives optimal levels of N-deacetylation and depolymerization to obtain a product that may be processed to bioengineered heparin that is substantially identical to pharmaceutical heparin.
  • the inventors have also identified relevant factors and algorithms for adjusting reaction conditions to obtain the desired N-deacetylated N-sulfonated product for further processing into bioengineered heparin from variable heparosan sources.
  • the invention is a method of making bioengineered heparin from heparosan, comprising incubating heparosan in NaOH under time and conditions sufficient to obtain (a) partial N-deacetylation to a N-acetyl content between 1 1 .5 and 1 8% and (b) partial depolymerization, to obtain a molecule number average molecular weight (M )of 9- 1 2.5 kDa, prior to further reaction.
  • M molecule number average molecular weight
  • MM may be measured by sodium docecyl sulfate polyacryl amide gel electrophoresis (SDS-PAGE) or by size exclusion chromatography (SEC), for example.
  • Suitable reaction conditions comprise incubating heparosan (i) in 1 .0 to 3.0M NaOH. (ii) for 2 to 4 h. (iv) at 50-70 °C.
  • the reaction conditions may comprise incubating heparosan (i) in 1.0 to 2.0 M NaOH,(ii) for 2.5 to 4 h, (iii) at 55-65°C.
  • high concentrations of heparosan may be used, such as about 10 g/L, although lower concentrations of heparosan may also be used.
  • reaction conditions to obtain the desired TV-acetyl content is calculated using equation (7):
  • Y, (%)) 1 5.02 - 23.793X, - 8.083 X 2 - 1 7.575X 3 + 10.527X, 2 + 4.579X 2 2 + 5.973 X 3 2 + 3.472X t X 2 + 6.941 X,X 3 + 3.433X 2 X (7) .
  • Y i is the N-acetyl content
  • X] is the molar NaOH concentration
  • X 2 is the reaction time in (hours)
  • X 3 is the reaction temperature (Celsius)
  • reaction conditions to obtain the desired depolymerization factor is calculated using the following equation:
  • Y 2 0.72963 - 0.03 1 75X, - 0.09502X 2 - 0.2227X 3 + 0.01088X, 2 + 0.02028X 2 2 + 0.01632X 3 2 - 0.0257X
  • equations (7) and (8) are solved together for Y ⁇ and Y 2 , to obtain conditions that are optimal for N-acetyl content and the depolymerization factor.
  • the product of the foregoing partial N-deacetylation and depolymerization is, in some embodiments, further processed. In some embodiments, this includes selective N-sulfonation with trimethylamine-sulfur trioxide complex, such as to obtain a ratio of N- acetylglucosamine and N-sulfoglucosamine equivalent to pharmaceutical heparin.
  • the product of N-sulfonation is further processed as follows: (1) C5- epimerization/2-O-sulfonation with an equi-unit mixture of C5-epimerase and 2-0- snlfntransferase; (2) 6-O-sulfonation with an equi-unit mixture of 6-O-sulfotransferase-l and -3 ; and (3) 3-O-sulfonation with 3-O-sulfotransferase- 1 in the presence of a PAPs regeneration system.
  • the bioengineered heparin has a clotting time substantially equivalent to pharmaceutical heparin.
  • the starting material for making bioengineered heparin is heparosan with Mn between 1 2.6 kDa and 24.6 kDa.
  • the invention also relates to composition and methods of use. Accordingly, the invention includes a pharmaceutical composition comprising the bioengineered heparin made by the foregoing methods. The invention also includes use of such a pharmaceutical composition of bioengineered heparin for the treatment of disease, and for the manufacture of a product for the treatment of disease.
  • Figure 1 Scheme for producing bioengineered anticoagulant heparin from E. coli K5 heparosan.
  • FIGS. 2A-2B Figures 2A-2B.
  • 2A 1H-NMR of vV-sulfo. vV-acetyl heparosan from 3 h N-deacetylated K5 heparosan.
  • 2B Remaining N-acetyl content as a function of N-deacetylation reaction time. Error bar represents the standard deviation.
  • FIGS 3A-3B Size exclusion chromatogram of K5 heparosan (top) and N-deacetylated K5 heparosan (bottom). M , MW and PDI calculated from the chromatogram for each sample are annotated on the figure. 3B: polyacrylamide gel electrophoresis gel of bovine lung heparin ladder (lane 1 ), bioengineered heparin (lane 2) and pharmaceutical porcine intestinal heparin (USP heparin) (lane 3). MN, MW and PDI calculated from the gel for each sample are on the figure.
  • FIGS 5A-5B Liquid chromatography-mass spectrometry of disaccharides the most commonly found in heparan sulfate/heparin.
  • 5A Extracted ion chromatogram of the bioengineered heparin
  • SB Extracted ion chromatogram of the commercial USP heparin (OS: AUA-GlcNAc, NS: AUA-GlcNS, 6S: AUA-GlcNAc6S, 2S: AUA2S-GlcNAc, NS6S: ⁇ - GlcNS6S, NS2S: AUA2S-GlcNS, 2S6S: AUA2S-GlcNAc6S, TriS: AUA2S-GlcNS6S)
  • Figure 6 Activated partial thromboplastin time assay of bioengineered heparin and pharmaceutical porcine intestinal heparin (USP heparin).
  • Figures 7A-7C 7A Response surface and corresponding contour plot of the effects of NaOH concentration and reaction time on the product N-acetyl content, with the reaction temperature fixed at the coded level 0 (actual level 60 °C).
  • 7B Response surface and corresponding contour plot of the effects of NaOH concentration and reaction temperature on the product N-acetyl content, with the reaction time fixed at the coded level 0 (actual level 3 h).
  • 7C Response surface and corresponding contour plot of the effects of reaction time and reaction temperature on the product N-acetyl content, with the NaOH concentration fixed at the coded level 0 (actual level 2 M).
  • FIGS 8A-8C 8A Response surface and corresponding contour plot of the effects of NaOH concentration and reaction time on the product depolymerization factor, with the reaction temperature fixed at the coded level 0 (actual level 60 °C). 8B Response surface and corresponding contour plot of the effects of NaOH concentration and reaction temperature on the product depolymerization factor, with the reaction time fixed at the coded level 0 (actual level 3 h). 8C Response surface and corresponding contour plot of the effects of reaction time and reaction temperature on the product depolymerization factor, with the NaOH concentration fixed at the coded level 0 (actual level 2 M).
  • Heparosan is a polysaccharide with the repeating dissacharide unit [GlcAa-(l -4)GlcNAcR(l - 4)k
  • pharmaceutical heparin' refers to porcine intestinal heparin that complies with United States Pharmacopeia requirements for heparin.
  • bioengineered heparin refers to heparin that is produced from heparosan, such as the heparosan obtained from microbial fermentation.
  • bioengineered heparin When bioengineered heparin is “substantially equivalent to pharmaceutical heparin” it means that, for a given property under consideration, the bioengineered heparin falls within the range of normal variability of that property, or is not statistically different from that property, when measured under the same conditions. Thus bioengineered heparin which is
  • bioengineered heparin that has a clotting time substantially equivalent to pharmaceutical heparin means that the clotting time of bioengineered heparin falls within the normal ranges of pharmaceutical heparin, under identical circumstances, or is not statistically different from pharmaceutical heparin under identical circumstances.
  • USP heparin monograph has no specific requirements for M , M W , PDI, N-acetylation, N-sul onation, or saccharide content, but is defined by features such as clotting time and absence of contaminants. Similar definitions are used for the European Pharmacopoeia.
  • the USP provides a physical sample (USP standard) that is a blend of commercially available heparins that qualify in all aspects of the USP requirements.
  • the 'W- acetyl content is the % content of vV-acetylglucosamine residues.
  • the "depolymerization factor” is the ratio of the MN of the final desired product over the MN of the original product. In the present invention, the depolymerization factor is not adjusted for the loss of N-acetyl groups because the loss of N-acetyl groups does not significantly impact MN.
  • Measures of molecular weight of polymers is often expressed as number average molecular weight (MN). weight average molecular weight (Mw) and polydispersity index (PDI).
  • MN number average molecular weight
  • Mw weight average molecular weight
  • PDI polydispersity index
  • M N number average molecular weight
  • the weight average molecular weight (Mw) is calculated by
  • TV is the number of molecules of molecular weight ;
  • MN and Mw may be measured by different means.
  • MN and Mw and other values, between USP heparin and bioengineered heparin the same method is used for both USP and bioengineered samples, to avoid variation in measured values that is caused
  • the polydispersity index (PDI) measures the degree of heterogeneity in a mixture. PDI is calculated as:
  • substantially pure means that the molecule is essentially free of other substances to an extent practical and appropriate for its intended use.
  • a substantially pure heparosan is at least 90% pure.
  • the material is greater than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even greater than 99%o free of contaminants. The degree of purity may be assessed by means known in the art.
  • Escherichia coli K5 describes variants of E. coli that produce the K5 exopolysaccharide. Suitable E. coli K5 strains may be obtained from public collections such as ATCC (American Type Culture Collection, USA), such as E. coli strain ATCC23506. Escherichia coli K5 strains may be also be isolated from clinical sources, and/or genetically modified.
  • E. coli in general, can grow in a wide variety of media and a range of pH, temperature, 0 2 and other conditions. As reported in WO 201 1 /028668, variations in growth conditions can affect (a) the rate of bacterial growth, (b) maximal cell density, (c) production of the K5 p vnnnlvsaccharide capsule, (d) the degree of modification of the K5 capsule by E. coli K5, hage resident within E. coli K5, and other factors, (e) the release of the 5 cxopolysaccharide capsule into medium, (f) whether the heparosan polysaccharide produced is of a size range suitable for processing into heparin, and (g) the amount and type of contaminants.
  • conditions that promote cell lysis may increase the yield of the K5 exopolvsaccharide in the culture supernatant, but may also increase the degradation the K5 cxopolysaccharide capsule and the number and amount of contaminants in the supernatant.
  • Heparosan as a polysaccharide, may copurify with contaminants such as lipolysaccharide (LPS), nucleic acids, non-heparosan polysaccharides, and derivatives of heparosan.
  • LPS lipolysaccharide
  • WO 201 1/028668 describes the heparosan obtained from the same E. coli K5 grown in different media.
  • the heparosan varied in its number average molecular weight (MM), weight average molecular weight (Mw) and polydispersity index (PDF) depending on the method on the medium, as shown in Table 1 .
  • MM number average molecular weight
  • Mw weight average molecular weight
  • PDF polydispersity index
  • WO 201 1/028668 also described a process for scalable production of high purity heparosan from E. coli K5.
  • the heparosan produced through larger scale fermentation had a number lolecular weight of about 58,000 Da, a weight average molecular weight of about a, and polydispersity index (PDI) of about 1.4. Purity was estimated at 95% or above.
  • the temperature was kept at around 37°C, and pH was kept between 6 and 8 by- adding NH 4 OH as pH goes down.
  • WO 201 1/028668 also described heparosan purification, which may comprise: (a) a step to prepare a culture supernatant or filtrate, (b) binding of heparosan to a solid phase, such as a resin, and elution of the heparosan therefrom, (c) precipitation with alcohol and (d) depyrogenation. Additional binding, precipitation and depyrogenation steps may be added. These are usually chosen to limit their impact on heparosan.
  • the final highly purified heparosan may then be further processed.
  • the distribution of chain lengths (as seen in number average molecular weight (M N ), weight average molecular weight (Mw) and polydispersity index (PD1)) may vary. Accordingly, any process for the production of bioengineered heparin that mimics USP porcine heparin must also take into account the variation in starting material.
  • K5 heparosan differs from in USP porcine heparin in the number and distribution of side groups, and in the length of the polymer chains.
  • the challenge is to identify conditions that can obtain the correct balance of side groups, and the correct size distribution.
  • the next step in the bioengineered heparin process is the chemical vV-deacetylation and N- sulfonation step ( Figure 1 ).
  • Figure 1 the chemical vV-deacetylation and N- sulfonation step.
  • the ratio of N-acetylglucosamine and N-sulfo- glucosamine must match those in porcine heparin. This requires fine control of the N- deacetylation reaction to preserve the appropriate proportion of N-acetyl groups. This is a critical step because the N-deacetylation reaction directly affects the N-acetyl content of the bioengineered heparin produced.
  • N-sulfo/N-acetyl heparosan afforded following N-sulfonation serves as the new backbone for subsequent enzymatic modifications, and the positioning of the N-sulfo and N-acetyl groups directly impacts the activity of the enzymes in affording the desired final structure of heparin.
  • base-catalyzed deacetylation may also reduce the molecular weight of
  • Heparin depolymerization (like other polysaccharides) is usually achieved through acid-catalyzed mechanisms, such as with Nitrous acid.
  • E. coli K5 heparosan was produced by E. coli K5 strain fermentation and purified from the culture supernatant as described previously in WO 201 1/028668.
  • Heparin lyases I, II, and III were cloned from the genomic DNA of Flavobacterium heparinum. The expression of the recombinant heparin lyases was also carried out for E. coli. (Zhang (2008)).
  • a 12% polyacrylamide gel of dimensions 0.75 mm x 6.8 cm x 8.6 cm was used in heparin molecular weight analyses.
  • Heparin samples (5 ⁇ g) were loaded onto gels and then subjected to electrophoresis (200 V for 25 min) and stained with Alcian blue for 0.5 h. and then destained in water. Gels were scanned and the resulting digital images were analyzed using UN-SCAN1T computer software following the manufacturer's user guide. Number average molecular weight (M N ), weight average molecular weight (M w ) and polydispersity index (PDI) were calculated. Bovine lung heparin oligosaccharide ladder was used as a standards for calculation of molecular weight.
  • G Size exclusion chromatography of K5 heparosan and N-deacetylated heparosan for molecular weight determination
  • Size exclusion chromatography was performed using a TSK-GEL® G3000PWxl size exclusion column with a sample injection volume of 20 ⁇ , and a flow rate of 0.8 ml/min on an apparatus composed of a Shimadzu® LC-l OAi pump, a Shimadzu® CBM-20A controller and a Shimadzu® RID- 1 OA refractive index detector.
  • the buffer consisted of 0.2 M Na 2 S04.
  • the column was maintained at 60°C with an Eppendorf® column heater during the chromatography.
  • the size exclusion chromatograms were recorded with the LCsolution® Version 1.25 software and analyzed with its "GPC Postrun" function to calculate MN, Mw and PDI. Dextrans of different molecular weights were used as calibrants.
  • Heparinase II was added into the heparin samples and incubated at 37°C for 24 h. The products were recovered by centrifugal filtration with a YM- 10 10 MWCO spin column, tes were freeze-dried and ready for liquid chromatography-mass spectrometry. Liquid chromatography-mass spectrometry was performed on an Agilent® 1200 LC/MSD instrument (Agilent Technologies, Inc. Wilmington, DE) equipped with an ion trap and a UV detector. The column used was Acquity ultraperformance liquid chromatography® BEH CI 8 column ( 1.7 ⁇ , 2, 1 X100 mm) (Waters Corporation).
  • Eluent A was water/acetonitrile (85/1 5, v/v), and eluent B was water/acetonitrile (35/65, v/v). Both eluents contained 1 2 mM tributylamine and 38 mM ammonium acetate with pH adjusted to 6.5 with acetic acid.
  • the column effluent entered the source of the electrospray ionization-mass spectrometry for continuous detection. The content of the disaccharides were calculated from the peak area of the extracted ion chromatogram calibrated to a standard curve for each disaccharide.
  • the TV-deacetylation reaction relied on NaOH treatment and was sampled every hour for 5 h.
  • the iV-deacetylated glucosamine residues were subsequently N-sulfonated with trimethylamine-sulfur trioxide.
  • the HI proton of the glucosamine was used for quantifying the N-sulfo/N- acetyl ratio, as it shows a peak at around 5.3 1 ppm as in N-acetyl glucosamine and shifts to around 5.55 ppm when the ⁇ -acetyl was replaced with an N-sulfo group (Figure 2A).
  • the polysaccharide obtained after N-sulfonation was used for the ⁇ -NMR study because we found that was more stable and had better solubility than the N-deacetylated heparosan, and e in H I proton chemical shift was more evident in the ⁇ -NMR.
  • the N-acetyl content decreased with prolonged reaction time.
  • the molecular weight of bioengineered heparin and pharmaceutical heparin are typically characterized by polyacrylamide gel electrophoresis, as size exclusion chromatography has been reported to be impacted by electrostatic repulsion between heparin and the column packing material in standard buffers.
  • the availability of defined heparin oligosaccharide molecular weight standards provides a reliable way to determine heparin molecular weight using polyacrylamide gel electrophoresis.
  • the molecular weights of pharmaceutical heparins determined by this polyacrylamide gel electrophoresis method range, for M N and M w values, from 9-12 KDa and 13-20 KDa, respectively.
  • a molecular weight of the ⁇ '-deacetylated hcparosan of - 10 KDa (9-12 kDa, as determined by polyacrylamide gel electrophoresis) represents the optimal starting material for the preparation of a bioengineered heparin closely matching the molecular weight properties of porcine intestinal heparin.
  • N-acetylglucosamine and N-sulfoglucosmine values bioengineered heparin presented in Table 2 are comparable to the values for the many USP heparins reported by our laboratory (Zhang et al. "Structural characterization of heparins from different commercial sources,' “ Analytical and Bioanalytical Chemistry, 401 , 2793-2803 (201 1)) and others (Guerrini et al. "Combined quantitative (1)H and (13)C nuclear magnetic resonance spectroscopy for characterization of heparin preparations," Semin Thromb Hemost 27:473-82 (2001)).
  • the bioengineered heparin showed a lower iduronic acid content and higher glucuronic acid content compared to commercial heparin, and both numbers fell outside of the previously reported normal iduronic acid and glucuronic acid content variability range of different USP heparin.
  • the controlled N-deacetylation of heparosan is especially important, not only because it affects the N-acetyl content and molecular weight of the final heparin produced, but it also has a profound impact on subsequent enzymatic steps.
  • the precursor polysaccharide sequences created by previous chemoenzymatic modification steps can impact the sequence of polysaccharides produced in the following enzymatic steps.
  • control of the chemical steps of heparosan N-deacetylation/A-sulfonation is critical.
  • N-deacetylation with NaOH when appropriately controlled, can provide an optimal N- acetyl/N-sulfo ratio in the resulting polysaccharide product as well as result in a partially depolymerized product of the desired molecular weight.
  • the reaction conditions of aqueous NaOH with heparosan was carefully controlled to obtain (1 ) the appropriate level of N-deacetylation necessary for an N-acetyl/N-sulfo ratio that matched pharmaceutical heparin and, simultaneously, (2) molecular weight of the polysaccharide was similarly controlled to obtain the desired range.
  • the anticoagulant polysaccharides reported by Kuberan in 2003 were completely N- deacetylated and had no TV-acetylglucosamine residues, no molecular weight data were reported, and no anticoagulant activity levels were measured to compare with commercial heparin, although a gel mobility shift assay indicated binding with antithrombin.
  • the chemoenzymatically synthesized polysaccharides reported by Chen in 2007 and Zhang in 2008 also used per-TV-sulfonated heparosan as starting material and thus do not resemble the commercial heparin due to the lack of N-acetylglucosamine residue in the polysaccharide chain.
  • the anticoagulant polysaccharides reported by Chen in 2005 were synthesized by first chemically desulfonating commercial heparin, and is therefore irrelevant to the production of bioengineered heparin.
  • bioengineered heparin presented here has comparable TV-sulfoglucosamine and TV-acetylglucosamine content to that of the pharmaceutical heparin and its molecular weight and anticoagulant activity closely match the pharmaceutical heparin. While the inventors have identified optimal conditions for TV-deacetylation, TV-sulfonation. and depolymerization, we observed differences between bioengineered heparin and pharmaceutical USP heparin. Our analysis is that these differences, associated with the fine structure, are due the enzymatic steps following the TV-deacetylation and N- sulfonation and will require further research to make bioengineered heparin that is in all respects equivalent to pharmaceutical heparin.
  • the chemical iV-deacetylation of heparosan with aqueous sodium hydroxide has two major effects on the heparosan chain precursor.
  • the heparosan MN may vary from batch to batch due to the complexity of bioprocesses, and the expression of a K5 lyase, which depolymerizes the heparosan chain.
  • a different degree of depolymerization is required, based on the different starting heparosan M , to produce bioengineered heparin of the M resembling USP heparin. This may be achieved by varying the N-deacetylation reaction conditions.
  • the reaction product must also meet another criterion, the appropriate N-acetyl content, and a third objective, a d product yield.
  • the goal of the heparosan N-deacetylation becomes a multi- objective optimization problem, in which three objectives are targeted: N-acetyl content, M N , and product yield.
  • the model accurately predicted the required reaction conditions for N-deacetylation and depolymerization with different number average molecular weight heparosan samples.
  • E. coli K5 heparosan was produced by E. coli K5 strain (ATCC #23506) fermentation and purified from the culture supernatant as described previously. More specifically, the K5 heparosan with MN of 14.9 KDa and 1 4.0 KDa was produced by two different batches of exponential feeding fed-batch culture (WO 201 1/028668) and the K5 heparosan with M N of 18.4 KDa was produced from a pH-stat fed-batch culture (Wang et al. (201 l a) Escherichia coli K5 heparosan fermentation and improvement by genetic engineering," Bioengineered Bugs 2, 63-67). All the heparosan batches were purified with DEAE Sepharose® Fast Flow anion exchange resin from GE Healthcare (Piscataway, NJ) WO 201 1 /028668.
  • K5 polysaccharide was dissolved in varying concentration of 1 ml NaOH solution and incubated at different temperatures for different lengths of time. The reaction mixture was then di luted to 5 ml, cooled on ice, and adjusted to pH 7 with HC1. Sodium carbonate (60 mg) and trimethylamine-sulfur trioxide complex (60 mg) were added in a single step, and the mixture was incubated for 12 h. An equal portion of sodium carbonate and trimethylamine- sulfur trioxide was again added after 12 h and the chemoselective TV-sulfonation was continued for an additional 12 h at 50 °C.
  • the solutions were then brought to room temperature, dialyzed overnight against disti lled water using a 3500 Da molecular weight cut- off (MWCO) cellulose membrane.
  • MWCO molecular weight cut- off
  • Commercial heparin typically has a M around 15 KDa, with very few chains below 3500 Da, thus, the use of a 3500 Da MWCO cellulose membrane can effectively remove salt from the sample and should not affect the product chains that are ir target range.
  • the dialyzate was lyophilized to obtain salt-free, TV-sulfo, TV-acetyl heparosan polysaccharide.
  • the starting heparosan material used for the factorial design and Box-Behnken design had a M N of 14.9 KDa.
  • N-acetyl content was calculated as the peak area of N- acetyl glucosamine HI proton divided by the sum of peak areas of N-acetyl glucosamine HI proton and N-sulfo glucosamine HI proton.
  • the peak areas were calculated using the "manual integration" or "line fitting" function of Mnova NMR software.
  • SEC is a useful method for determining the molecular weight properties of heparin.
  • SEC was performed using TSK-GEL® G3000PWxl or G4000PWxl size exclusion column with a sample injection volume of 20 ⁇ and a flow rate of 0.6 ml/min on an apparatus composed of a Shimadzu® LC-l OAi pump, a Shimadzu® CBM-20A controller and a Shimadzu® RID- 10A refractive index detector.
  • the mobile phase consisted of 0.1 M NaNC>3.
  • the column was maintained at 40°C with an Eppendorf® column heater during the chromatography.
  • the SEC chromatograms were recorded with the LCsolution® version 1.25 software and analyzed with its "GPC Postrun" function.
  • TSK- GEL® G4000PWxl size exclusion column was used, and hyaluronan standards of different molecular weights (30.6 kDa, 54 kDa, 125 kDa and 250 kDa ), purchased from Hyalose L.L.C. (Oklahoma City, Oklahoma), were used as calibrants for the standard curve.
  • A. Full factorial design A four factor, two-level full factorial design was generated with the Minitab® software from the tab "Stat -> DOE -> Factorial -> Create Factorial Design" and performed with the four factors being heparosan concentration, NaOH concentration, reaction time and reaction temperature. Each of the factors has two coded levels, and the corresponding uncoded values are illustrated in Fable 5a.
  • the full factorial design was based on the first-order model: where, Y is the response, ⁇ is the model intercept and ⁇ is the linear coefficient, and Xj is the level of the independent variable.
  • the purpose of the full factorial design was to identify the factors that significantly affect the product properties in terms of N-acetyl content, MN and yield, and further investigate their effects quantitatively for predicting the reaction conditions in the subsequent response surface design.
  • Another value of the full factorial design is to identify the insignificant factors and exclude them from the next stage of experimental design, thus reducing the number of runs needed for the next response surface design. These insignificant factors can be set to a level that favors the process economics to benefit the real production process.
  • the full factorial design was carried out in duplicate to be statistically reliable. The experimental data were analyzed with the software Minitab® 15 following the tab "Stat -> DOE -> Factorial -> Analyze Factorial Design".
  • the 15 jV-deacetylation experimental runs were conducted in 1 ml centrifuge tubes, and the reaction temperature was controlled with temperature-adjustable water bath.
  • the experimental data was analyzed with MiniTab® 15 following "Stat -> DOE -> Response Surface -> Analyze Response Surface Design" and fitted into a second-order equation.
  • MiniTab® 15 following "Stat -> DOE -> Response Surface -> Analyze Response Surface Design” and fitted into a second-order equation.
  • the quadratic equation model is as the following:
  • Y ⁇ + ⁇ PiXi + ⁇ , 2 + P X.Xj (2)
  • Y is the predicted response;
  • ⁇ is the offset term;
  • ⁇ ⁇ is the linear effect;
  • ⁇ is the squared effect;
  • ⁇ is the interaction effect, and
  • Xj and X j are the dimensionless coded value of the variable Xj and Xj.
  • X, ⁇ (3)
  • Table 5b represents the conditions for running the four-factor, two level full factorial design
  • Xi heparosan concentration
  • N-acetyl content Yl (%) 3.392 - 0.039 X, - 1.336 X 2 - 3.392 X 3 - 1 .322 X 4 (4)
  • Depolymerization factor Y 2 0.23 + 0.0137 X, - 0.0919 X 2 - 0.0484 X 3 - 0.1415 X 4 (5)
  • heparins typically have a N-acetyl content of 1 1 .9% - 17.6%, with an average N- acetyl content of 14.8% (Guerrini et al. (2001 ) "Combined quantitative ( 1 )1 1 and ( 13)C nuclear magnetic resonance spectroscopy for characterization of heparin preparations," Semin Throw b llemost 27:473-82).
  • MN MN ranging from 14.3 KDa to 1 5.9 KDa, with an average MN of 15. 1 KDa.
  • N-sulfo, /V-acetyl heparosan produced by heparosan N-deacetylation and vV-sulfonation needs to go through enzymatic C5 epimerization and 0-sulfonations to become bioengineered heparin ( Figure 1 ).
  • the targeted /V-sulfo, N-acetyl heparosan should have a N-acetyl content of ideally 14.8%, or within the range of 1 1 .9% - 17.6%; and the targeted M N of the N-sulfo, vV-acetyl heparosan should be ideally 1 1 .7 KDa, or within the range of 1 1.0 KDa - 12.3 KDa.
  • the reaction condition of 2 M NaOIl, reaction time 3 h. and reaction temperature 60 °C gives a product with properties that are very close to the targets.
  • reaction condition of 2 M NaOH, reaction time 3 h. and reaction temperature 60 °C was chosen as the center point for the Box- Behnken design.
  • the high and low levels of each factor was chosen empirically (Table 6a) and all reactions were conducted at the 10 mg/ml heparosan concentration level, as heparosan concentration is not significant and a high level can increase process throughput.
  • the Box- Behnken design and the responses are illustrated in Table 6b.
  • the Box-Behnken responses were analyzed with the Minitab® software, and the regression result is illustrated in Table 6c.
  • the P value for most of the variables and their quadratic terms are greater than 0.05, indicating they are not significant in affecting the product recovery within the experimental design range; only the term X and intercept have P values of less than 0.05.
  • the R " value is relatively low (0.823), indicating only 82.3% of the variability in the response can be explained by the model; moreover, the Analysis of Variance for the response "product recovery” with F test gives an F value of 2.59 and a P value of 0.154, indicating the regression model was not significant at the 95% significance level.
  • the variability in the response "product recovery” was considered to be caused by random error at the experimental design range and was not chosen as a subject for optimization.
  • the R 2 of the regression model for N-acetyl content is 0.995, and the F and P values were 1 19.87 and 0, respectively, indicating good fitting and significance of the regression model.
  • the regression model for depolymerization factor gave a R 2 of 0.960, F value of 13.47 and P value of 0.005, validating the fitting and significance of the regression model.
  • N-acetyl content Y, (%) 15.02 - 23.793Xi - 8.083X 2 - 17.575X 3 + 10.527X, 2 + 4.579X 2 2 + 5.973X 3 2 + 3.472X,X 2 + 6.941X,X + 3.433X 2 X 3 (7)
  • Depolymerization factor Y 2 0.72963 - 0.03175X, - 0.09502X 2 - 0.2227X 3 + 0.01088X, 2 + 0.02028X 2 2 + 0.01632X 3 2 - 0.0257X,X 2 - 0.02684X,X 3 - 0.04509X 2 X 3 (8)
  • the model was first tested on a K5 heparosan batch with a M N of 14.0 KDa, which is smaller than the K5 material used to build the response surface model and would require a depolymerization factor of 0.83 to produce the /V-sulfo, 7V-acetyl heparosan with the desired molecular weight of around 1 1.7 KDa.
  • a reaction condition in coded level of NaOH concentration at 0.535, reaction time at -1 and reaction temperature at 0.0777 should afford a vV-sulfo, N-acetyl with N-acetyl content around 14.8% and M around 1 1.7 KDa.
  • the reaction s correspond to 2.54 M NaOH concentration, 2 h reaction time and reaction temperature at 60.8 °C.
  • the reaction was carried out at these conditions and the N-sulfo.
  • N- acetyl heparosan product was analyzed to have a N-acetyl content of 15.6% and MN of 1 1.3 KDa, close to the targets and within the commercial heparins' variability range.
  • the model was then used to predict the reaction conditions for another K5 heparosan batch, which was larger than the heparosan used to build the model having a MN of 1 8.4 KDa.
  • the response surface model predicts a coded reaction level of NaOH concentration at -0.238, reaction time at 1 , and reaction temperature at 0.143 to produce the N-sulfo, N-acetyl product with the desired N-acetyl content and MN.
  • the reaction conditions correspond to 1.76 M NaOH concentration, 4 h reaction time, and reaction temperature at 61 .4 °C.
  • the reaction was carried out with the above conditions and a N-sulfo, N-acetyl heparosan product with 13.5% TV-acetyl content and 1 1.5 KDa MN was obtained.
  • the N-acetyl content and MN are close to the target and within the commercial heparins' variability range.
  • reaction with NaOH can be used in a single step process to partially depolymerize and N-deacetylate heparosan to obtain a product with the required degree of N- acetylation, M , MW and PDI for manufacturing bioengineered heparin.
  • M the degree of N- acetylation
  • PDI the degree of each reaction depends on time, temperature, and concentration of reactants.
  • heparosan obtained from E. coli K5 fermentation varies in chain length and distribution, depending on culture conditions.
  • a response surface model was established by a full factorial design and a Box-Behnken design experiments. This model provides guidance in choosing the reaction conditions to obtain N-sulfo, N-acetyl heparosan with desired properties.
  • the model is the basis for solving the multi-objective optimization problem in making the ideal V-sulfo, N- acetyl heparosan for bioengineered heparin production.
  • a single reaction condition that meets both the criteria of the right 7V-acetyl content and M of the product can be obtained from the model equations.
  • the model equation 7 was developed to control the properties of the N-acetyl content of the N-sulfo, N-acetyl heparosan product.
  • the starting heparosan material is 100% N-acetylated
  • cetylation step converts the N-acetyl group to V-amino group
  • the V-sulfonation step completely sulfonates the N-amino group to become N-sulfo group.
  • different remaining N-acetyl contents can be obtained in the N-deacetylation and A-sulfonation reactions.
  • N-acetyl heparosan to generate bioengineered heparin, 14.8% N-acetyl content is targeted, which is the reported average N-acetyl content of commercial heparins.
  • the model equation 8 was developed to control the product MN.
  • the term "depolymerization factor" was used to describe the extent of depolymerization occurred during the reaction.
  • the depolymerization during the heparosan N-deacetylation was assumed to be random and not dependent on the starting material chain length.
  • the extent of the depolymerization was assumed to be reaction condition dependent and, thus, can be varied by changing the reaction conditions to obtain different depolymerization extent for starting heparosan material with different MN-
  • the model converts the optimization of the heparosan N-deacetylation condition to the mathematical problem of solving the two equations 7 and 8, which can be easily achieved with Minitab® and Matlab® software.
  • Three factor Box-Behnken design has its best prediction ability when the predicted point falls within 2 radius from the center point (0,0,0).
  • Myers et al. (2002) “Response surface methodology - process and product using designed experiments," 2nd ed. John Wiley & Sons, New York, NY.
  • This range covers the heparosan starting material M n with a lower limit of 12.6 KDa and upper limit of 24.6 KDa.
  • the prediction variance may be large if the heparosan starting material has an M n outside of this range.
  • Heparosan the starting material for synthesis, is produced by bacterial fermentation. To process to heparin, the heparosan must be partially deacetylated to an appropriate level of N-acetylglucosamine residues. It must also be sulfonated, to the correct content of N-sulfoglucosamine. Finally, MN MW and PDI must be reduced from the typically larger molecules produced from fermentation, a problem that is magnified by great batch to batch variation in heparosan.
  • heparosan can be treated in a single step base-catalyzed reaction that gives optimal levels of N- deacetylation and depolymerization to obtain a product that may be processed to bioengineered heparin that is substantially identical to pharmaceutical heparin in several respects.
  • the inventors have also identified relevant factors and algorithms for adjusting reaction conditions to obtain the desired N-deacetylated N-sulfonated product for further processing into bioengineered heparin from variable heparosan sources. This method is surprising in that it produces a molecule with the desired N- acetylglucosamine, N-sulfoglucosamine, MN MW and PDI.
  • the method is highly suited to commercial production.
  • the N-deacetylation and depolymerization can be done in a single step, and is amenable to sulfonation in the same reaction vessel.
  • the reactions occur at high concentrations of heparosan, and with a very high and stable yield.

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Abstract

La production d'héparine biologiquement modifiée qui est chimiquement identique à l'héparine pharmaceutique reste un objectif difficile à atteindre. On a à présent montré que l'héparosane pouvait être traité dans une réaction en une seule étape catalysée par une base qui donne des niveaux optimaux de N-désacétylation et de dépolymérisation afin d'obtenir un produit susceptible d'être transformé en héparine biologiquement modifiée qui est sensiblement identique à l'héparine pharmaceutique. Les inventeurs ont également identifiés les facteurs et les algorithmes pertinents pour ajuster les conditions de réaction afin d'obtenir le produit N-sulfoné N-désacétylé désiré afin de le transformer ensuite en héparine biologiquement modifiée à partir de sources d'héparosane variables.
PCT/US2012/026081 2011-02-22 2012-02-22 N-désacétylation et dépolymérisation d'héparosane en une seule étape pour préparer de l'héparine biologiquement modifiée WO2012116048A1 (fr)

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WO2014204929A2 (fr) 2013-06-17 2014-12-24 The University Of North Carolina At Chapel Hill Molécules d'héparine à activité anticoagulante réversible et leurs méthodes de fabrication
EP3399044A4 (fr) * 2015-12-28 2019-08-07 Ajinomoto Co., Inc. Procédé de production de sulfate d'héparane à activité anticoagulante
WO2020013346A1 (fr) 2018-07-11 2020-01-16 Ajinomoto Co., Inc. Procédé pour la sulfurylation enzymatique d'alcools et d'amines utilisant une bactérie de la famille des enterobacteriaceae
JP2020510119A (ja) * 2017-03-10 2020-04-02 ザ ユニバーシティ オブ ノース カロライナ アット チャペル ヒルＴｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｎｏｒｔｈ Ｃａｒｏｌｉｎａ Ａｔ Ｃｈａｐｅｌ Ｈｉｌｌ 短時間作用型ヘパリンベースの抗凝集剤化合物及び方法
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US11473068B2 (en) 2019-01-15 2022-10-18 Optimvia, Llc Engineered aryl sulfate-dependent enzymes
US11542534B2 (en) 2019-07-09 2023-01-03 Optimvia, Llc Methods for synthesizing anticoagulant polysaccharides
US11591628B2 (en) * 2016-09-07 2023-02-28 Rensselaer Polytechnic Institute Biosynthetic heparin
US11633424B2 (en) 2018-06-20 2023-04-25 The University Of North Carolina At Chapel Hill Cell protective methods and compositions
US11865137B2 (en) 2017-11-03 2024-01-09 The University Of North Carolina At Chapel Hill Sulfated oligosaccharides having anti-inflammatory activity
US11993627B2 (en) 2017-07-03 2024-05-28 The University Of North Carolina At Chapel Hill Enzymatic synthesis of homogeneous chondroitin sulfate oligosaccharides

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JP2016523535A (ja) * 2013-06-17 2016-08-12 ザ ユニバーシティ オブ ノース カロライナ アット チャペル ヒルＴｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｎｏｒｔｈ Ｃａｒｏｌｉｎａ Ａｔ Ｃｈａｐｅｌ Ｈｉｌｌ 可逆的ヘパリン分子、その製法及びその使用方法
EP3011043A4 (fr) * 2013-06-17 2017-01-18 The University of North Carolina At Chapel Hill Molécules d'héparine à activité anticoagulante réversible
US9951149B2 (en) 2013-06-17 2018-04-24 The University Of North Carolina At Chapel Hill Reversible heparin molecules and methods of making and using the same
WO2014204929A2 (fr) 2013-06-17 2014-12-24 The University Of North Carolina At Chapel Hill Molécules d'héparine à activité anticoagulante réversible et leurs méthodes de fabrication
EP3399044A4 (fr) * 2015-12-28 2019-08-07 Ajinomoto Co., Inc. Procédé de production de sulfate d'héparane à activité anticoagulante
US10704068B2 (en) 2015-12-28 2020-07-07 Ajinomoto Co., Inc. Method of producing heparan sulfate having anticoagulant activity
US11591628B2 (en) * 2016-09-07 2023-02-28 Rensselaer Polytechnic Institute Biosynthetic heparin
JP2020510119A (ja) * 2017-03-10 2020-04-02 ザ ユニバーシティ オブ ノース カロライナ アット チャペル ヒルＴｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｎｏｒｔｈ Ｃａｒｏｌｉｎａ Ａｔ Ｃｈａｐｅｌ Ｈｉｌｌ 短時間作用型ヘパリンベースの抗凝集剤化合物及び方法
US11903963B2 (en) 2017-03-10 2024-02-20 The University Of North Carolina At Chapel Hill Short-acting heparin-based anticoagulant compounds and methods
JP7330893B2 (ja) 2017-03-10 2023-08-22 ザ ユニバーシティ オブ ノース カロライナ アット チャペル ヒル 短時間作用型ヘパリンベースの抗凝集剤化合物及び方法
US11993627B2 (en) 2017-07-03 2024-05-28 The University Of North Carolina At Chapel Hill Enzymatic synthesis of homogeneous chondroitin sulfate oligosaccharides
US11865137B2 (en) 2017-11-03 2024-01-09 The University Of North Carolina At Chapel Hill Sulfated oligosaccharides having anti-inflammatory activity
US11633424B2 (en) 2018-06-20 2023-04-25 The University Of North Carolina At Chapel Hill Cell protective methods and compositions
WO2020013346A1 (fr) 2018-07-11 2020-01-16 Ajinomoto Co., Inc. Procédé pour la sulfurylation enzymatique d'alcools et d'amines utilisant une bactérie de la famille des enterobacteriaceae
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