CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 61/463,713, filed February 22, 201 1 ; and U.S. Provisional Patent Application No. 61/572,229, filed July 13, 201 1.

GOVERNMENT LICENSE RIGHTS

This invention was made with U.S. Federal Government support under grant HL096972 awarded by the National Institutes of Health. The U.S. Federal Government has certain rights in the invention.

BACKGROUND

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. Lindahl et al. (2005) "Generation of "Neoheparin" from E coli K5 capsular polysaccharide" J Med Chem 48(2):349-352; Zhang et al. (2008) "Solution structures of chemoenzymatically synthesized heparin and its precursors" Journal of the American Chemical Society 130(39): 12998- 13007. At the same time, molecules produced by methods of the prior art are not structurally and biologically equivalent to porcine intestinal heparin. For example, the heparin analog of Zhang ( 2008) was almost completely free of N-acetyl groups. The present inventors have overcome a crucial barrier in the synthesis from heparosan of a heparin that is equivalent to porcine intestinal heparin.

The inventors have identified a single step reaction between heparosan and NaOH that gives optimal levels of Λ-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.

SUMMARY OF THE INVENTION

The production of 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.

According, in one aspect, 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. 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. For example, 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. Applicants have found that high concentrations of heparosan may be used, such as about 10 g/L, although lower concentrations of heparosan may also be used.

In other embodiments, the 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) _. wherein Y i is the N-acetyl content, X] is the molar NaOH concentration, X ₂ is the reaction time in (hours), and X ₃ is the reaction temperature (Celsius)

In other embodiments, the 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| X ₂ - 0.02684X] X ₃ - 0.04509X ₂ X ₃ (8) wherein Y ₂ is the depolymerization factor, X _\ is the molar NaOH concentration, X ₂ is the reaction time in (hours), and X ₃ is the reaction temperature (Celsius)

In some embodiments, 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. In further embodiments, 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.

This method produces bioengineered heparin which is substantially equivalent to

pharmaceutical heparin with respect to (i) N-acetylglucosamine and N-sulfoglucosamine levels and (ii) number average molecular weight (M _N ), weight average molecular weight

(M _\ v) and polydispersity index (PDI). In further embodiments, the bioengineered heparin has a clotting time substantially equivalent to pharmaceutical heparin.

In some aspects, wherein 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.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Scheme for producing bioengineered anticoagulant heparin from E. coli K5 heparosan.

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.

Figures 3A-3B. 3 A: 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. Figure 4. H-NMR of bioengineered heparin (A) and commercial USP heparin sample (B).

Figures 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: ΔϋΑ- 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). The basal activated partial thromboplastin time was 30.5 ± 0.25 seconds, data represents mean ± SD, n = 3. 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).

Figures 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). DE AILED DESCRIPTION OF THE I NVENTION

DEFINITIONS

Heparosan is a polysaccharide with the repeating dissacharide unit [GlcAa-(l -4)GlcNAcR(l - 4)k As used herein, "pharmaceutical heparin' ^" refers to porcine intestinal heparin that complies with United States Pharmacopeia requirements for heparin.

As used herein "bioengineered heparin" refers to heparin that is produced from heparosan, such as the heparosan obtained from microbial fermentation.

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

substantially equivalent to pharmaceutical heparin with respect to N-acetylglucosamine and N-sulfoglucosamine levels and number average molecular weight (MN), weight average molecular weight (Mw) and polydispersity index (PDI) falls within the normal range of pharmaceutical heparin with respect to N-acetylglucosamine level, N-sulfoglucosamine level, M _N , MW, and PDI. Similarly, 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.

A range of values is appropriate because the 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. To deduce the ranges for MN, Μ¾·, N-acetylation or N-sulfonation, seven -~ial heparin active pharmaceutical ingredients and one commercial low molecular 3m different manufacturers were characterized with a view profiling their physicochemical properties. All heparins had similar molecular weight properties as determined by polyacrylamide gel electrophoresis (Mn, 10-1 1 kDa; Mw, 1 3-14 kDa;

polydispersity (PDI), 1 .3-1.4) and by size exclusion chromatography (Mn, 14-16 kDa; Mw, 21- 25 kDa; PDI, 1.4—1.6). The ratio of N-sulfo groups to N-acetyl groups in glucosamine residues ranged from 4.4-6.9 in over a dozen USP heparins examined in the course of ongoing studies. This study provided the physicochemical characterization of USP heparins necessary for the appropriate design and synthesis of a generic bioengineered heparin. See Zhang et al, "Structural characterization of heparins from different commercial sources," Analytical and Bioanalytical Chemistry, 401 , 2793-2803, (201 1). A number of specific metrics are used herein to characterize biological molecules. 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). The number average molecular weight (M _N ), is determined by measuring the molecular weight of n polymer molecules, summing the weights, and dividing by n.

The weight average molecular weight (Mw) is calculated by

where TV; is the number of molecules of molecular weight _; .

Each of MN and Mw may be measured by different means. When comparing 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

:asurement method. The polydispersity index (PDI) measures the degree of heterogeneity in a mixture. PDI is calculated as:

As used herein, (1 ) C5-epimerization/2-0-sulfonation with an equi-unit mixture of C5- epimerase and 2-O-sulfotransferase; (2) 6-O-sulfonation with an equi-unit mixture of 6-0- sulfotransferase-1 and -3; and (3) 3-O-sulfonation with 3-O-sulfotransferase- l , in the presence of a PAPs regeneration system is according to Zhang (2008)

As used herein "a" or "an" means one or more, unless specifically indicated to mean only one. The term '"about ^" is used as understood by the person of skill in the art in the context to which the "about" modifies the stated term. For a numerical value, "about" may be considered to encompass a variation of 10% around the stated value.

As used herein "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. Preferably, 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.

FERMENTATION OF E. coli K5 FOR THE PRODUCTION OF HEPAROSAN

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. For example, 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.

The importance of any one contaminant is related to the ease by which it may be removed by subsequent purification and processing steps, whether the contaminant interferes with subsequent processing of heparosan into heparin, and whether the contaminant poses a danger to humans or animals. Heparosan, as a polysaccharide, may copurify with contaminants such as lipolysaccharide (LPS), nucleic acids, non-heparosan polysaccharides, and derivatives of heparosan.

For example, 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 .

Table 1. Number average molecular weight (MN), weight average molecular weight (Mw) and polydispersity index (PDI) of heparosan recovered from different media in 2.8 L shake flasks. From WO 201 1/028668.

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. However, even if it is highly pure, 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.

PRODUCTION OF HEPARIN FROM HEPAROSAN

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 ). To enable the final bioengineered heparin to resemble the structure of the USP porcine heparin, 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. Even more important, the 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.

At the same time, base-catalyzed deacetylation may also reduce the molecular weight of

1 through a limited depolymerization reaction whose chemistry is not well understood. Heparin depolymerization (like other polysaccharides) is usually achieved through acid-catalyzed mechanisms, such as with Nitrous acid.

Surprisingly, Applicants have found that certain reaction conditions with NaOH can, in a single step, achieve the desired degree of (1 ) N-acetylation, (2) molecular size and (3) polydispersity required for the preparation of a bioengineered heparin that is substantially equivalent to pharmaceutical heparin in M.N, MW, and PDI,. Even more surprisingly, the bioengineered heparin was substantial ly equivalent to pharmaceutical heparin in terms of N- acetylglucosamine and N-sulfoglucosmine levels, coagulation inhibition, and was close to the sugar composition of pharmaceutical heparin in several respects. The invention may be further understood by reference to the following examples, which are provided to illustrate certain embodiments of the invention are not intended to be limiting. The person of ordinary skill would understand that variations may be made without departing from the spirit of the invention.

EXAMPLES EXAMPLE 1 DEMONSTRATION OF BASE CATALYSED PARTIAL

DE ACETYL ATI ON AND DE-POLYMERIZATION IN A SINGLE STEP PERMITS PRODUCTION OF BIOENGINEERED HEPARIN THAT IS BIOEQUIVALENT TO USP PORCINE HEPARIN.

I. Materials and methods A. Preparation of Escherichia coli K5 heparosan

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.

B. iV-Deacetylation/iV-SuIfonation of K5 heparosan 5 polysaccharide ( 100 mg) was dissolved in 25 ml of 2 M NaOH, incubated for 5 h at 60°C, 5 ml aliquots were removed every hour, cooled to room temperature, and adjusted to pH 7 ... ττ ι E _acn tj _me points was then warmed to 45-50 °C, sodium carbonate (60 mg) and imine-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 selective Λ-sulfonation was continued for an additional 12 h at 50 °C. The solutions were then brought to room temperature, dialyzed overnight against distilled water using a 3500 Da molecular weight cut-off (MWCO) cellulose membrane. The dialyzate was lyophilized to obtain salt-free, yV-sulfo, N-acetyl heparosan polysaccharide. See Kuberan et al (2003a) "Chemoenzymatic synthesis of classical and non-classical anticoagulant heparan sulfate polysaccharides," J Biol Chem 278:52613-21.

C. Expression of heparan sulfate biosynthetic enzymes and heparin lyases The catalytic domains of human C5-epimerase, hamster 2-O-sulfotransferase, hamster 6-0- sulfotransferase-1 , mouse 6-0-sulfotransferase-3, and mouse 3- -sulfotransferase-l were recombinantly expressed in E. coli and purified as described previously (Zhang et al. (2008) "Solution structures of chemoenzymatically synthesized heparin and its precursors" Journal of the American Chemical Society 130(3 ) : 12998- 13007. Chen et al. (2005) "Enzymatic redesigning of biologically active heparan sulfate," J Biol Chem 280:42817-25). 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)).

D. Preparation of bioengineered heparin from iV-acetyl, N-sulfo heparosan by enzymatic modification Three enzymatic modification steps were used to convert N-acetyl, N-sulfo heparosan to a bioengineered anticoagulant heparin: (1 ) C5-epimerization/2-0-sulfonation (with an equi-unit mixture of C5-epimerase and 2-O-sulfotransferase); (2) 6-O-sulfonation (with an equi-unit mixture of 6- -sulfotransferase-l and -3); and (3) 3-O-sulfonation (with 3-0- sulfotransferase- 1 ). Each step was performed in buffer in the presence of a PAPs regeneration system. (Chen (2005), Zhang (2008)).

E. NMR analysis of iV-acetyl, N-sulfo heparosan

All ¹ H-NMR were conducted on a Briiker® 600 MHz NMR spectrometer. The samples were in 5 mm standard NMR tubes. Acquisition of the spectra was carried out using TOPSPIN® 2.0 software. All the spectra were acquired at the temperature of 298 K. A recycle delay time of 10 s was used. The acquired Ή-NMR spectra were processed with Mnova NMR software for phase and baseline correction. The peak areas were calculated with the "manual integration" or 'line fitting" function of Mnova NMR software. F. Polyacrylamidc gel electrophoresis molecular weight determination of pharmaceutical heparin and bioengineered heparin

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 μg) 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 μΐ, 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. H. Disaccharide composition analysis using liquid chromatography-mass spectrometry

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 μη , 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. I. In vitro anticoagulation activity of bioengineered and pharmaceutical heparin.

Stock solutions of both heparin samples were prepared in saline and added to platelet-poor pooled human plasma that had been collected over sodium citrate. Following re-calcification with calcium chloride and the addition of Platelin® reagent (BioMeriux, Durham, NC), clotting times were determined on an AMAX® coagulation analyzer (Sigma).

II. Results

A. Control of the /V-sulfo/iV-acetyl ratio in the /V-deacetylation and N- sulfonation step

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 resulting N-sulfo, N-acetyl heparosan was subjected to Ή-NMR study for determining the N-sulfo/iV-acetyl ratio of the products (N-sulfo/N-acetyl ratio = [1 - %N-acetylglucosamine]/%N-acetylglucosamine, where % N-acetylglucosamine = jV-acetyl content). 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 Λ-acetyl was replaced with an N-sulfo group (Figure 2A). The polysaccharide obtained after N-sulfonation was used for the Ή-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 Ή-NMR. The N-acetyl content decreased with prolonged reaction time. In treatment with 2 M NaOH at 60° C, a 3 h N-deacetylation reaction performed in triplicate yielded an N-acetyl content of 12.3-16.6% as determined from the NMR spectra. The mean value of N-acetyl content was 14.8%, with 95% confidence limits of 12.2% to 17.3% (Figure 2B). These values closely match the N-acetyl content reported for pharmaceutical heparins of 1 1.9-17.6%. Guerrini et al. (2001) "Combined quantitative (1 )H and (13)C nuclear magnetic resonance spectroscopy for characterization of heparin preparations," Semin Thromh Hemost 27:473-82.

B. The influence of the N-deacetylation step on the molecular weight of the polysaccharide The K5 heparosan obtained from fermentation and purification showed a number average molecular weight of 49 KDa, much larger than the molecular weight of commercial heparin (Figure 3). After a 3 h N-deacetylation reaction time, the number average molecular weight of the product jV-deacetylated heparosan decreased to 9.7 KDa as determined by size exclusion chromatography (Figure 3A). From this N-deacetylated heparosan, we demonstrated the production of a bioengineered heparin with molecular weight properties similar to those reported for porcine intestinal heparins, as shown Figure 3B, and further demonstrated below.

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. The values obtained for pharmaceutical heparin and bioengineered heparin in this study were M 13 & 14 KDa and M 18 KDa, respectively (Figure 3B). Because the chemical N-sulfonation and enzymatic O-sulfonation steps are under mild conditions, we reasoned that no further depolymerization should occur the molecular weight of the bioengineered heparin should be the molecular weight ;etylated heparosan plus the added sulfo groups. The increase in molecular weight from 9.7 Da for /V-deacetylated hcparosan to 13 Da lor bioengineered heparin after sul foliation confirms this hypothesis.

Therefore, a molecular weight of the Λ'-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.

By carefully selecting the appropriate N-deacetylation reaction conditions, we surprisingly obtained both the desired molecular weight properties (Figure 3) and the optimal .Y-sulfo .Y- acetyl ratio (Figure 2B and Figure 4). Heparin's binding with both antithrombin and thrombin to form a ternaiy complex is chain-size dependent, and so controlling the heparin molecular weight is critical in matching the anticoagulant activity of bioengineered heparin with pharmaceutical heparin.

C. Structural comparison of the bioengineered heparin with pharmaceutical heparin The ¹ H-NMR spectrum of the bioengineered heparin was similar to that of a pharmaceutical heparin prepared from porcine intestine (Figure 4). The sharp peaks from 3.4 to 3.8 ppm, not present in the pharmaceutical heparin spectrum, correspond to a glycerol impurity in the bioengineered heparin, which is carried over from the O-sulfo transferase enzymes where it is used as a cryopreservative. Bioengineered heparin has comparable N-sulfoglucosamine and N-acetylglucosamine content to that of the pharmaceutical heparin (Table 2).

The 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 values for 3-O-sulfoglucosamine, 2-O-sulfo iduronic acid, iduronic acid, glucuronic acid in bioengineered heparin were not similar to the values observed by our laboratory and others leparin (see Table 2). Table 2. Percent substitution of glucosamine and uronic acids for bioengineered heparin and

USP heparin as determined by Ή-NMR integration (Figure 4).

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.

Disaccharide analysis with high performance liquid chromatography-mass spectrometry after heparinase Π digestion afforded the disaccharide composition of the bioengineered heparin and commercial heparin (Figure 4 and Table 3). All major disaccharide components present in pharmaceutical heparin can also be found in the bioengineered heparin except for Δ UA2S-GIcNAc6S (Table 4).

Table 3. Structure of heparin-derived disaccharides prepared from bioengineered and commercial USP he arin samples using heparinase II.

Table 4. Disaccharide compositions of bioengineered heparin and commercial USP heparin.

D. Anticoagulant activity of the bioengineered and pharmaceutical heparins Activated partial thromboplastin time assays were used to assess the global in vitro anticoagulant activity of the heparin samples. The clotting times obtained at three concentrations of heparin showed statistically identical values (Figure 6).

III. Discussion

The controlled N-deacetylation of heparosan, the first chemical step in the chemoenzymatic modification of heparosan chain to afford anticoagulant heparin, 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. In an effort to build bioengineered heparin that closely resembles porcine intestinal heparin, control of the chemical steps of heparosan N-deacetylation/A-sulfonation is critical. We have shown that 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 structure, molecular weight and anticoagulant activity of this bioengineered heparin better match porcine intestinal heparin compared to previous versions of bioengineered heparin reported by Kuberan et al. (2003a) "Chemoenzymatic synthesis of classical and non-classical anticoagulant heparan sulfate polysaccharides," J Biol Chem 278:52613-21 ; Chen (2005) J Biol Chem 280:42817-25; Chen et al. (2007) "Using an enzymatic combinatorial approach to identify anticoagulant heparan sulfate structures" Chem Biol 14:986-93; and Zhang (2008) Journal of the American Chemical Society 130(39): 12998-13007)

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.

In contrast, the 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.

EXAMPLE 2: RESPONSE SURFACE OPTIMIZATION OF HEPAROSAN DEACETYLATION

I. Conversion of heparosan to heparin requires optimization of multiple input and output variables

The chemical iV-deacetylation of heparosan with aqueous sodium hydroxide has two major effects on the heparosan chain precursor. First, the A-acetyl groups of the N- acetylglucosamine residue are partially (or completely) removed to give unsubstituted amino groups. Second, the heparosan polysaccharide chain is partially depolymerized, reducing its molecular weight. Both of these must be controlled to obtain the desired level of N-acetyl substitution, molecular weight and distribution (M _\ y, MN, and PDI). Moreover, a high recovery is necessary for commercial production. There are four major factors that can be varied in the N-deacetylation/depolymerization step that can impact the product's N-acetyl content, average molecular weight and recovery yield. These are the initial concentration of heparosan, the NaOH concentration, the reaction time, and the reaction temperature. These four factors must be controlled to obtain a product with desired N-acetyl content, average molecular weight and a high recovery yield. An additional factor that must be considered is the variation in heparosan reactant. Heparosan produced from E. coli K5 fermentation varies in M due to variation in the medium composition and culture conditions. Even under a well-controlled fermentation operation, 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. However, the reaction product must also meet another criterion, the appropriate N-acetyl content, and a third objective, a d product yield. Thus, 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.

We developed a model to solve this complex problem. The model accurately predicted the required reaction conditions for N-deacetylation and depolymerization with different number average molecular weight heparosan samples.

I. Materials and Methods

A. Preparation of Escherichia coli K5 heparosan

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.

B. N-Deacerylation 7V-SuIfonation of K5 heparosan

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. 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.

C. NMR analysis of A-sulfo, A-acetyl heparosan

' H-NMR was conducted on a Briiker® 600 MHz NMR spectrometer. The samples were prepared in 5 mm standard NMR tubes after lyophilization in D ₂ 0. Acquisition of the spectra was carried out using TOPSPIN® 2.0 software. All the spectra were acquired at the temperature of 298 K. A recycle delay time of 10 s was used. The acquired Ή-NMR spectra were processed with Mnova NMR software for phase and baseline correction. The HI proton of the glucosamine was used for quantifying the N-acetyl content, as it shows a peak at around 5.31 ppm when N-acetyl glucosamine and shifts to around 5.55 ppm when N-sulfo glucosamine. A typical Ή-NMR spectrum of N-sulfo, N-acetyl heparosan produced is shown in Figure 2 with the peaks assigned. 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.

D. Size exclusion chromatography (SEC) of 5 heparosan and V-sulfo, N- acetyl heparosan for molecular weight determination

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 μΕ 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. For molecular weight determination of K5 heparosan, 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. For

- weight determination of N-sulfo, N-acetyl heparosan, TSK-GEL® G3000PWxl ision column was used, and heparin oligosaccharides of different molecular weights (dp 6. dp 10, dp 16 and dp 20) purchased from Huron (Manchester, UK) were used as calibrants for the standard curve.

II. Experimental design

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, βο is the model intercept and βι 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".

B. Box-Behnken design

Factor analysis along the Box-Behnken design (Box, G.E., Behnken, D.W (1960) "Some new three level designs for the study of quantitative variables. ^" ' Technometrics 2, 455-475) was generated with Minitab® software from the tab "Stat -> DOE -> Response Surface -> Create

Surface Design" and carried out to quantitatively model the effect of the variables on the N-acetyl, N-sulfo heparosan product's N-acetyl content, M _N and yield. The levels of the three significant factors were redefined (Table 6a) to be in the vicinity of the center point and the practically operational region. The Box-Behnken design has 15 experimental runs with three runs at the center point (Table 6b) to develop response surface models that describe the relationship of reaction variables with the product's N-acetyl content, MN and yield, respectively.

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. The quadratic equation model is as the following:

Y = βο +∑PiXi + ΣβπΧ, ² + P X.Xj (2) where Y is the predicted response; βο is the offset term; β _ί is the linear effect; βϋ is the squared effect; β^ is the interaction effect, and Xj and X _j are the dimensionless coded value of the variable Xj and Xj.

For statistical calculations, the relationships between the coded values and actual values are described by the following equation:

X, = ^ (3) where Xj is the dimensionless coded value of the independent variable x _\ ; x; is the actual value of that independent variable; x ₀ is the real value of the independent variable Xj at the center point and Axj is the step change.

III. Results

A. Full factorial design

Table 5b represents the conditions for running the four-factor, two level full factorial design

;sponses. The high and low levels of the factors were chosen according to previous reported conditions for polysaccharide N-deacetylation. The design responses were analyzed with the Minitab® software. Table 5c shows the first-order regression results and the significance levels of each factor. The P value level of 0.05 was used to determine whether a factor has significant effect on the responses. For N-acetyl content, factors X? (NaOH concentration). X3 (reaction time) and X ₄ (reaction temperature) all have a P value of less than 0.05, indicating they are significant in influencing the product iV-acetyl content at the tested levels. Xi (heparosan concentration) gives a P value of 0.946, greater than 0.05, indicating it does not have significant effect on product N-acetyl content. Similarly, X ₂ (NaOH concentration), X ₃ (reaction time) and X ₄ (reaction temperature) all have significant effect on the depolymerization factor and product recovery, while the P values of Xj (heparosan concentration) indicate heparosan concentration is not a significant factor in influencing the product depolymerization and recovery.

We were surprised to find that heparosan concentration was not significant in affecting product properties. This is beneficial, because it permits setting the heparosan to a high level ( 10 g/L) to increase the process throughput capacity, so that more material could be processed in a single batch reaction for prospective commercial production. The regression analysis with Minitab fits the full factorial design data into the following first-order equations:

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)

Product recovery Y3 (%) = 39.75 + 0.47 X, - 8.5 X ₂ - 4.01 X ₃ - 31.54 X ₄ (6)

The negative coefficients of X ₂ , X ₃ and X ₄ for all the three responses indicate they have negative effects on product N-acetyl content, depolymerization factor and product recovery.

B. Box-Behnken design experiment and response surface analysis The effects of the three significant variables NaOH concentration, reaction time and reaction temperature were further analyzed with a Box-Behnken design, aiming to build a quadratic surface model that describes the effects of these three variables on the product properties and can be used for predicting the reaction condition for heparosan with a different MN from the heparosan used for the factorial design and Box-Bchnken design.

Commercial 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). We found that commercial heparins have a MN ranging from 14.3 KDa to 1 5.9 KDa, with an average MN of 15. 1 KDa. The 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 ). According to the C5 epimerization and (9-sulfonation patterns, 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. From the result of the full factorial design, 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. Thus, the 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. For the response "product recovery", 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. However, 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. Thus, 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. Similarly, 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. The effects of the NaOH concentration, reaction time and reaction temperature on the product N-acetyl content and depolymerization factor can be explained with the following second- order polynomial equations: 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 effects of NaOH concentration, reaction time and reaction temperature on product N- acetyl content are illustrated with the three-dimensional response surface and contour plot in Figures 7a, b and c. The effects of NaOH concentration, reaction time and reaction temperature on product depolymerization factor are illustrated with the three-dimensional response surface and contour plot in Figures 8a, b and c. With the response surface model described with equation 7 and 8, the reaction condition that yield a product with both the right iV-acetyl content and a specific depolymerization factor according to the starting heparosan material M can be solved.

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. With the response surface model described by equation 7 and 8, 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.

IV. Discussion

We have found that 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. However, the degree of each reaction depends on time, temperature, and concentration of reactants. Moreover, 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. Furthermore, 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, and the V-sulfonation step completely sulfonates the N-amino group to become N-sulfo group. By varying reaction conditions described with equation 7, different remaining N-acetyl contents can be obtained in the N-deacetylation and A-sulfonation reactions. To prepare N-sulfo, 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- These assumptions are theoretically reasonable and validated experimentally with our model. Thus, the model could be used to control the product MN by varying the depolymerization factor. Product recovery was originally included in the study with the full factorial design, where it is found to be significantly affected by NaOH concentration, reaction time and reaction temperature. However, when the variation range of the reaction condition factors was decreased in the Box-Behnken design, we were surprised to find that the product recovery was effectively constant within those parameters. Thus, given specific input factors, we can obtain a known product at a known recovery rate, thus greatly simplifying application of the reaction to commercial production.

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. This was achieved with the Box-Behnken design, which allows an efficient estimation of the first-order and second-order coefficients with a relatively small number of experimental runs. 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.

To obtain heparosan starting material within this range, partial depolymerization with K5 lyase or acid may be required.

V. Conclusions

The production of bioengineered heparin that is chemically identical to pharmaceutical heparin remains elusive. 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. Surprisingly, it has now been shown that 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. It is further surprising that the method is highly suited to commercial production. For example, the N-deacetylation and depolymerization can be done in a single step, and is amenable to sulfonation in the same reaction vessel. Furthermore, the reactions occur at high concentrations of heparosan, and with a very high and stable yield.

By establishing of customized model that can optimize 3 key conditions despite high variability in the heparosan starting material, a relatively uniform product can be predictably j _anc | relatively few steps/ These result in establishment of a ifacturing step for commercialization of bioengineered heparin. Table 5a: Factors in actual and coded levels for the full factorial design

High level

Factors Symbol Low level (- 1 ) (+1)

Heparosan concentration (g/L) X] 4 10

NaOH concentration (M) X ₂ 2 10

Reaction time (h) X . 3 6

Reactio n temperature (°C) X _j 60 90

Table 5b. The full factorial design and the responses (depolymerization factor = product M _N / starting material _N ).

Product recover}' Y3

Run Xl X; X' X : N-acetyl content Yi (%) Depolymerization factor Yz (%)

1 -1 -1 -1 -1 17.88 0.722 83.39

2 1 -1 -1 -1 17.39 0.736 92.69

3 -1 1 -1 -1 4.67 0.294 77.34

4 1 1 -1 -1 3.43 0.306 60.47

5 -1 -1 -1 1 0.00 0.126 13.38

6 1 -1 -1 1 0.00 0.140 17.38

7 -1 1 -1 1 0.00 0.062 11.39

8 1 1 -1 1 0.00 0.065 4.47

9 -1 -1 1 -1 4.49 0.201 87.02

10 1 -1 1 -1 3.91 0.554 93.98

11 -1 1 1 -1 3.86 0.201 64.03

12 1 1 1 -1 3.68 0.189 48.64

13 -1 -1 1 1 0.00 0.096 7.45

14 1 -1 1 1 0.00 0.096 8.49

15 -1 1 1 1 0.00 0.061 6.75

16 1 1 1 1 0.00 0.064 4.57

17 -1 -1 -1 -1 10.82 0.557 73.47

18 1 -I -1 -1 13.91 0.618 99.87

19 -1 1 -1 -1 3.39 0.217 65.80

20 1 1 -1 -1 3.93 0.226 57.83

21 -1 -1 -1 1 0.00 0.135 17.79

22 1 -1 -1 1 0.00 0.133 17.35

23 -1 1 -1 1 0.00 0.059 4.36

24 1 1 -1 1 0.00 0.059 3.20

25 -1 -1 1 -1 3.46 0.374 64.03

26 1 -1 1 -1 3.79 0.462 84.52

27 -1 1 1 -1 6.33 0.139 48.10

28 1 1 1 -1 3.61 0.147 39.43

29 -1 -1 1 1 0.00 0.100 1.98

30 1 -1 1 1 0.00 0.100 9.17

31 -1 1 1 1 0.00 0.058 2.14

32 1 1 1 1 0.00 0.061 1.56

Table 5c. Effects of the factors from the full factorial design iV-acetyl content Depolymerization factor Recovery

Standard Standard Standard

Variable Coefficient error t value P value Coefficient error t value P value Coefficient error t value P va

Intercept 3.392 0.5763 5.89 0 0.23 0.01874 12.27 0 39.75 1.719 23.12 0

Xi -0.039 0.5763 -0.07 0.946 0.0173 0.01874 0.92 0.365 0.47 1.719 0.28 0.78

X ₂ -1.336 0.5763 -2.32 0.028 -0.0919 0.01874 -4.9 0 -8.5 1.719 -4.94 0

X ₃ -3.392 0.5763 -5.89 0 -0.0484 0.01874 -2.58 0.016 -4.01 1.719 -2.33 0.02 x ₄ -1.322 0.5763 -2.29 0.03 -0.1415 0.01874 -7.55 0 -31.54 1.719 -18.34 0

Table 6a: Factors in actual and coded levels for the Box-Behnken design

Factors Symbol Coded and actual level

-1 ^{~ ~~} 0 ^" 1

NaOH concentration (M) X] 1 2

Reaction time (h) X2 2 4

Reaction temperature (°C) X ₃ 50 60 70

Table 6b. The Box-Behnken design and the responses

N-acelyl content Yi Depolymerization factor

Run Xl X2 X3 (%) Y2 Product recovery Y3 (%)

1 -1 -1 0 66.23 0.848 77.77

2 1 -1 0 8.67 0.890 76.60

3 -1 1 0 44.64 0.682 75.22

4 1 1 0 0.97 0.622 71.97

5 -1 0 -1 76.92 1.047 63.81

6 1 0 -1 18.48 0.983 73.69

7 -1 0 1 30.67 0.585 91.50

8 1 0 1 0.00 0.413 68.65

9 0 -1 -1 56.82 0.990 83.29

10 0 1 -1 32.26 0.917 83.66

11 0 -1 1 12.02 0.706 83.00

12 0 1 1 1.19 0.452 74.51

13 0 0 0 15.20 0.739 101.16

14 0 0 0 14.62 0.761 91.56

15 0 0 0 15.24 0.688 87.14

Table 6c. Regression analysis of the Box-Behnken design

N-acetyl content Depolymcrizalion factor Recovery

Standard Standard Standard

Variable Coefficient error t value P value Coefficient error t value P value Coefficient error t value P v

Intercept 15.02 1.6074 9.345 0 0.72963 0.03683 19.813 0 93.2866 4.006 23.29 0

Xi -23.793 0.9843 -24.172 0 -0.03175 0.02255 -1.408 0.218 -2.1743 2.453 -0.886 0.4 x ₂ -8.083 0.9843 -8.212 0 -0.09502 0.02255 -4.214 0.008 -1.9135 2.453 -0.78 0.4

X ₃ -17.575 0.9843 -17.855 0 -0.2227 0.02255 -9.875 0 1.6511 2.453 0.673 0.5

X, ² 10.527 1.4489 7.266 0.001 0.01088 0.0332 0.328 0.756 -12.2983 3.611 -3.406 0.0

X2 ² 4.579 1.4489 3.16 0.025 0.02028 0.0332 0.611 0.568 -5.5986 3.611 -1.551 0.1

X3 ² 5.973 1.4489 4.122 0.009 0.01632 0.0332 0.491 0.644 -6.573 3.611 -1.821 0.1

XJXT 3.472 1.3921 2.494 0.055 -0.0257 0.03189 -0.806 0.457 -0.5214 3.469 -0.15 0.8

X1X ₃ 6.941 1.3921 4.986 0.004 -0.02684 0.03189 -0.842 0.438 -8.1826 3.469 -2.359 0.0

X2X3 3.433 1.3921 2.466 0.057 -0.04509 0.03189 -1.414 0.217 -2.2155 3.469 -0.639 0.5

R ² = 0.995 R ² = 0.960 R ² = 0.823