Rheologica Acta Rheol Acta 25:287-295 (1986) Shear-induced structure in enzymatically-synthesized dextran solutions J. Sabatié, L. Choplin, F. Paul, and P. Monsan Department of Chemical Engineering, Université Laval, Québec (Canada) and BioEurope, Z. I. Montaudran, Toulouse (France) Abstract: A number of dextran derivatives and low molecular weight dextrans have found outlets in different fields such as fine chemistry and pharmaceutical industry, whereas only a few native (high molecular weight) dextrans have found applications. Thus, theoretical studies have scarcely concerned the rheological properties of native dextran solutions. Among the different synthesis processes, only the enzymatic in vitro synthesis produces a pure dextran, thereby allowing the rheological behavior of dextran solutions to be interpreted in terms of molecular considerations. The shear viscosity curve reveals an irreversible threshold-type shear thickening at a specific critical shear rate ~*. The value of ?* depends on the temperature of experiments and on the dextran concentration. Transient experiments give a description of the shear-thickening as a function of time and allows us to study the effect of several parameters (temperature, solvents, etc.). The shear stress growth, during the strueture formation, is made of two different kinetics: the first one depends strongly on the molecular conformation; the second one is the result of intermolecular interactions of the hydrogen-type bonds. Key words: Dextran, enzymatic synthesis, shear flow, stress growth, structure formation I. Introduction Dextran is the collective name of a large class of bacterial polysaccharides composed exclusively of D- glucose units. Dextran synthesis is catalyzed by an exo- cellular enzyme, dextransucrase (EC 2.4.1.5, ct (1 ~ 6) D-glucan: D-fructose 2 glucosyl transferase) according to the reaction: n sucrose ~ (glucose)~ + n fructose. dextran Dextran is a branched polymer in which the linear parts result from linear condensation of D-glucopyra- nosyl units by c~ (1 ~ 6) osidic bonds, whereas branch- ing points result from ~t (1 ~ 3), « (1 -~ 2) or ~ (1 ~ 4) bonds. The degree, nature and length of branching is related to the strain of bacteria which produced the enzyme and the conditions of polymer synthesis [1]. Dextransucrase from Leuconostoc mesenteroides NRRL B 512 F, a non-pathogenic lactic bacterium, produces a low-branched dextran polymer containing 95% of c~ (1 ~ 6) bonds and 5% ~ (1 ~ 3) bonds, and which is 118 highly soluble in water until relatively high molecular weight (fig. 1). This explains the exclusive use of this strain for industrial production of dextran [2]. 2. Dextran applieations It is clear that most applications of dextran concern low molecular weight fractions (-< 100.000 daltons) as well as dextran derivatives, whereas few examples of native dextran applications in food industry, agricul- ture, coating and metallurgy are given in the literature [3-5]. Most applications are only potential [2]. The biggest outlets are in the pharmaceutical industry where well-controlled molecular weight fractions like 70,000 and 40,000 are used as plasma substitute and blood flow improver, respectively. A number of dex- tran derivatives have found commercial applications in pharmaceutical industry and fine chemistry: iron- dextran, dextran-sulphate (~rw 500,000), chemically modified dextrans used as chromatographic supports (sephadex, sepharose, etc.).
`
`PGR2020-00009
`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1076 - Page 1
`
`

`

`288 Rheologica Acta, Vol. 25, No. 3 (1986) H H %/ HO ~C -='==='='= C [ C= 0 I ° I ."/." ~/ù ù,ù H OH I/H ùo\~___11 o c5~o. --~~ ~~ ,H/! \ x H OH ,~"" "c, / ~- o ù "\f ù où ù~,N', ,/ o où/c,3~c2 /H où =/o~ x\ / c« c\ I\~ / H H ~C~~O r I H ~ C --H .,v~~, CONTINUING J POLYMER CHAIN ~ß Fig. 1. Primary structure of the dextran molecule ' 1.6 3. Rheology of native dextran solutions Because of the high molecular weight and aggrega- tion of native dextrans, their solutions exhibit complex rheological behavior. The rheological behavior is un- doubtfully a conditioning factor for industrial applica- tions. Therefore, without a careful study of the rheo- logical properties of native dextran solutions, their applications remain potential. Only some qualitative observations concerning the rheology of native dextran solutions are reported in the literature. Inverse thixotropy together with strong "fluid elasticity" were observed by De Waele [6] in 1945. He attributed this rheological behavior to the long linear and simultaneously branched structure. In 1957, Hartmann and Patat [7] studied the rheological properties of native dextran and found unusual behavior with a time-dependent shear-thickening phenome- non. The reversibility of this phenomenon was limited to small deformations and small deformation rates. They at- tributed this behavior to mechanically enforced coagulation of dextran rnolecules. Ebert [8] considered that this shear-thickening phenomenon was due to the aggregation of dextran molecules. He built a model of the aggregation process with the assumption that intermolecular hydrogen bonding occurs between helices (also an assumption on the conforrnation of dextran molecule, resulting from intramolecular hydrogen bonding) resulting in a super-structure. Sidebotham [9] pointed out that the work of Ebert [8], the observations of electron microscopy [10, 11], and the nature of the interactions between dextran and proteins [12-14] suggest that dextran molecules remain associated presumably in some form of network structure, even in dilute solution. Later, Fedin et al. [15] emphasized that, in concen- trated solutions, dextran molecules form aggregates which rest highly stable even when diluted. In 1973, Murphy and Whist- ler [3] argued that many properties of dextran solutions might be explained by the presence of motecular associations. On the other hand, it has been shown [16] that dextran does not tend to adopt specific ordered conformation in solution due to both their loosely jointed backbones and their branched and highly flexible structure. The flexibility of dextran mole- cule as a result of their high percentage of c~ (1 ~ 6) bonds is a barrier to liquid-crystal formation, but no birefringence mea- surement has been performed yet to eliminate this possibility. After the work of Hartmann and Patat [7], no careful detailed study of the time-dependent shear-thickening phenomenon exhibited by dextran solutions has been performed. The explanations given previously, for example the formation of aggregates or structure in- duced by shear, are tentative and no decisive interpre- tation has been given. Moreover, it could be claimed that the classical techniques used for dextran synthesis lead to a native dextran of relatively low purity and the impurities such as cellular debris can influence the occurrence of aggregate formation. Our purpose is first to use a method of dextran synthesis which gives us a dextran of high purity. Secondly, we will use this dextran to perform careful rheometrical measurements allowing us to study the occurrence of the time-depen- dent shear-thickening phenomenon as well as the in- fluencing parameters. 4. Dextran synthesis At the present time, extracellular dextransucrase is produced from batch cultures of L. mesenteroides NRRL B512F and dextran is made consecutively
`
`PGR2020-00009
`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1076 - Page 2
`
`

`

`Sabatié et al., Shear-induced structure in enzymatically-synthesized dextran solutions 289 either by in vivo or cell-free processes. Both processes lead to uncontrolled high molecular weight crude dextran. Alcoholic precipitation of the polymer, acid hydrolysis, alcoholic fractional precipitation and finally gel permeation chromatography steps are used to obtain the desired molecular weight dextran [2]. Dis- advantages of this method are related to the complexi- ty of the synthesis medium which tends to enlarge the molecular weight distribution to increase the difficulty and the cost of dextran purification and to decrease the yield of the whole process. We have developed a more refined synthesis thanks to the progress made by dextransucrase purification methods. The production of highly purified dextran- sucrase from L. mesenteroides NRRL B 512 F has been deve!oped using phase partition purification [17] and used for in vitro dextran synthesis. In this case, it is rauch easier to obtain a pure dextran because of the synthesis medium's simplicity and control. This dextran contains 5% of c~(1-~ 3) bonds which attach the side-chains to the backbone. Studies [18, 19] on the length of the side-chains show that 40% of the side-chains consist only of a single D-glucose unit, 45% contain two monomers and 15% contain more. Molecu- lar weights, with values as high as 6-188 daltons, have been reported by most authors from light-scattering and ultracentrifugation measurements [20-23]. Ebert et al. [24] point out that these values are the highest erer obtained for dissolved molecules and determine the molecular weight by four different methods which give them values of about 250,000 daltons. They attribute this difference to intermolecular interactions which form aggregates with very high stability. 5.3 Rheologieal measurements Aqueous solutions of dextran (from 2% to 15% dextran by weight) were made by gently stirring at 45 °C and remain stable and homogeneous during all experiments. These measurements have been per- formed with a "Rheometrics-System Four" rheometer, using the fluid mode with a cone-and-plate fixture (25 mm cone radius and 1 degree cone-angle). 6. Results and diseussion Results of stress growth and stress relaxation experi- ments are given in figure 2. For small shear rates, the shear stress approaches its steady-state value monoton- ically, whereas for larger shear rates, the shear stress growth function ~+(t; %) exhibits an overshoot which increases in magnitude as shear rate % increases. More- over, the time at which the steady-state value is reached deereases as the shear rate increases. All the characteristics of a classical non-linear viscoelastic behavior seem to be exhibited by this dextran solution. For higher values of the shear rate, however, the shear stress growth function presents a completely dif- ferent pattern (fig. 3). Such an evolution of the shear stress is typical of the formation of a shear-induced structure: initially, the shear stress growth curve ex- hibits a sharp overshoot. The time at which this over- shoot oceurs corresponds to a lower value than that shown in figure 2 for lower shear rates, in agreement with non-linear viscoelastic results. After this sharp overshoot the eurve goes through a "minimum" and starts to increase progressively. The build-up of struc- ture is made of two kinetics: the first one being rauch 5. Materials and methods 2.o 5.1 Dextran synthesis medium Sucrose (50g/L), sodium acetate buffer 20mM pH 5,2, sodium benzoate 5 g/L. Purified dextransucrase [17] was added to the synthesis medium to start the reaction at a final activity of 0,1 IU/mL. The tempera- tute was maintained at 10 ° C. 5.2 Dextran purification After consumption of sucrose, dextran was recovered by isopropyl alcohol addition directly to the synthesis medium up to 40% (v/v). Precipitations were repeated several times in order to eliminate fructose and other components. Alcohol was evaporated under reduced pressure and dextran was lyophilized. J.6 ~ -- ~ o~~«;!o. 0.4 10% Oextran (wlw)in water Tsynt h =/O°C ~ Texp =25°C Fig. 2. Shear stress growth and shear stress relaxation function data i, V.~ o ~-- ,-- .. 0 5 I0 15 20 25 3o Time » s
`
`PGR2020-00009
`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1076 - Page 3
`
`

`

`290 l~Jaeologica Acta, Vol. 25, No. 3 (1986) 180 144 IO8 ,2 .g, 72 36 ro __J l 10% Dextran (w/w) in water Texp= 25°C ; Tsynth=lO°C ; ~o=30s -I >)~~' -ja- . \ I !, \ ,,2 -- I [ I t 0 5 I0 15 20 t,min i03 i03 10% Dextran(w/w)in water ® ® Tsynfh = tO°C i Texp = 25 °C ® ® no a = * ~ I,®-L_~ I0 a OI " " • ,r~l ~~~ ." ~,I 2 Jo' '~ VJ ~BSF ~* ~ 0 ~ A ~ .~ _ -~ I0 0 ,oop~o:;:~o ........... ,,,----, ..... , ........... ,,,d , ' ,;; I°-'~o-~ ,o-' ,o ° ~* ~o k°' ~, s -I 500 400 2OO I00 0 25 300 g. 1 z Fig. 3. Evolution of shear stress and pri- mary normal-stress difference during structure formation, r 0 corresponds to a viscostiy value (r/0 = ~0/~0) which is locat- ed on the extrapolation of the BSF-curve (at 70 = 30 s-I), see figure 4 Fig. 4. Shear viscosity and primary normal-stress difference curves. BSF curve: Before Structure For- mation, ASF curve: After Structure Formation; zx, ©: increasing shear rate data, A, o: decreasing shear rate data more rapid than the second one. The equilibrium value is reached after a very long period of time (several hours). If we report the equilibrium values on a viscosity versus shear rate graph (see fig. 4), we then obtain a representation showing a jump in viscosity at a critical shear rate, denoted i*. This threshold-type shear- thickening divides the viscosity curve in two parts. The first part, denoted BSF (Before Structure Formation), exhibits a Newtonian plateau followed by a smooth transition (typical of natural polymers) to a shear-thin- ning behavior. The second part of the viscosity curve, denoted ASF (After Structure Formation), exhibits a more pronounced shear-thinning than in the first part. It is of primary interest to note that the extrapolation of the BSF curve (through ~ values higher than ,)*) gives values of viscosity which correspond to the minima exhibited by the shear stress growth curves after the sharp overshoot (see fig. 3). Such a singularity has been noticed for other polymeric solutions exhibit- ing threshold-type shear-thickening behavior [25, 26]. For the primary normal-stress difference, we found also a sharp discontinuity at the same critical shear rate ~*. But the normal stress growth experiment (fig. 3) exhibits a completely different evolution than that of the shear stress. The primary normal-stress
`
`PGR2020-00009
`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1076 - Page 4
`
`

`

`Sabatié et al., Shear-induced structure in enzymatically-synthesized dextran solutions 291 INPUT ~ LSI iJ I- *« *l OUTPUTS ~,,42~ '3 i ~- I00 80 60 40 20 0 600 480 o 360 n Z 240 ,-..-'-, .... T ù";,', i :: I t i I t i l 120 o; /"--"I-"~'\'\ I r \ ~ i ù. , k \, 6,6 I 3,2 t,mJn t9,8 26,4 33 Fig. 5. Schematic diagram illustrating the reversibility of the structure formation difference goes through a maximum and then ap- proaches the steady-state value. The occurrence of the maximum corresponds approximately to the maximum of the rate of shear stress growth (inflection point) during the first kinetics of the structure formation. At this stage, it is not possible to give an adequate inter- pretation of this coincidence without a detailed knowl- edge of what is occurring during the structure forma- tion. In order to study the reversibility of this threshold- type shear-thickening behavior, we performed a series of step-rate tests (with ~ higher than 9*) of increasing duration. Figure 5 reports the input signal together with the output in shear stress as well as the output in the primary normal-stress difference. We see that the reversibility is limited to moderate strain. When we wait for sufficient time in order to reach the transition zone between the first part and the second part of the shear stress growth curve, the phenomenon becomes partially irreversible and, at the same time, the magni- tude of the overshoot in the primary normal-stress growth curve decreases substantially. When the equi- librium is reached, stopping and starting the shear has no more effect and the equilibrium values are instan- taneously reached for both the shear stress and the primary normal-stress difference. The irreversibility of the structure formation results in the existence of two curves for each material function r/and NI (see fig. 4). The open symbols corre- spond to values obtained with increasing shear rate and the closed symbols to values obtained after structure formation with decreasing shear rate. We have con- structed first the BSF curve up to the critical shear rate ))*. At ;)% we allowed the structure to be formed. After reaching the steady-state value, we continued to con- struct the curves (ASF) with increasing shear rate. A reverse rate sweep (decreasing shear rate) gave us the extension of the ASF curves for the values of shear rate lower than ~*. The ASF viscosity curve seems to indicate that the solution exhibits a yield-stress where-
`
`PGR2020-00009
`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1076 - Page 5
`
`

`

`292 Rheologica Acta, Vol. 25, No. 3 (1986) as, initially (Before Structure Formation) a Newtonian plateau was obtained. In oscillatory shear flow experiments, with a strain value of 100%, the dynamic viscosity shows no discon- tinuity. This is not the case, however, with a strain value of 400% (not shown hefe), for which we extend beyond the linear domain. After the formation of the structure at a shear rate higher than the critical shear rate ~* in shear flow, we performed oscillatory shear flow experiments (100% strain) and we obtained an- other curve (ASF) for dynamic viscosity as shown by figure 6. The BSF and ASF curves for dynamic viscosi- ty have been compared with the corresponding curves for shear viscosity. The Cox-Merz rule appears to be followed in both cases for small values of shear rate and frequency whereas a relatively marked deviation occurs at larger shear rates and frequencies. The storage and loss moduli, G' and G" respectively, before and after structure formation are shown in figure 7. Note the change in slopes for both moduli as well as the fact that the structure formation has a greater effect on the storage elastic modulus than on the loss modulus, resulting in a practically complete elimination of the difference between both moduli after the structure formation. Moreover, the slope of the G'-ASF curve is considerably reduced at small fre- quencies, indicating the presence of a yield-stress. This agrees with what we found in the case of the ASF- shear viscosity curve (see fig. 4). The critical value of the shear rate ~*, corresponding to the onset of the shear-thickening behavior, depends on the temperature as illustrated in table 1, the higher the temperature, the higher the critical shear rate. We also found that ~* depends on the dextran concentra- tion. Further work is going on in order to obtain corre- lations for predicting the onset of the threshold-type shear-thickening behavior and will be the subject of a subsequent publication. Not only does the critical shear rate depend on tem- perature but also the kinetics of the structure forma- tion. We will concentrate our attention and efforts on the mechanism involved in the structure formation. In figure 8, we report the influence of the sample tem- perature on structure formation. In all cases, the critical shear rate is lower than 15 s -1. Both kinetics (the first one as well as the second one) are substantially de- creased when the temperature increases. This can be explained by an increase in the molecular free volume, which results in a reduction of the possibility of inter- molecular interactions. Because the dextran molecule is neutral, the only intermolecular interactions that might occur are hydrogen-type bonds and London attractive forces. tO 3 102 ? 0 G. "¢1 Œ 0 io ° Z~ z~ I I I0% Dextron (w/w) in woter Tsynth = IO°C , Texp : 25°C 16 j 16 2 io -~ i ~.~.Ó- ASF ~~'lh«A ~ ' • I I i iIIIII i i I I I IIII I01 i02 i0 ° ~',s "1 ond w,rod.s -! Fig. 6. Comparison of the shear viscosity ~/and the magnitude of the complexe viscosity r/* before (BSF) and after (ASF) the structure formation. All data were taken at 100% strain tO 3 I0 % Dex'~"]ran (w/w)inrwater i ^ Tsy"th =lO°C ,T,» =250C II BG" i0 ~ ~ • G' ~]~_ ùltl| oG '-~-- [ As~-x I_,. ! ~l z". ° ä~ ,4 I _ii=~ " I_a u . ~'~ ---~~g7.,,',~T'- _°i°J °- i °° '= 11 - A "I ~BSF 1 ,o-, F- ~o~__ d2 i0 "2 i0 -I i00 i01 i02 i05 o~~rod.s -I Fig. 7. Storage modulus G' and loss modulus G" as a function of frequency before (BSF) and after (ASF) the structure for- mation Table 1. Variation of the critical shear rate, ~*, with tempera- ture, Tex p re~ p (°C) i* (s -1) 15 6 20 8.5 25 l0 40 14
`
`PGR2020-00009
`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1076 - Page 6
`
`

`

`Sabatié et al., Shear-induced structure in enzymatically-synthesized dextran solutions 293 701. [ I 25 II ,0% Dextron (w/w)in .oter 6oH Tslmt h : I0 °C i i ? : 15 s-' / il Tex v = 15°C__J 22 50 " .-. I_=d ~olK.-,'---"~7~-~ .... ~'~ '21 / ,o O 5 IO 15 20 25 Time ,min Fig. 8. Influence of temperature on the evolution of shear stress during structure formation 40 I I IO% Dextron (w/w) in woter 35 Tsynth : IO°C ; Texp = 25°C i ~' =15 s -I « --/.-_,~'-~~'---I __~.~.---- ~o ir~~~~.~,~.- ~-'-nÕ J5 "'~" f I0~ I0 20 30 40 50 Timet min Fig. 9. Influence of salts on the evolution of shear stress during structure formation The structure formation is also influenced by the conformation of the dextran molecules in solution. We increased the hydration of the dextran molecule through the addition of salts. This is equivalent to increasing the ionic strength of the solution. Figure 9 illustrates that the more expanded the molecules are, the higher the shear stresses are during the structure formation. This result is consistent with the fact that expanding the molecule deals with an increase in molecular interactions. We also notice that the first kinetics is much more significantly influenced than the second orte (ref. the change in the slopes of shear stress versus time). T~th = IO°C i Top =25°c l ./ ,~ o. ,[ I I I /_ ' , o ~ ~ ,L ,L :~ ] , Or""i" IO 20 30 40 50 Time,min Fig. 10. Influence of urea on the evolution of shear stress during structure formation (~= 15 s-l). Evolution of the rate of stress growth with urea concentration during the second kinetics In order to determine to what extent hydrogen bond- ing is playing a role in the intermolecular interactions occurring during structure formation, we added urea to the dextran solution. The presence of urea in aqueous solutions is known to break many of the hydrogen bonds [27, 28]. Figure 10 shows the inftuence of the addition of urea on the structure formation. The first kinetics remains practically unchanged (the slopes of shear stress versus time are essentially the same), whereas the second one is substantially decreased as indicated by the variation of the rate of stress growth (Az~At) with urea concentration. We interpret the effect of urea as a decrease in the intermolecular interactions of the hy- drogen-type bonds created during the structure forma- tion. These interactions seem to occur principally during the second part of the structure formation. However, because of the presence of water as a solvent, hydrogen bonds cannot be completely eliminated by the addition of urea. A more decisive conclusion can be obtained with the use of dimethylsulfoxide (DMSO) as the solvent. DMSO is considered to be a good solvent for dextran [29]; the molecules have to be in an expanded confor- mation. Figure 11 illustrates the kinetics of structure formation for dextran in DMSO. We use, in this par- ticular case, a 4% (w/w) dextran solution in order to prevent the rupture of hydrogen bonds which can occur when the molecules experience too high shear stresses. With this 4% dextran solution, the shear stresses deve]oped are not sufficiently high to break hydrogen bonds. In figure 11, both effects previously
`
`PGR2020-00009
`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1076 - Page 7
`
`

`

`294 Rheologica Acta, Vol. 25, No. 3 (1986) 45 I [ Dextron Tsynth = IO°C • Texp = 25°C 3e I f~-4 Yo Dextran 27 --/--(w/w)inDM~ ~/ _~. "~ ""---10% Dextran (w/w) in watet 9 O0 5 i I b i -- 8SF I.r. spe¢trum ASF • I I I , 4000 3000cm-! 2(~0 [ I I0 15 20 "riete, min 25 Fig. 11. Influence of DMSO on the evolution of shear stress during structure formation ()?= 15s-]). Infrared speetra of dextran in DMSO before (BSF) and after (ASF) structure formation observed with the presence of salts (fig. 9) and with the presence of urea (fig. 10) are amplified. The first kinetics is considerably increased due to the DMSO whose presence contributes to the expansion of the dextran molecule; on the other hand, the second kinetics is completely eliminated. This last result sug- gests that, in DMSO, no hydrogen bond is created during structure formation. We also show in figure 11 the infrared spectrum of the dextran solution before and after the structure formation: there is no change between the two spectra at the specific wavelength of the hydrogen bonds (2= 3,340cm -I, [29]). We con- clude that, in DMSO, the structure formation is the result of a "chain-chain packing". No hydrogen bond- ing occurs and the equilibrium value of the shear stress is reached relatively rapidly. This "chain-chain packing" occurs during the first kinetics. The shear- induced structure formed by "chain-chain packing" of the expanded molecules is also irreversible provided the equilibrium value of the shear stress is reached. Further work is needed in order to give a more explicit interpretation of this "chain-chain packing"; physical aggregation, gelation, permanent network, etc. are possible explanations which need to be explored. We have also to take into account the correspondence between the maximum of the normal-stress growth curve and the maximum of the rate of shear stress growth (inflection point) during the first kinetics (see fig. 3), in the interpretation of this shear-induced struc- ture formation. We will study also the stability of this structure under thermal treatment. The molecular characterization of enzymatically- synthesized dextran is presently under investigation via intrinsic viseosity measurements, light-scattering, and hydrodynamic chromatography. We are also currently exploring other techniques for molecular characteriza- tion of these dextrans in order to complete the under- standing of the mechanism involved in the structure formation. 7. Conclusions Contrarily to the classical methods for dextran pro- duction, the in vitro synthesis using purified enzyme allows us to produce a pure dextran without cellular debris which are known to contribute to the formation of microgels in solutions. Therefore, the threshold-type shear-thickening phenomenon, which leads to the for- mation of a shear induced structure, can be studied in the light of molecular considerations through careful rheological measurements. The threshold-type shear-thickening occurs at a criti- cal shear rate which depends on the temperature and the potymer concentration. For shear rates higher than the critical shear rate, a shear induced structure is formed. Provided sufficient strain is involved, the structure formation is irreversible. Once the structure is formed, the rheological properties of the solution are completely changed: the solution exhibits a more pronounced shear-thinning than before the structure formation; the solution exhibits also a yield stress. Moreover, the responses in stress growth experiments are instantaneous. We have found that the formation of the structure is made of two distinct kinetics: the first one results from a "chain-chain packing" and depends strongly on the molecule conformation. The second one is due to intermolecular interactions which are essen- tially hydrogen bonds. Acknowledgements The authors wish to acknowledge financial support pro- vided by the Natural Sciences and Engineering Research Council of Canada and by the FCAR program of the Province of Quebec. References 1. Jeanes A, Haynes WC, Wilham CA, Rankin JC, Melvin EH, Austin MJ, Cluskey JE, Fisher BE, Tsuchiya HM, Rist CE (1954) J Amer Chem Soc 76:5041 2. Alsop RM (1983) Progress Ind Microbiol 18:1 3. Murphy PT, Whistler RL (1973) in: Whistler RL, DeMiller JN (eds) Industrial Gums - Polysaccharides and their derivatives, 2nd ed. Academic Press, New York, p 513
`
`PGR2020-00009
`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1076 - Page 8
`
`

`

`Sabatié et al., Shear-induced structure in enzymatically-synthesized dextran solutions 295 4. Jeanes A (1966) Encycl Polym Sci Technol 4:805 5. Jeanes A (1977) ACS Symposium series 45:284 6. De Waele A (1945) Chem Ind (London) 64:253 7. Hartmann J, Patat F (1957) Makromol Chemie 25:53 8. Ebert HK (1967) Monatshefte Chemie 98:1128 9. Sidebotham RL (1974) Adv Carbohydrate Chem Biochem 30:371 i 10. Becker WT, Milch RA (1967) Johns Hopkins Med J 121:234 11. Newbrun E, Lacy R, Christie TM (1971) Arch Oral Biol 16:863 12. Laurent TC (1963) Biochem J 85:253 13. Laurent TC (1963) Acta Chem Scand 17:2664 14. Laurent TC, Killander J (1964) J Chromatogr 14:317 15. Fedin EI, Tsitsishvili VG, Grinberg YA, Bakari TI, Tolstoguzov VB (1975) Carbohydr Res 39:193 16. Powell DA (1979) Microbial polysaccharides and poly- saccharases. Berkeley, Ellwood, Academic Press, London, plt7 17. Paul F, Monsan P, Auriol D (1983) French Patent 8307650 18. Abbot D, Bourne EJ, Weigel H (1966) J Chem Soc (C):827 19. Larm O, Lindberg B, Svesson S (1971) Carbohydr Res 20:39 20. Arond LH, Frank PH (1954) J Phys Chem 58:953 21. Bovey FA (1959) J Polymer Sci 35:167 22. Jeanes A (1965) in: Whistler RL, DeMiller JN (cds) Methods in Carbohydrate Chemistry, vol 5. Academic Press Inc, New York London, p 118 23. Senti FR, Hellman NN, Ludwigh NH, Babcock GE, Tobin R, Glass CA, Lamberts BL (1955) J Polym Sci 17:527 24. Ebert KH, Schenk G, Rupprecht G, Brosche M, Weng HW, Heinicke D (1965) Makromol Chem 96:206 25. Jackson KP, Walters K, Williams RW (1984) J Non-New- tonian Fluid Mech 14:173 26. Choplin L, Sabatié J, submitted to Rheol Acta 27. Southnick JG, Lee H, Jamieson AM, Blackwell J (1980) Carbohydr Res 84:287 28. Antonini E, Bollelli L, Bruzzesi MR, Caputo A, Chian- cone E, Rossi-Fanelli A (1964) Biopolymers 2:27 29. Casu B, Reggiani M (1966) Tetrahedron 22:3061 (Received June 19, 1985) Authors' addresses: J. Sabatié, Prof. L. Choplin Department of Chemical Engineering Université Laval Québec, QC, G1K7P4 (Canada) Dr. F. Paul, Dr. P. Monsan BioEurope 4, impasse Didier-Daurat Z. I. Montaudran F-31400-Toulouse
`
`PGR2020-00009
`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1076 - Page 9
`
`