Thermal stability of sodium hyaluronate in
`aqueous solution
`
`Karen M. Lowry and Ellington M. Beavers*
`Biocoat, Inc., 455 Pennsylvania Avenue, Suite 120, Ft. Washington, Pennsylvania 19034
`
`Since its identification 60 years ago as a ubiquitous compo(cid:173)
`nent of the body of mammals, hyaluronic acid has been
`widely studied, primarily in the fields of medicine and bi(cid:173)
`ology. On the other hand, our research has dealt with
`hyaluronic acid as a chemical intermediate in the synthesis
`of novel lubricious coatings, and in this connection data
`were needed on stability of aqueous solutions of the poly(cid:173)
`mer over a range of temperatures from 25-100°C. The in-
`
`vestigation reported here provides that information, ob(cid:173)
`tained by exposing samples in sealed ampules in baths at
`controlled temperatures and determining the resulting
`change in viscosity of the solutions. Data of this kind have
`not previously been reported on sodium hyaluronate freed
`from the proteins and other organics normally associated
`with the polymer in its natural environment. © 1994 John
`Wiley & Sons, Inc.
`
`INTRODUCTION
`
`Hyaluronic acid and its salts are ubiquitous in
`mammalian bodies, serving in aqueous solution as
`lubricants, as viscoelastic humectants, in wound
`healing, and in various physiologic roles. l
`-5 It has
`never been synthesized in vitro, and was first isolated
`in 1934 by Meyer and Palmer6 from bovine vitreous
`humor. It has since been isolated also from umbilical
`cord, rooster comb, and bacterial culture broths. In all
`of its natural sources, the acid and salts are closely
`associated with other carbohydrates, proteins, lipids,
`and unidentified impurities, and over the years of its
`recognition as a polymeric "entity," the problems of
`isolating and purifying the substance have been stud(cid:173)
`ied and processes refined. 7-9
`Partly because of its polymeric nature, but mainly
`because of its biologic importance, sodium hyaluro(cid:173)
`nate (NaHy) has been given greater attention in the
`fields of medicine and biology than by chemists. In
`our research of the past 10 years, however, its role
`has been that of a most unusual and valuable chem(cid:173)
`ical intermediate in the synthesis of novel, lubricious
`coatings. 10-18
`In the course of that research, the need arose early
`for reliable information on the thermal stability of
`
`*To whom correspondence should be addressed.
`
`aqueous solutions of NaHy. Published information
`on the subject proved not to be amenable to compar(cid:173)
`ison on a common basis, especially not for establish(cid:173)
`ing kinetics of the degradation over a range of tem(cid:173)
`peratures of interest. Furthermore, uncertainty about
`the purity of the polysaccharide used in studies be(cid:173)
`fore the past decade made the data unsuitable except
`in the most general sense.
`In this study, the viscosity (a function of molecular
`weight) of dilute aqueous solutions of an ultrapure
`grade of NaHy was monitored after controlled times
`of exposure to various temperatures ranging from'25-
`90°C in sealed containers.
`
`TABLE I
`Analytical Values for Sodium Hyaluronate,
`Pharmaceutical Grade
`
`Appearance
`Sodium chloride content
`Sodium hyaluronate content
`Sodium content
`Water content (Karl Fischer)
`Intrinsic viscosity
`Molecular weight
`pH (1 % aqueous solution)
`Proteins
`Sulfated ash
`Viable count
`Salmonella
`Escherichia coli
`
`White, odorless powder
`0.14%
`93%
`5.2%
`10.7%
`1970 mVmg
`1.3 x 106 d
`6.9
`0.05%
`16.1%
`Olg
`Negative
`Negative
`
`Journal of Biomedical Materials Research, Vol. 28, 1239-1244 (1994)
`© 1994 John Wiley & Sons, Inc.
`
`CCC 0021-9304/94/101239-06
`
`VAL0060240
`
`ALL 2040
`PROLLENIUM V. ALLERGAN
`IPR2019-01505 et al.
`
`

`

`1240
`
`LOWRY AND BEAVERS
`
`Time
`
`CFT
`
`0.0
`0.5
`1.0
`2.0
`4.0
`6.0
`7.7
`24.0
`48.0
`
`317.56
`303.18
`304.59
`337.81
`315.97
`316.12
`307.11
`302.53
`312.46
`
`Time
`
`CFT
`
`0.0
`1.0
`2.0
`4.0
`6.0
`24.0
`48.0
`96.0
`
`430.71
`516.86
`545.46
`535.07
`421.50
`498.70
`447.67
`485.71
`
`Time
`
`CFT
`
`0.0
`0.5
`1.0
`2.0
`4.0
`6.0
`24.0
`48.0
`54.3
`
`2308
`2502
`2332
`2318
`2485
`2538
`2168
`1883
`2033
`
`Time
`
`CFT
`
`0.0
`0.5
`1.3
`2.0
`4.0
`6.0
`24.0
`
`1055
`1015
`775
`640
`203
`374
`239
`
`50°C
`s2
`
`0.1876
`1.5567
`0.2680
`5.0330
`2.5658
`4.1398
`1.8452
`0.6268
`0.4024
`
`50°C
`s'1.
`
`0.4212
`6.9854
`0.4645
`6.7544
`1.7100
`7.6750
`9.1033
`0.5130
`
`50°C
`s'1.
`
`223.0
`474.9
`4090.0
`877.0
`8164.0
`5022.0
`364.0
`82.3
`12441.0
`
`90°C
`s'1.
`
`79.8
`16349.0
`66.6
`58.8
`3.2
`13.6
`20.8
`
`Ratio
`
`1.000
`0.9550
`0.9500
`1.0640
`0.9950
`0.9950
`0.9670
`0.9530
`0.9840
`
`Ratio
`
`1.0000
`1.2000
`1.2664
`1.2423
`0.9786
`1.1579
`1.0394
`1.1277
`
`Ratio
`
`1.0000
`1.0841
`1.0104
`1.0043
`1.0767
`1.0997
`0.9393
`0.8159
`0.8808
`
`Ratio
`
`1.0000
`0.9621
`0.7346
`0.6066
`0.1924
`0.3545
`0.2265
`
`TABLE II
`Experimental Data
`
`Series 1
`
`s2 x 105
`
`Time
`
`CFT
`
`0.3721
`1.7130
`4.3690
`5.2010
`2.7280
`4.2890
`2.0040
`0.7904
`0.3996
`
`0.0
`1.0
`2.0
`3.0
`4.0
`5.0
`24.0
`50.5
`
`317.56
`370.60
`355.73
`332.33
`297.20
`325.94
`301.90
`266.83
`
`Series 2
`
`s'1. x 105
`
`Time
`
`CFT
`
`0.0
`1.0
`2.0
`4.0
`6.0
`24.0
`48.0
`
`430.71
`509.98
`485.22
`504.45
`381.37
`502.65
`284.85
`
`0.4541
`4.0920
`0.6145
`3.9910
`1.1390
`4.4420
`5.1520
`0.5653
`
`Series 6
`
`8'1. x 105
`
`Time
`
`CFT
`
`0.0
`1.0
`2.0
`3.0
`5.0
`72.0
`96.0
`
`1055
`1153
`1080
`1152
`1363
`746
`636
`
`83.5
`58.0
`119.4
`58.6
`201.6
`144.7
`43.7
`29.3
`265.9
`
`52 x 105
`
`14.3
`1476.0
`9.9
`7.9
`0.6
`2.1
`2.2
`
`60°C
`s'1.
`
`0.1876
`42.8895
`0.7358
`30.2323
`75.0236
`20.0130
`3.1650
`7.3560
`
`70°C
`s'1.
`
`0.4212
`9.4345
`0.8233
`1.6825
`0.1873
`0.3925
`0.0616
`
`60°C
`s'1
`
`79.8
`495.7
`2.6
`495.7
`791.3
`9.4
`32.8
`
`Ratio
`
`1.0000
`1.1670
`1.1202
`1.0465
`0.9359
`1.0263
`0.9507
`0.8403
`
`Ratio
`
`1.000
`1.1840
`1.1266
`1.1712
`0.8854
`1.1670
`0.6613
`
`Ratio
`
`1.0000
`1.0929
`1.0237
`1.0919
`1.2919
`0.7545
`0.6028
`
`s'1. x 105
`
`0.3721
`42.7800
`0.9631
`30.1800
`74.5600
`20.0400
`3.3070
`7.4240
`
`s'1. x 105
`
`0.4541
`5.4040
`0.7320
`1.2180
`0.2790
`0.5208
`0.1325
`
`s'1. x 105
`
`14.3
`53.1
`7.7
`53.1
`83.1
`4.9
`5.6
`
`MATERIALS AND METHODS
`
`The NaHy used was of an ultrapure pharmaceuti(cid:173)
`cal grade, supplied by the Pharma Division of Dio(cid:173)
`synth, with the properties shown in Table I. Two
`liters of an 0.03% (wt/wt) solution in distilled water
`was prepared, filtered through Whatman no. 50 pa(cid:173)
`per, and stored in a closed container in a refrigerator
`at temperatures ranging from just above freezing to
`5°C. Under these experimental conditions, growth of
`microorganisms was inhibited.
`
`Fifteen-milliliter aliquots of this solution were pi(cid:173)
`petted into glass ampules and the necks sealed with
`oxygen torch. The desired number of sealed ampules
`were immersed in a stirred oil bath set at the temper(cid:173)
`ature of interest and controlled to within ±OSc. At
`the end of the desired exposure intervals, one or
`more ampules were removed, immediately immersed
`in ice-water for 5 min, and then transferred to the
`refrigerator. The ampules remained in the refrigera(cid:173)
`tor for approximately 15 min and were then removed
`one at a time and broken at the score, and the solu-
`
`VAL0060241
`
`

`

`HYALURONATE STABILITY
`
`Time
`
`CFT
`
`0.0
`0.5
`1.0
`2.0
`
`317.56
`340.54
`377.90
`340.36
`
`Time
`
`CFT
`
`0.0
`1.0
`2.0
`4.0
`6.0
`24.0
`48.0
`
`430.71
`428.44
`258.04
`318.41
`279.93
`122.08
`95.94
`
`Time
`
`CFT
`
`0.0
`0.5
`1.0
`2.0
`4.0
`5.5
`24.0
`
`1055
`1353
`1233
`1263
`1032
`1085
`752
`
`Series 1
`70DC
`s'1
`
`0.1876
`4.2314
`151.006
`108.650
`
`Series 2
`90DC
`s'1
`
`0.4212
`0.4900
`0.0032
`0.1540
`0.0456
`0.1477
`0.0481
`
`70DC
`s'1
`
`79.8
`12649.0
`250.7
`1597.0
`38.8
`1637.0
`166.7
`
`Ratio
`
`1.0000
`1.0724
`1.1900
`1.0718
`
`Ratio
`
`1.0000
`1.0179
`0.5991
`0.7393
`0.6499
`0.2834
`0.2227
`
`Ratio
`
`1.0000
`1.2825
`1.1687
`1.1972
`0.9782
`1.0284
`0.7128
`
`TABLE II
`Continued
`
`s'1 x 105
`
`Time
`
`CFT
`
`0.37
`4.41
`150.00
`108.00
`
`0.0
`2.0
`4.0
`23.3
`28.3
`46.3
`75.0
`
`534.07
`529.08
`521.82
`518.38
`517.68
`514.22
`513.61
`
`s'1 x 105
`
`Time
`
`CFT
`
`0.4541
`0.4994
`0.0832
`0.2071
`0.1205
`0.0979
`0.0372
`
`0.0
`1.0
`2.0
`4.0
`7.0
`12.0
`24.0
`28.0
`
`484.24
`467.07
`475.50
`481.18
`418.01
`385.15
`375.24
`351.41
`
`Series 6
`
`s'1 x 105
`
`Time
`
`CFT
`
`Series 2
`25DC
`s'1
`
`5.5297
`0.0809
`24.8954
`10.0093
`14.2699
`46.4422
`0.6242
`
`Series 3
`70DC
`s'1
`
`43.579
`0.826
`0.426
`0.410
`7.717
`0.260
`0.071
`0.320
`
`80DC
`s'1
`
`14.3
`1148.0
`32.3
`153.8
`10.3
`154.6
`18.6
`
`0.0
`1.0
`2.0
`3.0
`4.0
`5.0
`24.0
`48.0
`
`1055
`1070
`922
`903
`766
`855
`541
`306
`
`79.8
`2054.7
`232.9
`106.6
`10.1
`602.1
`255.0
`465.1
`
`Ratio
`
`1.0000
`0.9907
`0.9771
`0.9706
`0.9693
`0.9628
`0.9617
`
`Ratio
`
`1.0000
`0.9645
`0.9820
`0.9937
`0.8632
`0.7954
`0.7749
`0.7257
`
`Ratio
`
`1.0000
`1.0142
`0.8739
`0.8559
`0.7261
`0.8104
`0.5128
`0.2900
`
`1241
`
`s'1 x 105
`
`3.8770
`1.9310
`10.0580
`5.3360
`6.8240
`18.0800
`2.0120
`
`s'1 x 105
`
`37.170
`17.640
`18.100
`18.530
`17.140
`11.870
`11.190
`9.924
`
`s'1 x 105
`
`14.3
`192.0
`26.4
`14.8
`4.7
`58.8
`24.8
`42.4
`
`tion was filtered through a 30-fine fritted glass disk. A
`10-ml portion of the filtrate was pipetted into the vis(cid:173)
`cometer mounted in a constant-temperature bath at
`25°C. After a period of 30 min to allow equilibration to
`the test temperature, capillary flow times (eft) were
`determined in calibrated Cannon-Fenske Routine
`Viscometers. Three such observations were made for
`each sample, and the values were averaged (Table II).
`The flow times (seconds) could be converted to spe(cid:173)
`cific viscosities by using the eft for water; however,
`because our interest is in the change of viscosity as a
`
`function of exposure time, we analyzed the following
`quantities instead:
`
`Cftt
`r= -
`cfta
`cft~ 2
`I
`2
`2
`Sr = ~ Scft + ft4 Seft •
`cJta
`t coO
`In these equations Cftt is the eft after exposure time of
`t h, efta is the eft at zero exposure time, and 52 is the
`sample variance of the subscripted observation.
`
`VAL0060242
`
`

`

`1242
`
`1.30
`
`1.20
`
`1.10 -
`
`1.00
`
`0.90
`'ED 0.80
`----...
`'E 0.70 -
`0.60
`0.50 -
`
`0.40
`
`0.30
`
`0.20
`
`25°e (298°K)
`
`looe (343°K)
`
`LOWRY AND BEAVERS
`
`I'" Series 21
`
`o
`
`10
`
`30
`20
`Time (hours)
`
`40
`
`50
`
`1.30
`
`1.20
`
`1.10
`
`1.00
`
`0.90
`°
`'E 0.80
`----...
`'E 0.70
`0.60
`
`0.50
`
`0.40
`
`0.30
`
`0.20
`
`Q Series 6
`• Series 1
`............... Series 2
`.........• Series 4
`• Series 4 FiJt
`....• Series 3A
`.
`
`..
`
`0
`
`10
`
`20
`
`30
`Time (hours)
`
`40
`
`50
`
`Figure 1. Change of relative viscosity at 2S°C.
`
`Figure 4. Change of relative viscosity at 70°C.
`
`50 0 e (323°K)
`
`80 0 e (353°K)
`
`1.30
`
`1.20
`
`1.10
`
`1.00
`
`\ Q Series 61
`
`~
`
`0.90
`°
`'E 0.80
`----...
`'E 0.70
`0.60
`
`0.50
`
`0.30
`
`" "
`1.20
`1.10-2ff~
`..
`1.00-~~lIr~:
`' ~
`
`1.30
`
`II
`
`0.90 -
`'ED 0.80 -
`> U 0.70
`
`0.60
`
`0.50
`
`0.40
`
`0.30
`
`... c...
`
`Q Series 6
`• Series 1
`II Series 2
`
`0.20
`
`o
`
`!
`
`!
`
`10
`
`30
`20
`Time (hours)
`
`40
`
`50
`
`0.20
`
`Lc...
`0
`
`10
`
`'
`
`!
`
`30
`20
`Time (hours)
`
`40
`
`50
`
`Figure 2. Change of relative viscosity at SO°C.
`
`Figure 5. Change of relative viscosity at 80°C.
`
`60 0 e (333°K)
`
`90 0 e (363°K)
`
`1.30
`
`1.20
`
`1.10
`1.00
`
`0.90
`'ED 0.80
`----...
`'E 0.70
`0.60
`
`0.50
`
`0.40
`
`0.30
`
`0.20
`
`~' ...
`Jae •.... ~
`•
`
`•
`
`1.30 -
`
`1.20
`
`1.00
`
`0.90
`'ED 0.80
`----...
`'E 0.70
`
`o
`
`I
`
`!
`
`10
`
`,
`
`!
`
`40
`
`50
`
`30
`20
`Time (hours)
`
`0.50
`
`0.40
`
`0.30
`
`0.20
`
`o
`
`o
`
`10
`
`'.
`
`30
`20
`Time (hours)
`
`40
`
`50
`
`Figure 3. Change of relative viscosity at 60°C.
`
`Figure 6. Change of relative viscosity at 90°C.
`
`VAL0060243
`
`

`

`HYALURONATE STABILITY
`
`1243
`
`Thermal Stability of Na Hyaluronate
`
`60"C
`
`~,30 {
`1
`
`120 l
`it '.OG I
`
`j
`-'" 1.10 'I'
`'0
`
`I
`O.9C! t
`
`I
`0.80 L : ...... "~~-...... ,-""~ ...... --.. -'--.............. , .. --........ --, ...... -----.... -
`o
`20
`10
`rime (hcurs)
`
`Figure 9.
`
`Illustration of initial increase in viscosity.
`
`DATA ANALYSIS
`
`It is apparent in examining the data that the NaHy
`solution seemed to undergo a significant increase in
`viscosity for a period of time before the decline in
`viscosity associated with polymer chain degradation
`at all temperatures higher than 25°C. This is illus(cid:173)
`trated in Figure 9 for data at 60°C, where the length of
`the error bars corresponded to 1 SD on either side of
`the average for the point. If only thermally induced
`chain scission were taking place, none of the plotted
`numbers would be greater than 1.
`This phenomenon, if real, may be related to the
`well-known viscoelastic behavior of solutions of
`NaHy of molecular weight higher than about
`1,000,000,19,20 which has been attributed to extensive
`
`Thermal Degradation of 0.03%
`Na Hyaluronate Solutions
`
`100000
`
`10000
`
`1000
`
`100
`
`10
`
`0.1 L..c~~~_~~<----L-l.~~~~L~~.cJ
`20
`30
`40
`50
`60
`70
`80
`90
`
`Temperature ('C)
`
`Figure 10. Time required for reduction in viscosity by 10%
`and by 50% from initial values.
`
`VAL0060244
`
`Figure 7. Time of exposure relative to temperature and
`viscosity.
`
`The data were fitted to a model by means of an
`iterative numerical technique with use of the software
`"TI< Solver PlusRII supplied by Universal Technical
`Systems, Inc. The actual and fitted data at the tem(cid:173)
`peratures studied are shown in Figures 1 through 6.
`Error bars are shown for one of the data series (series
`6) but not for others because the limitations of mono(cid:173)
`chromatic printing would not allow one series to be
`distinguished from another. With use of the fitted
`model, Figure 7 is constructed to relate time of expo(cid:173)
`sure to temperature and to viscosity. Figure 8 is a
`topographic map of Figure 7.
`
`90
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`o 5 10 15 20 25 30 35 40 45 50
`
`Figure 8. Topographic map of stability data.
`
`

`

`1244
`
`LOWRY AND BEAVERS
`
`chain entanglement and chain-chain interaction of
`the highly hydrated polymer?1 On the other hand,
`we tend to favor the idea that temperatures higher
`than that at which the original dry polymer was put
`into solution induce further "dissolving" -that is,
`further separation of chain segments hydrogen(cid:173)
`bonded to each other as in the original solid. The
`effect on viscosity is tantamount to increasing the
`number and/or length of polymer molecules. The ad(cid:173)
`ditive effect would then be overtaken and decline as
`simultaneous chain scission due to thermal degrada(cid:173)
`tion continues at rates characteristic of the tempera(cid:173)
`ture.
`At 25°C, there was virtually no change in viscosity
`over the period studied. No net decline was seen at
`50°C, but at 60°C and above, there was a rapid in(cid:173)
`crease in viscosity followed by exponential rates of
`decline. Figure 10 shows the times required for 10
`and 50% degradations in viscosity calculated from the
`fitted data.
`
`CONCLUSIONS
`
`The rate of viscosity breakdown of an aqueous so(cid:173)
`lution of sodium hyaluronate increases exponentially
`with temperature. At 2SoC, a drop in viscosity of 10%
`requires many thousands of hours; at 90°C, the same
`drop occurs in <1 h.
`The practical conclusion is that NaHy solutions
`should be stored in the short term at temperatures no
`higher than 2S°C. Storage at O-soC would further im(cid:173)
`prove stable life by approximately 2 orders of magni(cid:173)
`tude, other factors being equal. A sterile solution of
`NaHy protected from inoculation by microorganisms
`and stored in the refrigerator is estimated on the basis
`of these results to be stable for many years.
`
`Mrs. Renee (Luckenbill) Keller contributed to the exper(cid:173)
`imental work described here. Dr. John D. Guerin (Turning
`Points Management Consulting, Inc.) supplied much of the
`data analysis.
`
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`
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`
`Received August 30, 1993
`Accepted March 31, 1994
`
`VAL0060245
`
`