US4517295A — Hyaluronic acid from bacterial culture

Claim 1

Independent Claim 72 words

A method of producing hyaluronic acid comprising: fermenting a broth culture of hyaluronic acid-producing streptococcus bacteria in a growth medium under CO 2 -enriched anaerobic conditions; separating the bacterial cells from the resulting broth; and isolating the hyaluronic acid from the remaining constituents of the broth by separating the acid and those broth consituents of similar or higher molecular weight and then separating the acid from those similar broth constituents by precipitation.

Claim 2

Independent Claim 72 words

A method of producing hyaluronic acid comprising: fermenting a broth culture of hyaluronic acid-producing streptococcus bacteria in a growth medium under CO 2 -enriched anaerobic conditions; separating the bacterial cells from the resulting broth; and isolating the hyaluronic acid from the remaining constituents of the broth by separating the acid and those broth consituents of similar or higher molecular weight and then separating the acid from those similar broth constituents by precipitation.

Claim 3

Dependent Claim 26 words

A method as in claim 1 wherein the growth medium is made pyrogen-free prior to inoculation by filtration thereof through a 10,000 molecular weight cutoff filter.

Claim 4

Dependent Claim 20 words

A method as in claim 1 wherein the bacterial cell separation step includes first adding trichloroacetic acid to the broth.

Claim 5

Dependent Claim 24 words

A method as in claim 3 wherein the trichloroacetic acid is added to make the fermentation mixture up to a final concentration of 5-6%.

Claim 6

Dependent Claim 20 words

A method as in claim 3 wherein the growth of the bacteria is terminated by said addition of trichloroacetic acid.

Claim 7

Dependent Claim 25 words

The method of claim 1 wherein the precipitation is accomplished by adding the hyaluronic acid and the remaining broth constituents to ethanol without substantial mixing.

Claim 8

Dependent Claim 36 words

The method of claim 1 wherein isolation is accomplished by separating the broth from the cells, diafiltering the broth and precipitating the acid from solution by adding the solution to an organic solvent without substantial mixing.

Claim 9

Dependent Claim 33 words

A method as in claim 1 wherein the fermentation step includes controlling the pH at a substantially constant value ±0.1 within the range of 6.5-7.5 and the temperature to 37±1° C. throughout fermentation.

Claim 10

Dependent Claim 21 words

A method as in claim 8 wherein the pH is controlled by the automatic addition of base with a pH probe/controller.

Claim 11

Dependent Claim 19 words

A method as in claim 1 wherein the dissolved CO 2 is maintained at a concentration of about 5-10%.

Claim 12

Dependent Claim 26 words

A method as in claim 1 wherein the bacteria is grown to a density of greater than 2×10 8 cells per ml before termination of growth.

Claim 13

Dependent Claim 13 words

The method of claim 1 wherein the baceria are Streptococcus pyogenes type 18.

Claim 14

Dependent Claim 43 words

A method as in claim 1 carried out by filtering the growth medium through a 10,000 molecular weight cutoff filter prior to inoculation with the bacteria culture and performing the remaining steps under conditions of a class 100 clean room using pyrogen-free containers.

Claim 15

Dependent Claim 52 words

A method as in claim 13 further comprising: refining the isolated hyaluronic acid by dissolution thereof in a mildly basic buffered solution, precipitation of sodium hyaluronate with a mixed alkyl trimethylammonium bromide, resuspending the precipitate in dilute sodium chloride solution and reprecipitating it as acid by adding it to ethanol without stirring.

Claim 16

Dependent Claim 76 words

In the method of producing hyaluronic acid involving production of same by streptococcus bacteria, the improvement comprising isolating the acid from the broth in which the bacteria grows by separating the acid and those broth constituents of similar or higher molecular weight from the liquid and then separating the acid from those similar broth constituents by precipitation, and wherein the precipitation is accomplished by adding the acid and the remaining broth constituents to ethanol without mixing.

Claim 17

Dependent Claim 76 words

In the method of producing hyaluronic acid involving production of same by streptococcus bacteria, the improvement comprising isolating the acid from the broth in which the bacteria grows by separating the acid and those broth constituents of similar or higher molecular weight from the liquid and then separating the acid from those similar broth constituents by precipitation, and wherein the precipitation is accomplished by adding the acid and the remaining broth constituents to ethanol without mixing.

Claim 18

Dependent Claim 13 words

The method of claim 15 wherein the bacteria are Streptococcus pyogenes type 18.

Claim 19

Dependent Claim 38 words

The method of claim 15 wherein isolation is accomplished by separating the broth from the bacteria, diafiltering the broth and precipitating the acid from resulting solution by adding the solution to an organic solvent without any substantial mixing.

Claim 20

Dependent Claim 33 words

In the method of producing hyaluronic acid involving production of same by streptococcus bacteria, the improvement involving the maintenance of CO 2 -enriched anerobic conditions in the environment of the bacteria growth medium.

Claim 21

Dependent Claim 33 words

In the method of producing hyaluronic acid involving production of same by streptococcus bacteria, the improvement involving the maintenance of CO 2 -enriched anerobic conditions in the environment of the bacteria growth medium.

Claim 22

Independent Claim 99 words

A method of producing hyaluronic acid comprising: growing a culture of hyaluronic acid-producing streptococcus bacteria in a growth medium under CO 2 -enriched anaerobic conditions; forming a liquid suspension of the bacteria and its by-products in the case where the formation of such a suspension is not inherent in the growth method being used; separating the bacterial cells from the suspension; and isolating the hyaluronic acid from the constituents of the remaining liquid by separating the acid and those broth constituents of similar or higher molecular weight and then separating the acid from those similar broth constituents by precipitation.

Claim 23

Independent Claim 99 words

A method of producing hyaluronic acid comprising: growing a culture of hyaluronic acid-producing streptococcus bacteria in a growth medium under CO 2 -enriched anaerobic conditions; forming a liquid suspension of the bacteria and its by-products in the case where the formation of such a suspension is not inherent in the growth method being used; separating the bacterial cells from the suspension; and isolating the hyaluronic acid from the constituents of the remaining liquid by separating the acid and those broth constituents of similar or higher molecular weight and then separating the acid from those similar broth constituents by precipitation.

Claim 24

Dependent Claim 33 words

A method as in claim 19 wherein the growth step includes controlling the pH at a substantially constant ±0.1 value within the range of 6.5-7.5 and the temperature to 37±1° C. throughout fermentation.

Claim 25

Dependent Claim 21 words

A method as in claim 19 wherein the pH is controlled by the automatic addition of base with a pH probe/controller.

Claim 26

Independent Claim 88 words

A method of producing hyaluronic acid comprising: growing a culture of acid-producing streptococcus bacteria under CO 2 -enriched anaerobic conditions on a solid growth medium; forming a liquid suspension of the bacteria and its by-products to separate same from the solid medium, separating the bacterial cells from the suspension; and isolating the hyaluronic acid from the constituents of the remaining liquid by separating the acid and those liquid constituents of similar or higher molecular weight from the liquid suspension and then separating the acid from those similar constituents.

Claim 27

Independent Claim 88 words

A method of producing hyaluronic acid comprising: growing a culture of acid-producing streptococcus bacteria under CO 2 -enriched anaerobic conditions on a solid growth medium; forming a liquid suspension of the bacteria and its by-products to separate same from the solid medium, separating the bacterial cells from the suspension; and isolating the hyaluronic acid from the constituents of the remaining liquid by separating the acid and those liquid constituents of similar or higher molecular weight from the liquid suspension and then separating the acid from those similar constituents.

Claim 28

Dependent Claim 24 words

The method of claim 22 wherein the precipitation is accomplished by adding the hyaluronic acid and the remaining constituents to ethanol without substantial mixing.

Claim 29

Dependent Claim 33 words

A method as in claim 22 wherein the fermentation step includes controlling the pH at a substantially constant value ±0.1 within the range of 6.5-7.5 and the temperature to 37±1° C. throughout fermentation.

Claim 30

Dependent Claim 19 words

A method as in claim 22 wherein the dissolved CO 2 is maintained at a concentration of about 5-10%.

Claim 31

Dependent Claim 26 words

A method as in claim 22 wherein the bacteria is grown to a density of greater than 2×10 8 cells per ml before termination of growth.

Claim 32

Dependent Claim 51 words

A method as in claim 22 further comprising: refining the isolated hyaluronic acid by dissolution thereof in a mildly buffered solution, precipitation of sodium haluronate with a mixed alkyl trimethylammonium bromide, resuspending the precipitate in dilute sodium chloride solution and reprecipitating it as acid by adding it to ethanol without stirring.

Claim 33

Independent Claim 116 words

A method of producing hyaluronic acid comprising: providing hyaluronic acid-producing streptococcus bacteria; combining the bacteria with a growth medium whereby the bacteria produces by-products including hyaluronic acid; maintaining CO 2 -enriched anaerobic conditions in the environment of the growth medium; forming a liquid suspension of the bacteria and its by-products in the case where the formation of such a suspension is not inherent in the growth method and medium being used; separating the bacteria from the suspension; and isolating the hyaluronic acid from the constituents of the remaining liquid by separating the acid and those liquid constituents of similar or higher molecular weight from the liquid suspension and then separating the acid from those similar constituents.

Claim 34

Independent Claim 116 words

A method of producing hyaluronic acid comprising: providing hyaluronic acid-producing streptococcus bacteria; combining the bacteria with a growth medium whereby the bacteria produces by-products including hyaluronic acid; maintaining CO 2 -enriched anaerobic conditions in the environment of the growth medium; forming a liquid suspension of the bacteria and its by-products in the case where the formation of such a suspension is not inherent in the growth method and medium being used; separating the bacteria from the suspension; and isolating the hyaluronic acid from the constituents of the remaining liquid by separating the acid and those liquid constituents of similar or higher molecular weight from the liquid suspension and then separating the acid from those similar constituents.

Claim 35

Dependent Claim 15 words

The method of claim 28 wherein the bacteria is initially provided in a resting suspension.

Claim 36

Dependent Claim 13 words

The method of claim 28 wherein the bacteria is Streptococcus pyogenes type 18.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1988-05-13	1988-11-12	1989-05-14	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	1992-05-13	1992-11-12	1993-05-14	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	1996-05-13	1996-11-12	1997-05-14	$7700.00	Window Closed / Lapsed

Hyaluronic acid from bacterial culture

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