EP0152072A2
Expired / Lapsed
EP
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Pulsed light selective photolysis process for treatment of biological media and products made thereby
Assignee: MOLECULAR BIOPHYSICS TECHNOLOGY Inc
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Filing: 1985-02-08
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Granted: —
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Expires: 2005-02-08
Abstract
Abstract (D A novel irradiation process and products made thereby are disclosed. Biological media such as blood, blood fractions, genetically engineered protein products and vaccine preparations are treated by said process, photolyzing nucleic acids in preference to proteins in said media. E.g., DNA- or RNA-containing pathogens are inactivated while leaving the proteins substantially intact or functional. In general, the process comprises irradiating the medium with pulsed light of wavelength and flux selected so that (1) the nucleic acids in their ground state absorb radiation and thereby rise to an excited state or states, (2) the nucleic acids in their excited states absorb radiation and thereby rise to higher energy states and undergo photolysis, and (3) the proteins in their ground or their excited states do not absorb sufficient radiation to undergo substantial photolysis. It is surprising and unexpected that nucleic acids in their excited states undergo efficient photolysis whereas proteins under the same conditions in the same medium are kept substantially intact.
Inventors
- 👤 Paul A. Cornelius
- 👤 Robin M. Hochstrasser
- 👤 Neville R. Kallenbach
- 👤 Harvey Rubin
- 👤 George J. Todaro
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-07 07:47
CPC Classifications
A; A61; A61L; A61L2/00; A61L2/02; A61K; A61K31/00; A61K31/13; A61K31/14; A61K35/00; A61K35/12; A61K35/14; A61K39/00; A61L2/08; A61L2/10; A61L2103/00; A61L2103/05
Claims Analysis
Total Claims: 43
Claim 1
Independent Claim
93 words
Process for treating a biological medium to photolyze nucleic acids in preference to proteins present therein, characterized in that it comprises irradiating said medium with pulsed light of wavelength and flux selected so that (1) the nucleic acids in their ground state absorb radiation and thereby rise to an excited state or states, (2) the nucleic acids in their excited states absorb radiation and thereby rise to higher energy states and undergo photolysis, and (3) the proteins in their ground or their excited states do not absorb sufficient radiation to undergo substantial photolysis.
Claim 2
Dependent Claim
93 words
Process for treating a biological medium to photolyze nucleic acids in preference to proteins present therein, characterized in that it comprises irradiating said medium with pulsed light of wavelength and flux selected so that (1) the nucleic acids in their ground state absorb radiation and thereby rise to an excited state or states, (2) the nucleic acids in their excited states absorb radiation and thereby rise to higher energy states and undergo photolysis, and (3) the proteins in their ground or their excited states do not absorb sufficient radiation to undergo substantial photolysis.
Claim 3
Dependent Claim
38 words
The process of claim 1, characterized in that the biological medium is a solution selected from blood, blood fractions, genetically engineered proteinacous products of mammalian cells, and vaccine preparations containing viral DNA or RNA in a protein coat.
Claim 4
Dependent Claim
28 words
The process of claim 2, characterized in that the photolysis reduces nucleic acid activity by at least 10 4 and reduces protein functionality by no more than 40%.
Claim 5
Dependent Claim
117 words
The process of claim 3, characterized in that the biological medium is selected from whole blood, blood plasma, blood serum, blood factor VIII, and blood factor XI comprising nucleic acids in the form of DNA- or RNA-containing pathogens, genetically engineered proteinacous products of mammalian cells comprising nucleic acids in the form of active mammalian DNA or RNA, 4ndcvaccine preparations comprising nucleic acids in the form of viral DNA or RNA in a protein coat; and that the photolysis reduces nucleic acid activity (viral activity) by at least 10 4 , and preferably by at least 10 6 , and reduces functionality of the protein coat by no more than 40%, and preferably by no more than 35%.
Claim 6
Dependent Claim
38 words
The process of claim 4, characterized in that the biological medium is an aqueous solution comprising tryptophan-containing proteins; and that the photolysis reduces substantially all of the nucleic acid activity and reduces substantially none of the protein functionality.
Claim 7
Dependent Claim
46 words
The process of claim 1 for treating a biological medium containing nucleic acids and proteins, characterized in that it comprises irradiating said medium with a plurality of laser pulses of wavelength and intensity selected so that said nucleic acids are photolyzed in preference to said proteins
Claim 8
Dependent Claim
23 words
The process of claim 1 or 6, characterized in that the pulsed light is comprised of laser pulses having substantially the same wavelength.
Claim 9
Dependent Claim
115 words
.8. The process of claim 7, characterized in that each of said pulses has a wavelength within the range of 180 to 295 nm, and preferably of 220 to 280 nm, a duration less than 1 x 10 -5 seconds, and preferably in the range of 5 x 10 -9 to 1 x 10 -15 seconds, and most preferably of 1 x 10 -12 seconds, and a flux greater than 1 x 10 15 photons per cm2, and preferably in the range of from 1 x 10 to 1 × 10 18 , and most preferably in the range of from 1 × 10 17 to 1 x 10 18 photons per cm 2 .
Claim 10
Dependent Claim
19 words
The process of claim 1, characterized in that the pulsed light is comprised of laser pulses having different wavelengths.
Claim 11
Dependent Claim
53 words
The process of claim 1 or 9, characterized in that said laser pulses comprise one or more pulse(s) of a first wavelength and flux sufficient to raise a substantial portion of said nucleic acids from their ground state to an excited state, yet not sufficient to photolyze a substantial portion of said proteins.
Claim 12
Dependent Claim
48 words
The process of claim 10, characterized in that said laser pulses further comprise a second pulse which is preferentially absorbed by the nulceic acids in said excited state, but not substantially by said proteins, to thereby cause photolysis of said nucleic acids while minimizing photolysis of said proteins.
Claim 13
Dependent Claim
91 words
The process of claims 9, 10 or 11, characterized in that said laser pulses comprise one or more first pulse(s) of wavelength and flux selected so that said nucleic acids in their ground state absorb radiation and thereby rise to an excited state or states, one or more additional pulse(s) (second pulses) of wavelength and flux selected so that said nucleic acids in their excited state or states absorb radiation and thereby undergo photolysis, and said proteins do not absorb sufficient radiation from any of the pulses to undergo substantial photolysis.
Claim 14
Dependent Claim
43 words
The process of claim 12, characterized in that said excited states of nucleic acids are selected from the singlet state and the triplet state, and that said additional pulse or pulses are applied during the lifetime of said excited states of nucleic acids.
Claim 15
Dependent Claim
18 words
The process of claim 12, characterized in that said first and second pulses are simultaneously or sequentially applied.
Claim 16
Dependent Claim
43 words
The process of claim 12, characterized in that each of said second pulses is applied within 1 microsecond after each of said first pulses, and said first and second pulses are applied in alternating sequences of one or more than one pulse each.
Claim 17
Dependent Claim
190 words
The process of claim 12, characterized in that each of said first pulses has a wavelength within the range of 180 to 350 nm, preferably of 180 to 295 nm, a duration of less than 1 × 10 -5 seconds, and preferably in the range of 1 x 10 -10 to 10 -14 seconds, and most preferably in the range of 10 -5 to 10 -10 seconds, and a flux less than 1 x 10 18 , and preferably within the range of 1 x 10 13 to 5 x 10 14 photons per cm 2 ; and that each of said second pulses has a wavelength within the range of 300 to 700 nm, preferably of 500 to 560 nm, or of 300 to 450 nm, a duration of less than 1 × 10 -5 seconds, and preferably within the range of 1 × 10 -10 to 1 × 10 -12 seconds, and most preferably of 5 x 10 -9 to 1 x 10 -12 seconds, and a flux of greater than 1 x 10 , and preferably not exceeding 1 x 10 18 photons per cm 2 .
Claim 18
Dependent Claim
24 words
- The process of claim 16, characterized in that each second pulse is applied within 3 × 10 -12 seconds of each first pulse.
Claim 19
Dependent Claim
23 words
The process of claim 16, characterized in that each second pulse is applied within 1 × 10 -6 seconds of each first pulse.
Claim 20
Dependent Claim
257 words
A process for sterilizing a biological fluid comprising proteins, which fluid is suspected of carrying DNA- or RNA-containing pathogens, characterized in that it comprises the steps of (a) irradiating said fluid with one or more first light pulse(s) of wavelength(s) within a first wavelenth range of 220 to 280 nm, said first pulse(s) each having a duration of less than 2 x 10 -8 seconds, and preferably between 9 × 10 -9 and 1 × 10-12 seconds per pulse; and having a combined flux within said first wavelength range of between about 1 × 10 13 to 1 × 10 16 , and preferably between 1 × 10 14 and 5 × 10 14 photons per cm 2 ; and (b) irradiating said fluid with one or more second higher intensity light pulse(s) of wavelength(s) within a second wavelength range above 300 nm, preferably from 350 to 410 or 510 to 560 nm, said second pulse(s) each having a duration of less than 2 × 10 -8 seconds, and preferably between 9 × 10 -10 and 1 × 10 12 seconds, and each having a flux within said second wavelength range of at least about 1 × 10 15 , and preferably of 1 x 10 17 to 1 x 10 18 photons per cm 2 , each of said second pulse(s) being applied to said fluid within up to one microsecond after each of said first pulse(s), whereby RNA or DNA-containing pathogens in the fluid are substantially inactivated while proteins in the fluid are substantially unaltered.
Claim 21
Dependent Claim
257 words
A process for sterilizing a biological fluid comprising proteins, which fluid is suspected of carrying DNA- or RNA-containing pathogens, characterized in that it comprises the steps of (a) irradiating said fluid with one or more first light pulse(s) of wavelength(s) within a first wavelenth range of 220 to 280 nm, said first pulse(s) each having a duration of less than 2 x 10 -8 seconds, and preferably between 9 × 10 -9 and 1 × 10-12 seconds per pulse; and having a combined flux within said first wavelength range of between about 1 × 10 13 to 1 × 10 16 , and preferably between 1 × 10 14 and 5 × 10 14 photons per cm 2 ; and (b) irradiating said fluid with one or more second higher intensity light pulse(s) of wavelength(s) within a second wavelength range above 300 nm, preferably from 350 to 410 or 510 to 560 nm, said second pulse(s) each having a duration of less than 2 × 10 -8 seconds, and preferably between 9 × 10 -10 and 1 × 10 12 seconds, and each having a flux within said second wavelength range of at least about 1 × 10 15 , and preferably of 1 x 10 17 to 1 x 10 18 photons per cm 2 , each of said second pulse(s) being applied to said fluid within up to one microsecond after each of said first pulse(s), whereby RNA or DNA-containing pathogens in the fluid are substantially inactivated while proteins in the fluid are substantially unaltered.
Claim 22
Dependent Claim
25 words
The process of claim 19, characterized in that said first pulse(s) is (are) applied at a frequency of between 10 and 1.000.000 pulses per second.
Claim 23
Dependent Claim
17 words
The process of claim 19, characterized in that said first and second pulses are applied substantially simultaneously.
Claim 24
Dependent Claim
26 words
The process of claim 19, characterized in that said first and second pulses are applied in alternating sequences of one or more than one pulse each.
Claim 25
Dependent Claim
29 words
The process of claim 19, characterized in that it comprises placing said fluid in a target region of a thin layer and irradiating said target region with said pulses.
Claim 26
Dependent Claim
22 words
The process of claim 23, characterized in that said layer has a thickness of less-than 0,5 mm, and preferably of 0,2 mm.
Claim 27
Dependent Claim
28 words
The process of claim 23, characterized in that said fluid is passed through said target region at a rate which exposes each portion thereof to said combined fluxes.
Claim 28
Dependent Claim
28 words
The process of claim 25, characterized in that said fluid is passed through each millimeter of said target region at a rate of about 5 milliliters per second.
Claim 29
Dependent Claim
14 words
The process of claim 19, characterized in that said light pulses are laser pulses.
Claim 30
Dependent Claim
17 words
The process of claim 27, characterized in that said light pulses are pulses from a single laser.
Claim 31
Dependent Claim
48 words
The process of claim 27, characterized in that the fluid is selected from the group consisting of blood, blood fractions such as blood plasma, blood factors or blood serum, genetically engineered proteinaceous products of mammalian cells, and vaccine intermediates containing viral DNA or RNA in a protein coat.
Claim 32
Dependent Claim
30 words
The process of claim 29, characterized in that said pulses are applied from a plurality of directions to strike substantially all of the plasma and serum present in said fluid.
Claim 33
Dependent Claim
222 words
The process of claims 1 to 4 for preparing a killed virus vaccine, characterized that it comprises the steps of (a) providing a virus containing solution, said virus comprising a nucleic acid portion and a tryptophan-containing protein coat; (b) irradiating a target region of a thin layer of said virus containing solution with one or more first light pulse(s) of wavelength(s) within a first wavelength range of 220 to 280 nm, said first pulse(s) each having a duration of less than 2 x 10 seconds per pulse; and having a combined flux within said first wavelength range of between about 1 x 10 13 to 1 x 10 16 photons per cm z ; and (c) irradiating said target region of said layer with one or more second higher intensity light pulse(s) of wavelength(s) within a second wavelength range above about 350 nm, said second pulse(s) each having a duration of less than 2 x 10 -8 seconds, and each having a flux within said second wavelength range of at least about 1 x 10 15 photons per cm 2 , each of said second pulse(s) being applied to said layer within up to one microsecond after each of said first pulse(s), whereby said nucleic portion of said virus is inactivated while alteration of the structure of said protein coat is minimized.
Claim 34
Independent Claim
88 words
A method of producing tryptophan-containing proteins, characterized in that it comprises the steps of (a) providing cells of mammalian origin which, when cultured, produce said tryptophan-containing proteins; (b) culturing said cells; (c) releasing said protein into a medium; and (d) inactivating the nulceic acid components in said medium by exposing it to a plurality of pulses of high intensity laser light of different wavelengths which are differentially absorbed by said nucleic acid components in their ground and excited states to produce nucleic acid inactive, protein rich end products.
Claim 35
Independent Claim
88 words
A method of producing tryptophan-containing proteins, characterized in that it comprises the steps of (a) providing cells of mammalian origin which, when cultured, produce said tryptophan-containing proteins; (b) culturing said cells; (c) releasing said protein into a medium; and (d) inactivating the nulceic acid components in said medium by exposing it to a plurality of pulses of high intensity laser light of different wavelengths which are differentially absorbed by said nucleic acid components in their ground and excited states to produce nucleic acid inactive, protein rich end products.
Claim 36
Dependent Claim
56 words
The method of claim 32, characterized in that _ said inactivating step further comprises irradiating said medium with a first light pulse of a first wavelength of sufficient flux to raise a portion of said nucleic acid components from their ground state to an excited state, yet not sufficient to inactivate the proteins in said medium.
Claim 37
Dependent Claim
70 words
The method of claim 33, characterized in that said inactivating step further comprises irradiating said nucleic acids while in said excited state with a second light pulse which is absorbed by nucleic acids in said excited state, but not substantially by said proteins in their ground state, to raise said acids to higher energy states to thereby cause photolysis of said nucleic acids while minimizing the photolysis of said proteins.
Claim 38
Dependent Claim
16 words
A biological medium treated by the process of one or more of claims 1 to 18.
Claim 39
Dependent Claim
16 words
A biological fluid sterilized by the process of one or more of claims 19 to 30.
Claim 40
Dependent Claim
14 words
A blood fraction or blood sterilized by the process of claim 29 or 30.
Claim 41
Dependent Claim
20 words
A virus vaccine prepared by the process of one or more of claims 1 to 18 and 31 to 34.
Claim 42
Dependent Claim
16 words
A protein composition prepared by the method of one or more of claims 1 to 34.
Claim 43
Dependent Claim
15 words
A blood fraction comprising plasma proteins prepared by the process of claims 1 to 30.
References Cited (Backward Citations)
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