Glycols as pathogen inactivating agents

Information

  • Patent Grant
  • 11553712
  • Patent Number
    11,553,712
  • Date Filed
    Friday, December 23, 2011
    12 years ago
  • Date Issued
    Tuesday, January 17, 2023
    a year ago
Abstract
The disclosure relates to uses, methods and compositions for the inactivation of pathogens in biological compositions, using a glycol as a pathogen inactivating agent.
Description
FIELD OF THE INVENTION

The present disclosure relates to uses, methods and compositions for the inactivation of pathogens in biological compositions.


BACKGROUND OF THE INVENTION

Use of biological compositions is important for developing and producing therapeutics (e.g., the production of recombinant proteins). Biological compositions, such as blood compositions, save many lives by blood transfusion for instance, for patients having a blood disease, a haemorrhage, or undergoing a surgical procedure. However, the presence of pathogens in biological compositions presents a significant health risk.


Methods to inactivate pathogens in biological compositions have been developed. Classical pathogen inactivation methods include approaches based on heat treatment, solvent and/or detergent treatment, gamma irradiation, UV treatment, and leukodepletion. However, the efficiency and effectiveness of said methods varies because of the different sensitivities of pathogens and incompatibility of some methods with specific biological compositions.


There is a need for new pathogen inactivation methods and agents.


SUMMARY OF THE INVENTION

The present disclosure relates to uses, methods, agents and compositions for the inactivation of pathogens in biological compositions.


In one aspect the disclosure relates to the use of a glycol as a pathogen inactivating agent. In some embodiments the glycol is propylene glycol.


In one aspect the disclosure relates to methods for inactivating a pathogen in a biological composition, said method comprising contacting said biological composition with a glycol. In some embodiments for inactivating a pathogen in a biological composition, the glycol is propylene glycol. In some embodiments for inactivating a pathogen in a biological composition, said biological composition is a blood composition or a milk composition. In some embodiments for inactivating a pathogen in a biological composition, said pathogen is selected from the group consisting of viruses, bacteria, fungi, protozoa, parasites, and prions. In some embodiments said virus is selected from the group consisting of X-MuLV, PRV, BVDV and TGEV virus. In some embodiments for inactivating a pathogen in a biological composition, said method results in a pathogen elimination equal or greater than 4 Log10TCID (Tissue Culture Infective Dose) according to the methods of Kärber and/or Spearman-Kärber. In some embodiments for inactivating a pathogen in a biological composition, the concentration of glycol after the contacting step is between 40 and 50% (w/w) of the biological composition. In some embodiments for inactivating a pathogen in a biological composition, the concentration of glycol after the contacting step is between 40 and 50% (v/v) of the biological composition. In some embodiments for inactivating a pathogen in a biological composition, said method is performed at a temperature between 15 and 25° C. In some embodiments for inactivating a pathogen in a biological composition, said method is performed at a pH between 7.0 and 8.0.


In one aspect the disclosure relates to a biological composition comprising a glycol, wherein said biological composition is obtained by any of the methods described herein In some embodiments said glycol is at a concentration between 40 and 50%. In some embodiments said glycol is propylene glycol. In some embodiments the biological composition is a milk composition or a blood composition.





BRIEF DESCRIPTION OF THE DRAWINGS

The drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The figures are illustrative only and are not required for enablement of the disclosure.



FIG. 1 shows TEGV inactivation in an affinity chromatography eluate containing 45% of propylene glycol.



FIG. 2 shows BVDV inactivation in an affinity chromatography eluate containing 45% propylene glycol.





DETAILED DESCRIPTION OF THE INVENTION

In one aspect the disclosure relates to the use of a glycol as a pathogen inactivating agent. In one aspect the disclosure relates to methods for inactivating pathogens in a biological composition, said method comprising contacting said biological composition with a glycol. In one aspect the disclosure relates to a biological composition comprising glycol. In some embodiments said biological composition comprising glycol is obtained by any of the methods described herein.


In some embodiments of the uses, methods and compositions described herein, the glycol is vicinal glycol. In some embodiments the vicinal glycol is propylene glycol or ethylene glycol.


The term “glycol” (or “diol”) refers to a chemical compound containing two hydroxyl groups (—OH). The term “vicinal glycol” refers to a glycol with two hydroxyl groups attached to adjacent atoms (e.g., in vicinal position).


In some embodiments the glycol used in the methods and compositions described herein is a vicinal glycol comprising between two and six carbons and having a chemical formula R1R2—(C—OH)2—R3R4, wherein R1, R2, R3 and R4 may be identical or different and are each either a hydrogen atom or an alkyl group, wherein the combination of R1, R2, R3 and R4 contains at most two carbon atoms. Examples of vicinal glycols are propylene glycol, ethylene glycol, 1,2-butanediol and 1,2-pentanediol.


In some embodiments of the uses, methods and compositions described herein, the glycol is propylene glycol or ethylene glycol.


The term “propylene glycol”, also called “1,2-dihydroxypropane” or “methyl glycol”, refers to propane-1,2-diol and has the structural formula (I) represented below.


The term “ethylene glycol”, also called “1,2-dihydroxyethane”, refers to ethane-1,2-diol and has the structural formula (II) represented below.




embedded image


In some embodiments of the uses, methods and compositions described herein, the glycol is a geminal glycol. Geminal glycols have two hydroxyl groups attached to the same carbon atom and include 1,2-methane diol, 1,2-ethane diol and 1,2-propanediol. In some embodiments of the uses, methods and compositions described herein, the glycol is a diol wherein the hydroxyl groups are not on the same or adjacent carbon atoms. Examples of such glycols are 1,3-butanediols, 1,4-pentanediols, and 1,3-benzenediol.


In one aspect the disclosure relates to pathogen inactivating agents, compositions that comprise such agents and uses thereof.


The term “pathogen” refers to any biological agent (e.g., any nucleic acid containing agent or proteinaceous infectious particle such as a prion) that can cause disease in a mammal, such as a human. The term pathogen includes unicellular and multicellular microorganisms, with DNA or RNA as genetic material, in single-stranded or double-stranded form. The term particularly includes viruses, bacteria, fungi, protozoa and prions. Examples of bacteria include, but are not limited to, Streptococcus, Escherichia and Bacillus species; examples of viruses include, but are not limited to, the Human Immunodeficiency Virus (HIV) and other retroviruses, the herpesviruses, the paramyxoviruses, the poxviruses, the togaviruses, the cytomegaloviruses and the hepatitis viruses (HAV, HBV, HCV); examples of parasites include, but are not limited to, malaria parasites (Plasmodium species) and trypanosomal parasites.


In some embodiments of the disclosure, said pathogen to be inactivated is selected from the group consisting, of viruses, bacteria, fungi, protozoa, parasites and prions.


In some embodiments said pathogen is a virus.


In some embodiments said virus is an enveloped virus or a non-enveloped virus.


Enveloped viruses are viruses that have a host-cell-like “envelope” and include for example, but are not limited to, mammalian or avian Leukemia viruses, Herpes viruses, Pox viruses, Hepadnaviruses, Flaviviruses, Togaviruses, Coronaviruses, Hepatitis viruses, Retroviruses, Orthomyxoviruses, Paramyxoviruses, Rhadoviruses, Bunyaviruses, Filoviruses and Reoviruses. Non-enveloped viruses, also called naked viruses, are well known in the art and include, but are not limited to, adenoviruses, norovirus, rotavirus and human pappillomavirus.


In a some embodiments the virus is X-MuLV, PRV, TGEV or BVDV. The term “X-MuLV” for “Xenotropic murine leukemia virus-related virus” refers to a gammaretrovirus. The term “PRV” refers to a pseudorabies virus. The term “TGEV” for “Transmissible Gastroenteritis Coronavirus” refers to a species of animal virus belonging to the family of Coronaviruses. The term “BVDV” for “Bovine viral diarrhea virus” is a pestivirus from the family of flaviviruses.


In one aspect the disclosure relates to methods for inactivating pathogens in a biological composition, said method comprising contacting said biological composition with a glycol.


As used herein, the term “contacting” refers to a process of bringing into contact at least two distinct compositions or components such that they can interact.


The term “biological composition” refers to a composition (or a material) originating from a biological organism, including mammals. Examples of biological compositions include, but are not limited to, blood compositions, milk (such as milk from transgenic mammals), clinical samples such as urine, sweat, sputum, feces and spinal fluid, cellular and tissue extracts, cell culture medium, etc. As used herein, biological compositions also include synthetic compositions that can function as biological compositions, such as blood substitutes, and compositions that have undergone one or more purification or separation steps.


According to the disclosure a blood composition includes, but is not limited to, whole blood and blood products. The term “blood product” refers to one or more components that may be separated from whole blood, and encompasses cellular blood component (such as erythrocytes or red blood cells, platelets, leukocytes and concentrates thereof), blood proteins (such as blood clotting factors, enzymes, albumin, plasminogen, immunoglobulins) and blood fluid components (such as plasma, fractions of blood plasma and serum). In some embodiments the blood composition is leukodepleted (e.g., depleted in leukocytes).


In some embodiments the blood composition to be treated is selected from the group consisting of whole blood, erythrocytes concentrates, platelets concentrates, plasma and fractions of blood plasma.


In some embodiments the biological composition is a milk composition. In some embodiments the milk composition to be treated is derived from milk of a transgenic animal that produces a protein of interest secreted in said milk.


In some embodiments the method is performed on an eluate in a process of purification, such as by affinity chromatography, of a biological composition, such as a milk composition.


The terms “pathogen inactivation” or “inactivating pathogens”, as used herein, refer to the suppression or inhibition of the replication (or reproduction) of said pathogens, and/or their destruction or elimination. Typically, a pathogen inactivating agent severely or at least substantially hampers the ability of the pathogen to replicate or reproduce under appropriate conditions.


Methods for determining if a particular method results in the suppression or inhibition of replication of pathogens are well known in the art. Typically such methods include the steps of determining the number of (active) pathogens prior to treatment with a pathogen inactivating agent and determining the number of (active) pathogens after treatment. The particular method for determining the number of active pathogens will depend on the nature of the pathogen and includes colony forming assays (for determining the number of active bacteria) and infective assays (for determining the number of “active” viruses). One measure of the number of active viruses is the Tissues Culture Effective Dose (TCID), which can be determined for instance by the Kärber and/or Spearman-Kärber methods. (See e.g., Karber, G. (1931). Arch. J. Exper. Path. u. pharmakol., 162, 480; Spearman (1908). Brit. J. Psychol., 2:227-242)


In some embodiments the methods of the disclosure result in pathogen elimination higher or equal to 4 Log10TCID. The pathogen elimination may be calculated according to the methods of Kärber and/or Spearman-Kärber as explained in the examples.


In some embodiments the biological composition, after the contacting step, will contain an amount of glycol sufficient to inactivate, eliminate or lower the amount of pathogen, for example, below a desired level. In some embodiments the glycol concentration in the biological composition after the contacting step is between 10% and 75% (w/w), between 15% and 70% (w/w), between 20% and 65% (w/w), between 25% and 60% (w/w), between 30% and 60% (w/w), between 35% and 55% (w/w), or between 40% and 50% (w/w) of the composition. In some embodiments the glycol concentration in the biological composition after the contacting step is between 10% and 75% (v/v), between 15% and 70% (v/v), between 20% and 65% (v/v), between 25% and 60% (v/v), between 30% and 60% (v/v), between 35% and 55% (v/v), or between 40% and 50% (v/v) of the composition.


While not required, generally, it is expected that the pathogen inactivation increases with the length of exposure of the biological composition comprising the pathogen to the glycol. In some embodiments the biological composition is contacted with the glycol for a duration that permits pathogen elimination greater than or equal to 4 Log TCID (Tissue Culture Infective Dose)


In some embodiments the biological composition is contacted with the glycol for at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 1200 minutes, at least 150 minutes, at least 180 minutes, at least 210 minutes, at least 240 minutes, at least 300 minutes, at least 360 minutes, at least 2500 minutes, at least 1000 minutes, or more. In some embodiments the biological composition is contacted with the glycol for a duration between 15 and 360 minutes, between 60 and 240 minutes, or between 90 and 180 minutes. In some embodiments the glycol is removed from the biological composition after a specific amount of pathogen inactivation has been achieved. In some embodiments the glycol remains present in the biological composition after the inactivation of the pathogen.


In some embodiments of the methods described herein are performed at a temperature between 10 and 30° C., between 12 and 28° C., or between 15 and 25° C.


In some embodiments the methods described herein are performed at a pH between 4 and 11, between 5 and 10, between 6 and 9, between 6.5 and 8.5, or between 7 and 8. In some embodiments the methods described herein are performed at a pH of around 7.5. In some embodiments the methods described herein are performed at a pH of 7.5. A person of ordinary skill in the art can rely on the literature to determine which pH range is acceptable for a particular biological composition.


In some embodiments the methods comprise a further step of viral elimination such as nanofiltration.


In some embodiments the methods are performed during an elution phase in a process of purification, such as affinity chromatography, of a biological composition. In some embodiments the glycol is added to an affinity elution buffer. In some embodiments an affinity elution buffer comprises 50 mM tris, 45% (w/w/) propylene glycol and 1.5M NaCl and has a pH of 7.5. In some embodiments an affinity elution buffer comprises 50 mM tris, 45% (v/v/) propylene glycol and 1.5M NaCl and has a pH of 7.5.


In some embodiments the methods described herein do not comprise a step of contacting the biological composition with cruciferous oil or with arginine in a significant amount.


A significant amount of arginine, as used herein, corresponds to an arginine concentration of at least 0.2M, at least 0.01M, or at least 0.001M, after the contacting step.


A significant amount of cruciferous oil, as used herein, corresponds to a concentration of at least 0.1% of said cruciferous oil, at least 0.01%, or at least 0:001%, after the contacting step.


In one aspect the disclosure relates to a biological composition comprising glycol for inactivating pathogens, wherein said biological composition is obtained by contacting a biological composition with a glycol, such as by any of the methods described herein.


In some embodiments said glycol is propylene glycol or ethylene glycol.


In some embodiments the glycol concentration in the biological composition is between 10% and 75% (w/w), between 15% and 70% (w/w), between 20% and 65% (w/w), between 25% and 60% (w/w), between 30% and 60% (w/w), between 35% and 55% (w/w), or between 40% and 50% (w/w) of the composition. In some embodiments the glycol concentration in the biological composition is between 10% and 75% (v/v), between 15% and 70% (v/v), between 20% and 65% (v/v), between 25% and 60% (v/v), between 30% and 60% (v/v), between 35% and 55% (v/v), or between 40% and 50% (v/v) of the composition.


In some embodiments the biological composition also comprises a detergent such as TWEEN 20 or TWEEN 80. In some embodiments the biological composition also comprises a solvent such as TNBP (Tri-N-butyl Phosphate). In some embodiments the biological composition comprised a detergent prior to contacting with glycol. In some embodiments the biological composition is contacted with a detergent prior to contacting with glycol. In some embodiments the biological composition is contacted with a detergent simultaneously with contacting with glycol. In some embodiments the biological composition is contacted with a detergent after contacting with glycol.


In some embodiments the biological composition does not comprise arginine in a significant amount.


In some embodiments the biological composition does not comprise cruciferous oil in a significant amount.


In some embodiments the biological composition does not comprise either arginine or cruciferous oil in a significant amount.


EXAMPLES

The following examples describe some embodiments of making and practicing the methods and compositions of the disclosure. However, it should be understood that the examples are for illustrative purposes only and are not meant to limit the scope of the disclosure. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.


Material and Methods


The inactivation of two enveloped viruses (TGEV and BVDV) by propylene glycol (PG) present at 45% (v/v) in an affinity chromatography eluate was evaluated. The eluate was generated during the production of a transgenic protein of interest (in a milk composition derived from a transgenic animal producing said protein, which is secreted in its milk).


Cytotoxicity, Viral Interference and Quenching.


The cytotoxicity, viral interference and quenching parameters of the starting material (the eluate) were determined prior to the incubation assay in presence of PG. The assays to determine the cytotoxicity, viral interference and quenching were done on the eluate sample of an affinity chromatography.


Cytotoxicity.


The cytotoxicity parameters of the starting material were evaluated using the conditions in Table I:









TABLE I







samples for evaluation of the cytotoxicity and tested dilutions.











Dilution
Cells and
Observation post-


Sample to inoculate
range
associated virus
inoculation





Starting matrix
Undiluted to
ST (swine testis)
Day +3/6


(eluate)
1/243
TGEV



(range of 3)
MDBK (Madin-
Day +3/6




DarbyBovine




Kidney)




BVDV









The non cytotoxic concentration of the sample matrix is defined as the first dilution of the sample matrix that does not involve any destruction of the cell coat of cells incubated into the matrix.


The cytotoxicity parameters obtained at Day +3 in this assay were used to determine the viral interference conditions and were confirmed at Day +6.


Viral Interference Control and Sample Quenching.


The viral interference and the sample quenching parameters were determined simultaneously.


The viral interference parameters of a sample for the titration system were evaluated. The assay consists of a dilution titration of the viruses BVDV and TGEC in a sample matrix (first dilution point: non-cytotoxic matrix, as determined above) compared with a titration in a culture medium. Before determining the appropriate dilutions of the viruses, a 30 minutes incubation period at 4° C. was performed in order to mimic the assay environment, as the actual assay has latency times of 15-30 minutes prior to the titration of fractions at T0, T5 and T15.


The potential interference of the matrix with both titration systems (ST and MDBK cells) was evaluated according to the operating conditions shown in Table II.









TABLE II







operating conditions of interference.















Post-inoculation


Cells
Virus
Diluent
Dilution range of the diluent
observation





MDBK
BVDV
Starting matrix
1st timepoint: non-cytotoxic
D +6




(eluate) at non
matrix + 3 other dilution points




toxic
(growing) at a range of 3.




concentration




Culture medium
undiluted
D +6


ST
TGEV
Starting matrix
1st timepoint: non-cytotoxic
D +6




(eluate) at non
matrix + 3 other dilution points




toxic
(growing) at a range of 3.




concentration




Culture medium
undiluted
D +6









The viral interference/quenching by the matrix was determined to be significant if a difference >1.0 log10 TCID50/mL was observed between the titration in sample matrix (the eluate) and the titration in culture medium.


Process


Operating Conditions.


The kinetics of the inactivation of the enveloped viruses (TGEV and BVDV) by the propylene glycol (PG) was evaluated by contacting the viruses for six hours at 20° C. (±5° C.) with the eluate of the affinity chromatography containing 45% (v/v) PG as obtained during the purification process of the transgenic protein. The virus was added to the sample at a concentration of 5% (v/v). For each virus, the assay was performed in duplicate.


Material.


The starting material was an affinity chromatography eluate. The starting material was spiked with virus at 5% (v/v).


The conditions of the viral suspensions of TGEV and BVDV used in this assay are described in Table III (TEGV) and Table IV (BVDV).









TABLE III





TGEV spikes.


















Virus used
TEGV (clarified supernatant)



Medium
Cell Culture medium ST



Aliquots
5 × 5 mL, 4 × 1 mL, 81 × 80 μL



Titer
8.53log10 TCID50/mL ± 0.5log10



Storage
<−65° C.

















TABLE IV





BVDV spikes.


















Virus used
BVDV (clarified supernatant)



Medium
Cell Culture medium MDBK



Aliquots
10 × 3 mL, 1 × 11 mL, 81 × 0.2 mL



Titer
6.08log10 TCID50/mL ± 0.5log10



Storage
<−65° C.










Respective cell Culture media (for ST and MDBK cells) were used during the neutralization step. This neutralization was performed at concentrations determined in the assays on the cytotoxicity, the viral interference and the matrix quenching.


Assay.


A beaker was placed in a heating device at 20° C.±5° C. The beaker was placed on a magnetic stirrer and maintained at 20° C.±5° C. before treatment.


For each assay, an aliquot of starting material (20 mL) was thawed in a water bath at 20° C.±5° C. After thawing, the temperature was checked.


Each aliquot (≥1 mL) of viral suspension was thawed at ambient temperature. An aliquot of about 0.1 mL was stored at a temperature lower than −65° C. The viral suspension was used to create a sample of the starting material containing 5% of virus.


The treatment consist of spiking 1 mL (5%) of viral suspension in 19 mL of matrix containing 45% of PG (obtained from the eluate of the affinity chromatography).


After a rapid homogenization and a check of the temperature of the mixture, a sample aliquot (1 mL) was taken and quenched with culture medium (ST cell culture medium or MDBK cell culture medium, depending on the cell line used). The added volume of cell culture medium depended on the data obtained in the cytotoxicity, interference and viral quenching study. This sample constituted the “T0”.


The virus-spiked material (containing PG at 45% (v/v) was incubated for six hours at 20° C.±5° C. in the same manner as for the “T0” sample, sample aliquots of 1 mL were taken (and immediately diluted) after incubation periods of: T=5 min, T=15 min, T=60 min, T=180 min, T=360 min.


The samples collected during the different incubation assays of the eluate matrix “FVII Select” (that contain PG at 45%) are summarized in the Table V.









TABLE V







Designation of the collected samples during assays.










TGEV treatment
BVDV Treatment











Fractions
Assay A
Assay B
Assay A
Assay B





Spike
Spike A TEGV
Spike B TEGV
Spike A BVDV
Spike B BVDV


Incubation T = 0 min
T0A TGEV
T0B TGEV
T0A BVDV
T0B BVDV


Incub. T = 5 min
T5A TGEV
T5B TGEV
T5A BVDV
T5B BVDV


Incub. T = 15 min
T15A TGEV
T15B TGEV
T15A BVDV
T15B BVDV


Incub. T = 60 min
T60A TGEV
T60B TGEV
T60A BVDV
T60B BVDV


Incub. T = 180 min
T180A TGEV
T180B TGEV
T180A BVDV
T180B BVDV


Incub. T = 360 min
T360A TGEV
T360B TGEV
T360A BVDV
T360B BVDV









The samples, after titration, were stored at a temperature below −65° C. In addition, controls were generated with low, average and high viral load, by spiking of the matrix diluted in a non-cytotoxic and non-interfering concentration with the TGEV and BVDV viruses.


The incubation assays of the matrix containing 45% PG were considered successful if the following conditions were satisfied:

    • a temperature of 20° C.±5° C. and a incubation period of 6 hours,
    • taking of the sample aliquots as planned.


Titration of the Samples of Process.


The titration of the samples generated during the above-described assays was done on the same day.


Titration Protocol.


The titration of viruses of the samples shown in Table III was performed according to the study L-50 for TGEV and L-319 for BVDV.


The titration was done in three steps: seeding of the 96-wells plates, infection of said plates in standard titration or LVP (Large Volume Plating) and determination of the titer.


Seeding conditions of the 96-wells plates for the titration of each of the viruses are described in Table VI.









TABLE VI







seeding conditions of the 96-wells plates for the titration of BVDV


and TGEV.









Seeding features
BVDV
TGEV











Support
96-wells plates









Cells
MDBK
ST


Number of cells/well
1000
3000








Cell volume/well
100 μL









Culture medium
DMEM +
OptiMEM +



2% HS + P/S +
5% SVF + P/S +



NEAA
NEAA + NaPyruvate








Incubation period of plates
18 hours ± 6 hours (overnight)









For each virus:

    • samples obtained by the first run (Table V) were first titrated by the standard protocol,
    • fractions obtained by the first run for which no virus was detected with the standard protocol were analyzed in Large Volume Plating (LPV) similar to samples obtained by the second run,
    • if no virus was detected in the standard protocol, the first sample and the last sample (sample collected after 6 hours of incubation) were minimally titrated using LVP.


The titrations were performed immediately after the treatment assays; without freezing the samples.


Standard Titration.


The culture supernatant was removed and replaced by 20 μL of the sample to be titrated.


After a one-hour incubation at 37° C., 130 μL of culture medium was added to each well. Viral propagation resulted in a total or partial destruction of the cell coat.


For each dilution, 12 infection replicates were performed in order to permit a statistic analysis according to the Kärber and/or Spearman-Kärber methods, (See e.g., Chapter 5 of “Virology Labfax”, Bios Publishers (plus Academic Press (US), or Blackwell non-US, 1993; Karber, G. (1931). Arch. J. Exper. Path. u. pharmakol., 162, 480; Spearman (1908). Brit. J. Psychol., 2:227-242).


LVP Titration.


The viral titration method “Large Volume Plating” in “n” replicates allows for an increase in tested sample volume and thus an increase in the detection limit. The protocol is identical to the standard titration, except that the analysis was done using only one sample dilution, placed in all the wells of one or more 96-wells plate(s). The statistic analysis was done according to the method of Spearman-Kärber.


Controls.


In parallel with the sample titration, the following controls were performed:

    • a negative control was used for each titration series. This control consists of a titration of the culture medium (used in the titration series) with the conditions used in the sample titration.
    • A positive control was also used for each titration series. In this study, BVDB and TGEV were used as a positive control. The titer of these positive controls was 6.08 log10 TCID50/mL and 6.41 log10 TCID50/mL±0.5 log10 TCID50/mL.


Validity of a Titration Assay.


A titration assay was considered valid if:

    • No destruction of the cell coat was observed with the negative control.
    • The sample titration shows a rate of positive wells between 0 and 100% for at least three successive dilutions.
    • For at least the last dilution of the sample, a positive well rate equal to 0% is recognized.


Calculation of Titers, Charges and Reduction Factor.


After an incubation period of six days (for each of the viruses), for each well of each of the dilutions, the number of cells that had a total or partial destruction of the cell coat were quantified (with a microscope at size ×40 and/or ×100). The virus titer in each well was determined according to the Kärber formula, expressed in TCID50/mL (in log10).


The titer of viral suspension was calculated according to the Karber method. The titration of a virus was given with an uncertainty of ±0.5 log10 TCID50/mL and was calculated with the formula:







IC

(

α
=

5

%


)


=

1.96
×



Σ


(


p
i



xq
i


)



(

n
-
1

)


2







wherein: pi is the rate of positive wells at dilution i.

    • qi is the rate of negative wells at dilution i.


However, if the virus was only observed at the first tested dilution of the sample, and its infection rate was lower than 100%, the logarithmic concentration of virus in TCID50/mL was calculated according to the formula of the method of Spearman Kärber:








Log
10






C

=

log






10


[



log
e



(

n

n
-
r


)



v
.


log
e



(
2
)




]








wherein

    • C is the virus concentration in TCID50/mL,
    • v is the inoculum volume per well
    • n is the number of inoculated wells for each dilution
    • r is the number or infected wells.


With the titers and viral loads expressed here in decimal value, the total viral load in a sample was calculated with the titer and the sample volume according to this formula:

Total viral load=titer×sample volume(mL).


The reduction factor (RF) was calculated compared to the viral load in the <<T0>> sample.

RF=(total viral load in “T0”)/(total viral load in sample taken at a later time).

Results


TGEV Study on the Affinity Chromatography Eluate (in Presence of 45% PG).


The results are described in Table VIII and illustrated in FIG. 1.
















TABLE VIII





Timepoint
Volume
Titre
Correction

Reduction
Clearance



Sample
(ml)
(log10TCID50/ml)
(cytotoxicity)
Log10TICD50
Factor (log)
Factor (log)
Titration















TGEV - Run 1














Spike
1
7.45
N/A
7.45
NA
NA
Standard


T = 0
20
5.12
9
7.38
NA
0.07
Standard


Load Sample


T = 5
20
3.95
9
6.21
1.17
1.24
Standard


T = 15
20
2.78
9
5.04
2.34
2.41
Standard


T = 60
20
1.32
9
3.58
3.8
3.87
Standard


T = 180
20
0.8
9
3.06
4.32
4.39
Standard


T = 360
20
<0.8
9
<3.06
>4.32
>4.39
Standard







TGEV - Run 2














Spike
1
7.62
N/A
7.62
NA
NA
Standard


T = 0
20
5.28
9
7.54
NA
0.08
Standard


Load Sample


T = 5
20
4.28
9
6.54
1
1.08
Standard


T = 15
20
3.2
9
5.46
2.08
2.16
Standard


T = 60
20
0.8
9
3.06
4.48
4.56
Standard


T = 180
20
−0.12
9
2.14
5.4
5.48
1LVP (96 wells)


T = 360
20
<−0.12
9
<2.14
>5.40
>5.48
1 LVP (96 wells)









BVDV Study on the Affinity Chromatography Eluate (in Presence of 45% PG).


The results are described in Table IX and illustrated in FIG. 2.
















TABLE IX





Timepoint
Volume
Titre
Correction

Reduction
Clearance



Sample
(ml)
(log10TCID50/ml)
(cytotoxicity)
Log10TICD50
Factor (log)
Factor (log)
Titration















BVDV - Run 1














Spike
1
6.28
NA
6.28
NA
NA
Standard


T = 0
20
3.12
27
5.85
NA
0.43
Standard


Load Sample


T = 5
20
1.9
27
4.63
1.22
1.65
Standard


T = 15
20
2.25
27
4.98
0.87
1.3
Standard


T = 60
20
2.25
27
4.98
0.87
1.3
Standard


T = 180
20
2.25
27
4.98
0.87
1.3
Standard


T = 360
20
0.8
27
3.53
>4.32
2.75
Standard







BVDV - Run 2














Spike
1
6.2
NA
6.20
NA
NA
Standard


T = 0
20
3.03
27
5.76
NA
0.44
Standard


Load Sample


T = 5
20
1.59
27
4.32
1.44
1.88
Standard


T = 15
20
1.32
27
4.05
1.71
2.15
Standard


T = 60
20
1.59
27
4.32
1.44
1.88
Standard


T = 180
20
<0.8
27
<3.53
>2.23
>2.67
Standard


T = 360
20
<−0.12
27
<2.61
>3.15
>3.59
1 LVP









X-MuLV and PRV Studies on the Affinity Chromatography Eluate (in Presence of 45% PG).


A similar study was done on a affinity chromatography eluted with 45% PG using the X-MuIV and PRV viruses and including the determination of standard deviations.


Results are described in Tables X and XI.









TABLE X







X-MuLV study.












Titer
Volume
Volume
Viral Load


Sample
(TCID50/ml)
(ml)
correction
(log10)





Spiking Positive control
8.03 ± 0.36





Theoretical load (5% spike)
6.71 ± 0.36
20

8.01 ± 0.36


Control T = 0 h (w/o SD)
4.80 ± 0.42
20
10
7.10 ± 0.42


Control T = 6 h (w/o SD)
0.59 ± 0.91
20
10
2.89 ± 0.91


T = 0 h (+SD)
≤0.78*
20
100
≤4.08


T = 1 h (+SD)
≤0.78*
20
100
≤4.08


T = 3 h (+SD)
≤0.78*
20
100
≤4.08


T = 6 h (+SD) (Standard titration)
≤0.78*
20
100
≤4.08


T = 6 h (+SD) (LVP)
≤−1.13*
20
100
≤2.17


T = 6 h (+SD) (LVP + ST)
≤−1.13*
20
100
≤2.17
















TABLE XI







PRV study.












Titer
Volume
Volume
Viral Load


Sample
(TCID50/ml)
(ml)
correction
(log10)





Spiking Positive control
8.64 ± 0.32





Theoretical load (5% spike)
7.32 ± 0.32
20

8.62 ± 0.32


Control T = 0 h (w/o SD)
2.17 ± 0.30
20
10
4.47 ± 0.30


Control T = 6 h (w/o SD)
≤0.78*
20
10
≤3.08


T = 0 h (+SD)
≤1.48*
20
10
≤3.78


T = 1 h (+SD)
≤1.48*
20
10
≤3.78


T = 3 h (+SD)
≤1.48*
20
10
≤3.78


T = 6 h (+SD) (Standard titration)
≤1.48*
20
10
≤3.78


T = 6 h (+SD) (LVP)
**
20
10
NA









The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

Claims
  • 1. A method for inactivating and/or eliminating an enveloped virus for the production of a recombinant protein, said method comprising conducting affinity chromatography on a biological composition containing a recombinant protein, wherein the recombinant protein is eluted during the affinity chromatography with an elution buffer comprising propylene glycol, wherein the biological composition is a blood composition, a milk composition, urine, sweat, sputum, feces, spinal fluid, or cellular or tissue extracts, wherein the concentration of propylene glycol in the elution buffer is between 40 and 50% (v/v), and wherein the propylene glycol is not combined with arginine.
  • 2. The method of claim 1, wherein said method is performed at a temperature between 15 and 25° C.
  • 3. The method of claim 1, wherein said method is performed at a pH between 7.0 and 8.0.
  • 4. The method of claim 1, wherein the affinity chromatography column is washed with a wash buffer prior to elution of the recombinant protein, wherein the concentration of propylene glycol in the wash buffer is between 30 and 40% (v/v).
  • 5. The method of claim 1, wherein recombinant protein is maintained in the elution buffer with a concentration of propylene glycol between 40 and 50% (v/v) for less than 6 hours.
  • 6. A method for inactivating and/or eliminating a virus during a purification of a protein from a biological composition, said method comprising contacting said biological composition during the purification of the protein with a glycol to inactivate the virus, wherein the concentration of glycol after the contacting step is between 40 and 50% (v/v) of the biological composition, and wherein the glycol is not combined with arginine.
  • 7. The method of claim 6, wherein said glycol is propylene glycol.
  • 8. The method of claim 6, wherein said biological composition is a blood composition or a milk composition.
  • 9. The method of claim 6, wherein the virus is an enveloped virus.
  • 10. The method of claim 6, wherein said method results in a viral elimination equal or greater than 4 Log10TCID, wherein the TCID is according to the methods of Kärber and/or Spearman-Kärber.
  • 11. The method of claim 6, wherein said method is performed at a temperature between 15 and 25° C.
  • 12. The method of claim 6, wherein said method is performed at a pH between 7.0 and 8.0.
  • 13. A method for inactivating a virus during the purification of a protein from a biological composition, said method comprising contacting an eluate with a glycol during the process of purification of the protein from the biological composition to inactivate and/or eliminate the virus, wherein the concentration of glycol after the contacting step is between 40 and 50% (v/v) of the elute, and wherein the glycol is not combined with arginine.
  • 14. The method of claim 13, wherein said glycol is propylene glycol.
  • 15. The method of claim 13, wherein said biological composition is a blood composition or a milk composition.
  • 16. The method of claim 13, wherein the virus is an enveloped virus.
  • 17. The method of claim 16, wherein said enveloped virus is selected from the group consisting of X-MuLV, PRV, BVDV and TGEV virus.
  • 18. The method of claim 13, wherein said method results in a viral elimination equal or greater than 4 Log10TCID, wherein the TCID is according to the methods of Kärber and/or Spearman-Kärber.
  • 19. The method of claim 13, wherein said method is performed at a temperature between 15 and 25° C.
  • 20. The method of claim 13, wherein said method is performed at a pH between 7.0 and 8.0.
RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 of international application PCT/IB2011/003271, filed Dec. 23, 2011, which claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application Ser. No. 61/428,416, filed Dec. 30, 2010, the disclosure of which is incorporated by reference herein in its entirety.

PCT Information
Filing Document Filing Date Country Kind 371c Date
PCT/IB2011/003271 12/23/2011 WO 7/23/2013
Publishing Document Publishing Date Country Kind
WO2012/090067 7/5/2012 WO A
US Referenced Citations (157)
Number Name Date Kind
3910999 Moyer Oct 1975 A
4020183 Asculai et al. Apr 1977 A
4299828 Wang et al. Nov 1981 A
4420398 Castino Dec 1983 A
4612169 Iwasaki et al. Sep 1986 A
4764369 Neurath et al. Aug 1988 A
5145663 Simmons Sep 1992 A
5356651 Degen et al. Oct 1994 A
5576040 Moller et al. Nov 1996 A
5648253 Wei Jul 1997 A
5756687 Denman et al. May 1998 A
5827690 Meade et al. Oct 1998 A
5843705 DiTullio et al. Dec 1998 A
5849992 Meade et al. Dec 1998 A
6210736 Echelard et al. Apr 2001 B1
6268487 Kutzko et al. Jul 2001 B1
6441145 DiTullio et al. Aug 2002 B1
6448469 Smith Sep 2002 B1
6472584 Smith Oct 2002 B1
6528699 Meade et al. Mar 2003 B1
6545198 Echelard et al. Apr 2003 B1
6548653 Young et al. Apr 2003 B1
6580017 Echelard et al. Jun 2003 B1
6593463 Chen et al. Jul 2003 B1
6727405 Gordon et al. Apr 2004 B1
6743966 Smith Jun 2004 B2
7019193 Ditullio et al. Mar 2006 B2
7026154 Gaillac et al. Apr 2006 B1
7045676 Gordon et al. May 2006 B1
7087719 Visuri et al. Aug 2006 B2
7101971 Meade et al. Sep 2006 B2
7354594 Chen et al. Apr 2008 B2
7501553 Chen et al. Mar 2009 B2
7531632 Perreault May 2009 B2
7550263 Meade et al. Jun 2009 B2
7632980 Chen et al. Dec 2009 B1
7651686 Chen et al. Jan 2010 B2
7928064 DiTullio et al. Apr 2011 B2
7939317 Gordon et al. May 2011 B1
8173860 Meade et al. May 2012 B2
8252227 Bardat Aug 2012 B2
9029316 Bardat et al. May 2015 B2
9102762 Christensen et al. Aug 2015 B2
9358275 Bardat et al. Jun 2016 B2
9511087 Frieling et al. Dec 2016 B2
10034921 Chen et al. Jul 2018 B2
10174110 Meade et al. Jan 2019 B2
10611826 Paolantonacci et al. Apr 2020 B2
20020131957 Gavin et al. Sep 2002 A1
20020144299 Chen et al. Oct 2002 A1
20020155998 Young et al. Oct 2002 A1
20030005468 Meade et al. Jan 2003 A1
20030033618 Smith Feb 2003 A1
20030036637 Fulton Feb 2003 A1
20030046716 Echelard et al. Mar 2003 A1
20030096974 Ditullio et al. May 2003 A1
20030177513 Echelard et al. Sep 2003 A1
20030204860 Melican et al. Oct 2003 A1
20030213003 Meade et al. Nov 2003 A1
20040006776 Meade et al. Jan 2004 A1
20040025193 Echelard et al. Feb 2004 A1
20040092719 Birck-Wilson et al. May 2004 A1
20040097710 Visuri et al. May 2004 A1
20040098755 Mulroy et al. May 2004 A1
20040102380 Fulton et al. May 2004 A1
20040109847 Chen et al. Jun 2004 A1
20040117863 Edge et al. Jun 2004 A1
20040121303 Gavin et al. Jun 2004 A1
20040133931 Gavin et al. Jul 2004 A1
20040143857 Young et al. Jul 2004 A1
20040148648 Behboodi et al. Jul 2004 A1
20040167320 Couto et al. Aug 2004 A1
20040192595 Murakami et al. Sep 2004 A1
20040205832 Meade et al. Oct 2004 A1
20040226052 Meade et al. Nov 2004 A1
20040226053 Meade et al. Nov 2004 A1
20050006307 Jones et al. Jan 2005 A1
20050013811 Chen et al. Jan 2005 A1
20050029195 Gibson et al. Feb 2005 A1
20050060766 Chen Mar 2005 A1
20050071890 Chen et al. Mar 2005 A1
20050097625 Meade et al. May 2005 A1
20050158832 Young et al. Jul 2005 A1
20050160483 Meade et al. Jul 2005 A1
20050169908 Murakami et al. Aug 2005 A1
20050177882 Gavin et al. Aug 2005 A1
20050181482 Meade et al. Aug 2005 A1
20050186608 Olsen Aug 2005 A1
20050192226 Enkhbaatar et al. Sep 2005 A1
20050193431 Echelard et al. Sep 2005 A1
20050197496 Perreault Sep 2005 A1
20050208000 Bernstein et al. Sep 2005 A1
20050235371 Chen et al. Oct 2005 A1
20050245444 Echelard et al. Nov 2005 A1
20050260672 Couto et al. Nov 2005 A1
20060026695 Edge et al. Feb 2006 A1
20060105347 Meade et al. May 2006 A1
20060121004 Echelard et al. Jun 2006 A1
20060123500 Echelard et al. Jun 2006 A1
20060130159 Masiello et al. Jun 2006 A1
20060168671 Gavin et al. Jul 2006 A1
20060174359 Melican et al. Aug 2006 A1
20060178309 Visuri et al. Aug 2006 A1
20060179493 Meade et al. Aug 2006 A1
20060179500 Meade et al. Aug 2006 A1
20060182744 Strome et al. Aug 2006 A1
20060191025 Echelard et al. Aug 2006 A1
20060191029 Gavin et al. Aug 2006 A1
20060286548 Liposky et al. Dec 2006 A1
20070037192 Ziomek et al. Feb 2007 A1
20070192878 Perreault Aug 2007 A1
20080004212 Echelard et al. Jan 2008 A1
20080019905 Strome et al. Jan 2008 A9
20080063780 Meade et al. Mar 2008 A1
20080118501 Schindler et al. May 2008 A1
20080176786 Ditullio et al. Jul 2008 A1
20090068193 Chen et al. Mar 2009 A1
20090246194 Meade et al. Oct 2009 A1
20090311239 Chtourou et al. Dec 2009 A1
20100021612 Meade et al. Jan 2010 A1
20100056757 Perreault Mar 2010 A1
20110070167 Enkhbaatar et al. Mar 2011 A1
20110082083 Magneson et al. Apr 2011 A1
20110229460 Meade Sep 2011 A1
20120058047 Strome et al. Mar 2012 A9
20130149301 Meade Jun 2013 A1
20130324619 Chtourou Dec 2013 A1
20140046033 Schindler et al. Feb 2014 A1
20140194360 Frieling et al. Jul 2014 A1
20140206617 Frieling et al. Jul 2014 A1
20140228301 Meade et al. Aug 2014 A1
20140242182 Evans et al. Aug 2014 A1
20140296490 Faid et al. Oct 2014 A1
20150152162 Boulange et al. Jun 2015 A1
20150175983 Bataille et al. Jun 2015 A1
20150368334 Meade et al. Dec 2015 A1
20150368357 Meade et al. Dec 2015 A1
20150374801 Chen et al. Dec 2015 A1
20160002330 Meade Jan 2016 A1
20160039913 Meade et al. Feb 2016 A1
20160089422 Chtourou et al. Mar 2016 A1
20160129115 Magneson et al. May 2016 A1
20160158676 Hawkins et al. Jun 2016 A1
20160168229 Paolantonacci et al. Jun 2016 A1
20160326547 Meade et al. Nov 2016 A1
20170121402 Chtourou May 2017 A1
20170129966 Masiello May 2017 A1
20170190753 Abache Jul 2017 A1
20180139938 Chen May 2018 A1
20180169297 Chtourou et al. Jun 2018 A1
20180355034 Mondon et al. Dec 2018 A1
20190254276 Chtourou Aug 2019 A1
20190309057 Meade et al. Oct 2019 A1
20190309058 Meade et al. Oct 2019 A1
20200255518 Schindler et al. Aug 2020 A1
20200331994 Chtourou et al. Oct 2020 A1
20210275668 Plantier et al. Sep 2021 A1
Foreign Referenced Citations (31)
Number Date Country
962514 Feb 1975 CA
101065118 Oct 2007 CN
1829736 Sep 2009 CN
0638242 Jan 1995 EP
0 819 968 Jan 1998 EP
0804074 Jan 2005 EP
2350271 Jan 2016 EP
S46-6912 Mar 1971 JP
S55-164627 Dec 1980 JP
S59-175879 Oct 1984 JP
H04-503067 Jun 1992 JP
H07-126109 May 1995 JP
H09-206362 Aug 1997 JP
H10-507367 Jul 1998 JP
2008-531700 Aug 2008 JP
2012-509081 Apr 2012 JP
WO 9008559 Aug 1990 WO
WO 9429334 Dec 1994 WO
WO 1995002393 Jan 1995 WO
WO 9502393 Jan 1995 WO
WO 9614737 May 1996 WO
WO 9964462 Dec 1999 WO
WO 1999064441 Dec 1999 WO
WO 2002058747 Aug 2002 WO
WO 2004011023 Feb 2004 WO
WO 2004092360 Oct 2004 WO
WO 2006093924 Sep 2006 WO
WO 2009110940 Sep 2009 WO
WO 2010009388 Jan 2010 WO
WO 2010059232 May 2010 WO
WO 2013066251 May 2013 WO
Non-Patent Literature Citations (27)
Entry
Dunham, W. B., and W. J. MacNeal. “Inactivation of Influenza Virus by Mild Antiseptics.” Journal of Immunology 49.2 (1944): 123-8.
Scientific Report of EFSA, Available data on notified biocides efficacy under field conditions (compared to sodium hydroxide and sodium carbonate), European Food Safety Authority, European Food Safety Authority (EFSA), EFSA Journal 2009; 7 (10):259 (Year: 2009).
Jack A. Ragheb, The Amphotropic and Ecotropic Murine Leukemia Virus Envelope . . . , Journal of Virology, Nov. 1995, p. 7205-7215 (Year: 1995).
S. Davison, C. E. Benson, A. F. Ziegler, and R. J. Eckroade, Evaluation of Disinfectants with the Addition of Antifreezing Compounds Against Nonpathogenic H7N2 Avian Influenza Virus, Avian Diseases 43:533-537, 1999 (Year: 1999).
The pressure washing forum (dated 2009, downloaded Aug. 22, 2019) (Year: 2009).
O. H. Robertson, M.D.,The Bactericidal Action of Propylene Glycol Vapor on Microorganisms Suspended in Air. I, J Exp Med. Jun. 1, 1942; 75(6): 593-610 (Year: 1942).
I. Olitzky, Antimicrobial Properties of a Propylene Glycol Based Topical Therapeutic Agent, Journal of Pharmaceutical Sciences, vol. 54, No. 5, May 1965, 787-788 (Year: 1965).
Wikipedia page for Protein purification (Year: 2021).
PCT/IB2011/003271, dated May 10, 2012, International Search Report and Written Opinion.
PCT/IB2011/003271, dated Jul. 11, 2013, International Preliminary Report on Patentability.
Burnouf et al., Nanofiltration of plasma-derived biopharmaceutical products. Haemophilia. Jan. 2003;9(1):24-37.
Hill et al., Guidelines on the selection and use of therapeutic products to treat haemophilia and other hereditary bleeding disorders. Haemophilia. Jan. 2003;9(1):1-23.
Mollerup et al., The Use of RP-HPLC for measuring activation and cleavage of rFVIIa during purification. Biotechnol Bioeng. Dec. 5, 1995;48(5):501-5.
Pedersen et al., Auto activation of human recombinant coagulation factor VII. Biochemistry. Nov. 28, 1989;28(24):9331-6.
Tomokiyo et al., Large-scale production and properties of human plasma-derived activated Factor VII concentrate. Vox Sang. Jan. 2003;84(1):54-64.
Aranha-Creado et al., Cumulative Viral Titer Reduction Demonstrated by Sequential Challenge of a Tangential Flow Membrane Filtration System and a Direct Flow Pleated Filter Cartridge. PDA J Pharm Sci Technol. Sep. 1997;51(5):208-212.
Bryant et al., Pathogen Inactivation: The Definitive Safeguard for the Blood Supply. Arch Pathol Lab Med. May 2007;131(5):719-33.
Chang, Transfusion therapy in critically ill children. Pediatr Neonatol. Apr. 2008;49(2):5-12. doi:10.1016/S1875-9572(08)60004-2.
Cohn et al., Preparation and Properties of Serum and Plasma Proteins. IV. A System for the Separation into Fractions of the Protein and Lipoprotein Components of Biological Tissues and Fluids 1a,b,c,d J Am Chem Soc. Mar. 1946;68:459-75.
Oncley et al., The Separation of the Antibodies, Isoagglutinins, Prothrombin, Plasminogen and β1-Lipoprotein into Subfractions of Human Plasma. J Am Chem Soc. Feb. 1949;71(2):541-50.
[No Author Listed] The pressure washing forum. Apr. 9, 2009. Retrieved from the internet on Aug. 22, 2019. 1 page.
Davison et al., Evaluation of disinfectants with the addition of antifreezing compounds against nonpathogenic H7N2 avian influenza virus. Avian Dis. Jul.-Sep. 1999;43(3):533-7.
Lorenz et al., Use-Dilution Test and Newcastle Disease Virus. Appl Microbiol. Jan. 1964;12:24-6.
Robertson et al., The bactericidal action of propylene glycol vapor on microorganisms suspended in air. I. J Exp Med. Jun. 1, 1942;75(6):593-610.
Extended European Search Report for Application No. EP 20173998.4 dated May 14, 2021.
Anthony et al., A strategy to estimate unknown viral diversity in mammals. mBio. Sep. 3, 2013;4(5):e00598-13. doi: 10.1128/mBio.00598-13.
EP 20173998.4, dated May 14, 2021, Extended European Search Report.
Related Publications (1)
Number Date Country
20130324619 A1 Dec 2013 US
Provisional Applications (1)
Number Date Country
61428416 Dec 2010 US