A NEW PROCESS OF PURIFICATION OF PROTEINS FROM HEN EGG WHITE AND THE USE OF THE SAME AS ANTIVIRAL AGENT AGAINST SARS-COV-2

Abstract

The present invention describes a novel process for the production of lysozyme HCl from hen egg white (HEW) with high chemical purity, and the use of said antiviral agent against SARS-CoV-2, optionally combined with other antivirals and/or immunosuppressants and ovotransferrin.

Claims

1. Process for the purification of lysozyme HCl from hen egg white which comprises: a) isolating crude lysozyme base from undiluted hen egg white using a weakly acidic cationic resin under stirring, followed by eluting with an aqueous saline solution; b) preparing a crude solution of lysozyme HCl by treating the aqueous saline solution eluted in step a) with an aqueous inorganic base until a final pH value ranging between 10 and 11 is reached, at a temperature ranging between 0 and 8° for 4-24 hours, to obtain the lysozyme base which is first recovered by filtration, then dissolved in an aqueous solution of hydrochloric acid until a final pH interval of 2.5-3.5 is reached; c) removing inorganic salts; d) viral inactivating/antiviral activating; e) isolating amorphous lysozyme HCl with a spray-drying technique; f) heat treating the lysozyme HCl obtained in step e).

2. Process according to claim 1, wherein in step a), a weakly acidic polyacrylic macroporous cationic resin with a particle size range of 300-1600 μm, preconditioned to pH 7.0-9.0, is used.

3. Process according to claim 2, wherein in step a), the pH is adjusted with a 15% w/w aqueous solution of sodium carbonate.

4. Process according to claim 1, wherein in step a), the relative ratio between undiluted hen egg white and resin ranges between 8-12 l/l.

5. Process according to claim 1, wherein in step a), the resin is maintained under stirring at a stirring speed of up to 60 rpm and a temperature ranging between 25° C. and 40° C.

6. Process according to claim 1, wherein in step a), the weakly acidic cationic resin is Purolite® C106EP or equivalent, having a total capacity ≥2.7 eq/l.

7. Process according to claim 1, wherein in step a), the resin is eluted with a 2 to 7% NaCl solution at a temperature ranging between 25 and 40° C.

8. (canceled)

9. Process according to claim 1, wherein the aqueous inorganic base in step b) is a 4-8% w/v aqueous solution of sodium hydroxide.

10. Process according to claim 1, wherein in step b), the crude lysozyme base is dissolved in demineralised water at a relative ratio ranging between 1/30 and 1/60v/v relative to the initial amount of hen egg white.

11. Process according to claim 1, wherein in step c), the crude lysozyme hydrochloride solution is corrected to a final pH interval of 3.0-4.0, using a 2-8% aqueous solution of hydrochloric acid, and ultrafiltered and/or diafiltered to remove the inorganic salts.

12. Process according to claim 1, wherein step c) is conducted with ultrafiltration and diafiltration membranes with a cut-off of 10 kdaltons.

13. Process according to claim 1, wherein in step d), the ultrafiltered/diafiltered aqueous solution is heated to a temperature ranging between 40° C. and 100° C. for a period of up to 7 days.

14. Process according to claim 1, wherein in step f), the heat treatment of lysozyme hydrochloride is performed on the powder obtained in step e), at a temperature ranging between 40° C. and 100° C. for a period of up to 7 days.

15. Process according to claim 13, wherein the heat-treated lysozyme exhibits unchanged chromatographic purity and enzyme activity at the end of the treatment (±1.0%).

16. Process according to claim 1, wherein in step e), the solution obtained in step c) or d) is heated at 40° C. and treated with a spray-dryer.

17. Process according to claim 14, wherein, in step e), the temperature of the desolvation chamber ranges from 160 to 220° C. to provide pure amorphous lysozyme hydrochloride.

18. A method of protecting patients from SARS-CoV-2 infection, said method comprising treating said patients with the lysozyme hydrochloride obtained by the process according to claim 1.

19. The method according to claim 18, wherein said lysozyme hydrochloride is administered in combination with antivirals and/or immunosuppressants selected from chloroquine, favilavir, remdesivir, avigan, tocilizumab, cyclosporin A, sirolimus, everolimus, temsirolimus, mycophenolate mofetil and pimecrolimus.

20. The method according to claim 19, wherein said lysozyme hydrochloride is administered in combination with ovotransferrin.

21. A method of preventing or treating SARS-CoV-2 in patients in need thereof, said method comprising administering to said patients a pharmaceutical composition for oral, topical, inhalation, injectable, intravenous, gastrointestinal, intraperitoneal, intrapleural, intrabronchial, nasal or rectal administration, said pharmaceutical composition, comprising an effective antiviral amount of lysozyme hydrochloride, optionally combined with ovotransferrin with suitable carriers and/or excipients.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0042] The present invention will now be described in detail, by means of a sample embodiment reported below, on the proviso that variations in the subject-matter, conditions and parameters within the limits defined in the independent claims are comprised in the present invention.

[0043] The lysozyme HCl produced according to the invention is isolated from HEW by a purification process involving the following sequence of purification procedures: isolation of crude lysozyme base from other ovoproteins present in HEW, preparation of crude lysozyme HCl solution, removal of inorganic salts, viral inactivation and isolation of solid lysozyme HCl by spray-drying technique, followed by heat treatment.

[0044] The lysozyme HCl produced according to said purification process has exhibited antiviral activity against SARS-CoV-2 infection. In particular, lysozyme HCl exhibited a preventive cytoprotective action against SARS-CoV-2 infection on uninfected cells, and antiviral activity on already infected cells.

[0045] The crude HEW lysozyme was purified by the following purification process: undiluted HEW was loaded onto a polyacrylic cationic resin with a particle size ranging between 300 and 1600 μm and a capacity of >2.7 eq/l, preconditioned in a pH interval of 9.0-7.0. The relative ratio between HEW and resin ranges between 8 and 12 l/l, and the resin is washed twice with a 1.0-1.5 bed volume of distilled water. The resin was then washed with a 1.5-2.1 bed volume of an aqueous solution of NaCl at a concentration ranging between 2 and 7% and a temperature ranging between 25 and 40° C. When said steps are performed, the resin can optionally be maintained under stirring at a stirring speed of up to 60 rpm.

[0046] The fractions eluted with aqueous solution of NaCl were basified at a final pH value ranging between 10 and 11 with an aqueous solution of 4-8% w/v sodium hydroxide, and the resulting mixture was maintained under stirring at 0-8° C. for 4-24 hours. The resulting precipitate was recovered by suction. The wet solid was dispersed under stirring in demineralised water (relative ratio between demineralised water and initial HEW 1/60- 1/100 l/l), and the resulting mixture was maintained under stirring at a temperature ranging between 20 and 50° C. for 30 minutes to 2 hours and corrected to a final pH interval of 2.5-3.5 with a 4-8% w/v aqueous solution of hydrochloric acid. The resulting solution was then heated under stirring at a temperature ranging between 20 and 60° C. for 30 minutes to 2 hours, then cooled, and the final pH value corrected to an interval ranging between 8.0 and 10 by adding a 1-4% w/v aqueous solution of sodium hydroxide. Said solution was treated under stirring for 1-4 hours with activated charcoal (powder) and then filtered (the filter can optionally be washed with demineralised water). The filtrate (and optionally the washing water) was then corrected to a final pH interval of 3.0-4.0 with a 2-8% aqueous solution of hydrochloric acid, and ultrafiltered and/or diafiltered to remove the inorganic salts. The resulting aqueous solution was then optionally heated to a temperature ranging between 40° C. and 100° C. for a period of up to 7 days, then cooled to 40° C., or heated directly to 40° C., and treated with a spray-dryer (desolvation chamber temperature 160-220° C.) to provide pure amorphous lysozyme hydrochloride as an ivory powder (>99% recovery). The above-mentioned step involving heat treatment of lysozyme hydrochloride in solution at a temperature ranging between 40° C. and 100° C. for a period of up to 7 days (preferably at 74° C. for 1 hour) can also alternatively/simultaneously be conducted on powdered lysozyme hydrochloride obtained after spray-drying treatment.

[0047] Said controlled heat treatments give the lysozyme prepared by the manufacturing process described herein an antiviral/virucidal activity against SARS-CoV-2 without denaturing the lysozyme which, after said treatments, exhibits unchanged HPLC purity and enzyme activity. In particular, said heat treatments that do not denature the lysozyme are preferably: [0048] 1. on solid lysozyme hydrochloride: a treatment at 99° C. for 40 minutes or at 74° C. for 1 hour [0049] 2. on lysozyme hydrochloride in aqueous solution: a heat treatment at 90° C. for between 2 and 6 minutes.

[0050] The lysozyme HCl obtained by said process is free of avian viruses such as Avian avulavirus 1 and influenza viruses H5N1, H7N1 and H7N9, and exhibits an enzyme assay value on dry matter (enzymatic turbidimetric method)>97.0%, preferably >98.0%, and HPLC purity>99.0%, preferably>99.5%.

[0051] Said lysozyme HCl exhibited high antiviral activity against SARS-CoV-2 infection in the in vitro test at a concentration ranging between 0.75 and 1.5 mg/ml.

[0052] The activity of lysozyme HCl has been confirmed to be cytoprotective, at practically the same concentrations, on uninfected cells exposed to viral infection and on already infected cells. In fact, at a lysozyme HCl concentration ranging between 0.75 and 1.5 mg/ml, the percentage viral replication was very low, and close to zero.

[0053] Moreover, the in vitro antiviral activity of lysozyme HCl combined with ovotransferrin at three different concentrations (1.25, 2.5 and 5 mg/l), discovered by us, proved to be synergic. Under said conditions, the concentration of 1.25 mg/ml of ovotransferrin exhibited the highest synergic effect. The synergic antiviral effect obtained is dose-dependent, and completely eliminated viral replication at the lysozyme concentration of 0.75 mg/ml (at this concentration of lysozyme HCl, and in the absence of ovotransferrin, viral replication was inhibited by about 62%). Ovotransferrin alone was tested at different concentrations, ranging from 10 mg/ml to 1.25 mg/ml, but no antiviral activity was detected in said interval.

[0054] The antiviral activity of lysozyme HCl against SARS-CoV-2 is closely correlated with heat treatment of lysozyme HCl (both in solution and in powder form), and exhibited both antiviral and virucidal activity.

Materials and Methods

[0055] The ovotransferrin (product code 501P2001090) used to conduct the in vitro tests is produced by Bioseutica BV (Landbouwweg 83 3899 Zeewolde BD (Netherlands)).

HPLC Method for Determination of Chromatographic Purity of Lysozyme HCl

[0056] HPLC column: TSKgei reverse-phase HPLC column (polymer base, Phenyl-5PW RP), ID 4.6 mm×7.5 cm (10 μm); Tosoh Bioscience [0057] Detector: UV [0058] Wavelength: 281 nm [0059] Preparation of sample: weight 80 mg of lysozyme HCl, diluted to 20 ml with water (4 μg/μl) [0060] Injection volume: 25 μl [0061] Mobile phase A: water/acetonitrile=90/10 v/v with 0.2% trifluoroacetic acid [0062] Mobile phase B: water/acetonitrile=30/70 v/v with 0.2% trifluoroacetic acid [0063] Elution: according to the following composition

TABLE-US-00001 Time Mobile phase A Mobile phase B 0 100 0 35 0 100 35.10 100 0 42.00 100 0 [0064] Flow rate: 1 ml/min
Determination of assay value (enzymatic turbidimetric method using Micrococcus lysodeikticus cells): according to the FIP method (Pharmaceutical Enzyme, Ed 1997, 84, 375) and J Pharm Pharmacol. 2001; 53 (4); 549-54.

EXAMPLES

Example 1

Chromatographic Purification of HEW

[0065] Absorption stage. 30 litres of HEW were loaded at a feed rate of 3 1/hour into a dryer with a Nutsche filter containing 2.9 litres of weakly acidic macroporous cationic polyacrylic resin having a particle size ranging between 300 and 1600 μm and a capacity≥2.7 eq/l, pre-conditioned with an aqueous solution of sodium carbonate (15% w/w) to pH 8.0, washed with water and flushed with nitrogen, the eluate being collected by gravity. 1 bed volume of demineralised water was then loaded, maintaining the resin under stirring, and the resulting suspension was maintained under stirring for 20 min, after which the water was drained by gravity; said treatment was repeated once more under the same experimental conditions.

[0066] Elution stage. The aqueous solution of 3.5% NaCl at 30-35° C. (1.8 bed volumes) is eluted by gravity.

[0067] Precipitation of crude lysozyme base. An aqueous solution of 6% NaOH (w/v) was added to the preceding 2% aqueous solution of crude lysozyme base under stirring at 20-25° C. until a final pH value of 10.5±2 was reached. The resulting solution was then cooled to 4° C. in 12-18 hours, and maintained at said temperature under stirring for 6 hours. The resulting precipitate was recovered by suction.

[0068] Preparation of amorphous powder of purified lysozyme hydrochloride. The wet solid obtained in the preceding step was dispersed under stirring at 35° C. in demineralised water (0.66 1) for 1 hour, and a 6% w/v aqueous solution of hydrochloric acid was then added until a final pH value of 2.9±0.1 was reached. The resulting solution was then heated under stirring at 40° C. for 1 hour, then cooled, and a 2% w/v aqueous solution of sodium hydroxide was added to reach a final pH value ranging between 8.5 and 9.0. Charcoal (powder) (2.5 g) was added to said solution, and the resulting mixture was stirred at room temperature for 2 hours; the resulting suspension was then filtered by suction, and the filter was washed with water (16 ml). The filtrate and washing water were collected, and a 6% w/v aqueous solution of hydrochloric acid was added at a temperature of less than 32° C. until a final pH value of 3.6±0.2 was reached. The resulting solution was then ultrafiltered (cut-off 10 kdaltons) to obtain a 30% w/v lysozyme hydrochloride content and diafiltered (cut-off 10 kdaltons) to remove the inorganic salts present. The resulting aqueous solution was then heated at 74° C. for 1 hour or alternatively at 90° C. for 2-6 minutes, then cooled to 40° C. and treated with a spray-dryer (desolvation chamber temperature 180° C.) to provide pure amorphous lysozyme hydrochloride as an ivory powder (105 g; recovery efficiency 99%).

[0069] The resulting product has an assay value (turbidimetric method) of 98.6% and an HPLC purity of 99.7%.

Evaluation of in vitro Antiviral Activity of Lysozyme HCl Against SARS-CoV-2

Determination of Non-Toxic Concentration of Lysozyme HCl

[0070] Cell toxicity was monitored by establishing the effect of lysozyme HCl against Vero cells (epithelial cells of monkey kidneys). The cells were seeded in 96-well plates at the concentration of 1×10.sup.4 cells/well. 24 hours after seeding, the cells were treated with serial dilutions of lysozyme HCl or chloroquine (as control), in a final volume of 200 in triplicate. After 72 hours' incubation at 37° C. with 5% CO.sub.2, cell viability was measured by MTT assay (D'Alessandro, M. et al., Differential effects on angiogenesis of two antimalarial compounds, dihydroartemisinin and artemisone: Implications for embryotoxicity, Toxicology. 241 (2007) 66-74. Doi: 10.1016/j.tox.2007.08.084).

[0071] The data were calculated as percentage cell viability using the formula:

[(absorbance of sample−blank of cell-free sample)/mean absorbance of control of media]×100.

[0072] The 50% cytotoxic concentration (CC50) that causes the death of 50% of Vero cells compared with the untreated control cells was determined. Visible morphological changes were observed with optical microscopy.

[0073] The CC50 of lysozyme HCl for Vero cells was determined as 13.3 mg/ml.

Determination of Non-Toxic Concentration of Ovotransferrin and Chloroquine

[0074] The cytotoxicity of ovotransferrin and chloroquine (CQ) was measured by MTT assay, similarly to the method used for lysozyme HCl. Ovotransferrin proved non-toxic at the maximum concentration used (10 mg/ml) (100% viability compared with control cells). The CC50 and CC10 of CQ proved to be 95.3±18 and 20.93±4.39 respectively.

Isolation of SARS-CoV-2 from Nasopharyngeal Swabs SARS-CoV-2 was isolated from 500 μl of nasopharyngeal swabs, added to Vero cells at 80% confluence; the inoculum was removed after 3 hours' incubation at 37° C. with 5% CO.sub.2, and the cells were incubated at 37° C., 5% CO.sub.2, for 72 hours, when the cytopathic effects were evident.

[0075] The numbers of viral copies in the cell supernatant were quantified by specific quantitative real-time RT-PCR (qRT-PCR) ((World Health Organization (WHO). Coronavirus disease (COVID-19) technical guidance: Laboratory testing for 2019-nCoV in humans. US CDC Real-time RT-PCR Panel for Detection 2019-Novel Coronavirus (28 January 2020). Available at: https://www.fda.gov/media/134922/download [last access 20 March 2020].

[0076] SARS-CoV-2 was precipitated with PEG according to the manufacturer's instructions, and the viral load was determined by Plaque Assay, using dilution factors ranging between 10 and 10^9.

[0077] The virus was used with a multiplicity of infection (MOI) of 0.05 in the following experiments.

Cell Infection and Treatment of Compounds

[0078] Vero cells were seeded in 96-well plates at a density of 1×10.sup.4 cells/well and cultured for 24 hours at 37° C. with 5% CO.sub.2, they were then infected with an MOI of 0.05 (1000 PFU/well) and incubated for 2 hours at 37° C. with 5% CO.sub.2. The virus was then removed, and the cells were treated with medium containing lysozyme HCl or chloroquine (as control) at different concentrations, and incubated for 72 hours at 37° C. with 5% CO.sub.2. An additional protocol was conducted, adding a pre-incubation step: the virus (MOI 0.05) was incubated for 1 hour at 37° C. in the presence of various concentrations of lysozyme HCl before addition to the cell monolayer.

Evaluation of Antiviral Activity of Lysozyme HCl

[0079] The numbers of viral copies in the cell supernatant were quantified by specific quantitative real-time RT-PCR (qRT-PCR).

[0080] The results (Table 1) were expressed as percentage viral replication, taking account of the replication in the untreated infected Vero cells, amounting to 100%.

[0081] Moreover, to verify the virucidal activity of the compound, a plaque test was conducted after inoculation into the cells of the virus plus the compound, plated in a 6-well plate. Briefly, after inoculation, the cells were covered with 0.3% agarose, dissolved in the cell medium and incubated for 72 hours at 37° C. with 5% CO.sub.2. After removal of the agarose, the cells were fixed with a 4% formaldehyde solution and then stained with methylene blue. The plaques in each well were counted, and the results were expressed as plaque-forming units (PFU)/mL and as the ratio between PFUs in the untreated control and the treated cells.

TABLE-US-00002 TABLE 1 Evaluation of antiviral activity of lysozyme HCl Lysozyme HCl Percentage viral replication concentration (mg/ml) Cells pre-treated with Lysozyme added to tested lysozyme HCl already infected cells 0.00 100 100 0.06 / 98 0.17 / 100 0.19 100 97 0.37 96 87 0.50 / 54 0.75 33 37 1.00 5 7 1.50 0 0 3.00 0 0

[0082] Each experiment was conducted in duplicate or triplicate, and two independent experiments were conducted.

Antiviral Activity of Lysozyme HCl and Ovotransferrin Against SARS-CoV-2

[0083] Ovotransferrin alone was tested at different concentrations, ranging from 10 mg/ml to 1.25 mg/ml, but no antiviral activity was detected in said interval. Table 2 summarises the antiviral activity of lysozyme HCl combined with three different concentrations of ovotransferrin, expressed as ACt and percentage viral replication (average of three experiments). In all conditions, ovotransferrin increased the antiviral activity of lysozyme HCl. Interestingly, the lowest concentration of ovotransferrin (1.25 mg/ml) exhibited the highest synergic effect. The synergic antiviral effect obtained is dose-dependent.

[0084] Table 3 shows the comparative percentage viral replication data between lysozyme HCl and lysozyme HCl combined with ovotransferrin.

TABLE-US-00003 TABLE 2 Antiviral activity of lysozyme HCl and ovotransferrin against SARS- CoV-2 expressed as percentage ΔCt and percentage viral replication Lysozyme HCl Ovotransferrin Percentage concentration concentration replication of (mg/ml) (mg/ml) ΔCt SARS-CoV-2 0.32 1.25 −9.3 62 2.5 −6.9 75 5 −4.3 83 0.42 1.25 −11.7 33 2.5 −12.0 54 5 −6.3 79 0.56 1.25 −17.9 25 2.5 −17.2 27 5 −11.8 54 0.75 1.25 −24.6 0 2.5 −17.9 25 5 −19.0 29

TABLE-US-00004 TABLE 3 Comparison between lysozyme HCl and lysozyme HCl combined with ovotransferrin; data expressed as percentage viral replication Percentage replication of Percentage Lysozyme HCl Ovotransferrin SARS-CoV-2 replication of concentration concentration (lysozyme + SARS-CoV-2 (mg/ml) (mg/ml) ovotransferrin) (lysozyme only) 0.32 1.25 62 93 2.5 75 5 83 0.42 1.25 33 89 2.5 54 5 79 0.56 1.25 25 93 2.5 27 5 54 0.75 1.25 0 62 2.5 25 5 29
Virucidal Activity To confirm the virucidal activity of lysozyme HCl, an assay was conducted on plaque using heat-treated lysozyme HCl (at 99° C. for 40 minutes) or non-heat-treated lysozyme HCl. Table 4 shows the results obtained, expressed as mean PFU/ml (average of three experiments). The infectivity of SARS-CoV-2 was reduced by 57.2%, 58.9% and 69.6%, using 0.42 mg/ml, 0.56 mg/ml and 1 mg/ml respectively. The non-heat-treated lysozyme HCl did not reduce the infectivity of SARS-CoV-2 at any of the doses used (Table 4).

TABLE-US-00005 TABLE 4 Virucidal activity of lysozyme HCl and heat-treated lysozyme HCl Untreated mean PFU/ml (average of three experiments) infected cells 0.42 mg/ml 0.56 mg/ml 1 mg/ml Lysozyme 73.33 (95.5%)   70 (83.9%) 61.65 (84.1%) 75 HCl (<100%) Heat-treated 102.5 (100%)  58.61 (57.2%) 60.41 (58.9%) 71.32 lysozyme  (69.6%)

[0085] In Table 5, the enzyme activity and HPLC purity of samples of lysozyme HC1 heat-treated under different experimental conditions were comparatively analysed, evaluating different temperatures, dry treatment times (on the solid product) or treatment times in aqueous solution, by comparison with the activity expressed as degree of viral replication on an average of 9 experiments per sample. The data obtained confirm that all the heat treatments conducted give rise to high antiviral activity, and confirm the absence of thermal degradation (denaturation) in all the samples examined, except for the samples treated in aqueous solution for times exceeding 6 minutes, wherein a marked reduction in HPLC purity (−3 to −4%) and enzyme activity (−8 to −12%) was observed.

[0086] The absence of thermal degradation in the samples examined, confirmed initially by HPLC analysis and enzyme activity, was further confirmed by HSQC NMR (heteronuclear single quantum correlation nuclear magnetic resonance) analysis of the samples in D20 solution. The .sup.1H—.sup.13C—HSQC spectra of a sample of native lysozyme (not heat-treated) and a sample of lysozyme subjected to dry heat treatment (99° C. for 40 min) proved identical, confirming that the heat-treated lysozyme was not denatured.

TABLE-US-00006 TABLE 5 Lysozyme HCl heat-treated under different experimental conditions vs. percentage viral replication and degree of denaturation % viral Enzyme Types Compound (test replication activity of heat concentration in (average of 9 HPLC (turbidimetric treatment mg/ml) replications) purity method) No heat Lys HCl (0.75 mg/mL) 25.51 99.7 98.7 treatment Lys HCl (0.5 mg/mL) 58.13 Lys HCl (0.33 mg/mL) 79.72 Heat treat- Lys HCl for 3 minutes 15.55 99.4 97.3 ment in (0.75 mg/mL) aqueous Lys HCl for 3 minutes 67.97 solution (0.5 mg/mL) (90° C. Lys HCl for 3 minutes 87.85 for the (0.33 mg/mL) times Lys HCl for 6 minutes 9.88 99.0 97.4 indicated) (0.75 mg/mL) Lys HCl for 6 minutes 51.62 (0.5 mg/mL) Lys HCl for 6 minutes 87.96 (0.33 mg/mL) Lys HCl for 20 minutes 18.30 97.0 92.1 (0.75 mg/mL) Lys HCl for 20 minutes 36.10 (0.5 mg/mL) Lys HCl for 20 minutes 78.00 (0.33 mg/mL) Lys HCl for 25 minutes 2.62 96.4 88.8 (0.75 mg/mL) Lys HCl for 25 minutes 24.61 (0.5 mg/mL) Lys HCl for 25 minutes 42.06 (0.33 mg/mL) Dry heat Lys HCl (0.75 mg/mL) 16.58 98.8 98.1 treatment Lys HCl (0.5 mg/mL) 35.56 (99° C. Lys HCl (0.33 mg/mL) 60.32 for 40 min) Dry heat Lys HCl (0.75 mg/mL) 11.31 98.9 98.8 treatment Lys HCl (0.5 mg/mL) 42.05 (74° C. Lys HCl (0.33 mg/mL) 83.24 for 60 min)