Intranasal Antiviral Therapy for Mucosal Protection Against Virus Infections

Abstract

A topical intranasal antiviral composition for protecting against virus infections. The composition comprises an antigen binder and a pharmaceutical suspender material to allow effective delivery into the nasal cavity. Examples of materials that could be used in the pharmaceutical suspender are microcrystalline cellulose or sodium carboxymethylcellulose (Na⋅CMC). One particular target for the antigen binder could be SARS-CoV-2. For example, the antigen binder could target the S1 subunit of SARS-CoV-2. The composition could be made by a process in which the pharmaceutical vehicle is prepared, and then the antigen binder is added to the pharmaceutical vehicle to make a bioactive mixture, and then adding solid sodium chloride to the bioactive mixture. Also disclosed are methods of protection against virus infection using the intranasal antiviral composition. For example, in the case of SARS-CoV-2, the antigen binder would block the virus particles from attaching to ACE2 receptors on host cells of the nasal mucosa or nasopharynx. This blocking action would protect against virus infection.

Claims

1. A method of making a topical intranasal antiviral composition, comprising: having a preparation of antigen binder; making a pharmaceutical vehicle comprising a pharmaceutical suspender; heat sterilizing the pharmaceutical vehicle; cooling the pharmaceutical vehicle; adding the antigen binder to the pharmaceutical vehicle to make a bioactive mixture; adding solid sodium chloride to the bioactive mixture.

2. The method of claim 1, wherein the antigen binder preparation is a liquid containing the antigen binder at a concentration of 5-150 mg/ml.

3. The method of claim 1, wherein the pharmaceutical vehicle comprises a homogenous aqueous mixture containing the pharmaceutical suspender.

4. The method of claim 3, wherein the homogenous aqueous mixture is made by adding the pharmaceutical suspender to water or an aqueous solution.

5. The method of claim 4, wherein the pharmaceutical suspender comprises microcrystalline cellulose and sodium carboxymethylcellulose.

6. The method of claim 5, wherein the pharmaceutical suspender is added to the water or aqueous solution as a powder blend consisting essentially of microcrystalline cellulose and sodium carboxymethylcellulose.

7. The method of claim 1, wherein the pharmaceutical suspender is a powder blend comprising microcrystalline cellulose and sodium carboxymethylcellulose.

8. The method of claim 7, wherein the powder blend is 4-20 wt % sodium carboxymethylcellulose and 75-95 wt % microcrystalline cellulose.

9. The method of claim 8, wherein the amount of microcrystalline cellulose in the blend relative to the amount of sodium carboxymethylcellulose (by weight) is in the range of 20:1 to 3:1 (MCC:Na⋅CMC).

10. The method of claim 9, wherein the antiviral composition comprises 0.04-0.70 wt % Na⋅CMC.

11. The method of claim 8, wherein the powder blend has a viscosity of 20-200 centipoise.

12. The method of claim 1, wherein the antigen binder is polyclonal IgY antibodies.

13. The method of claim 1, wherein the antigen binder is targeted to a coronavirus spike protein.

14. The method of claim 13, wherein the antigen binder is targeted to an S1 subunit of coronavirus spike protein.

15. The method of claim 13, wherein the coronavirus is SARS-CoV-2.

16. The method of claim 1, wherein the antiviral composition has a viscosity of 15-120 centipoise.

17. The method of claim 1, wherein the osmolarity of the antiviral composition is in the range of 260-325 mOsm.

18. The method of claim 1, further comprising adding a preservative.

19. The method of claim 1, wherein the sodium chloride is added after making the bioactive mixture.

20. The method of claim 4, wherein the step of adding the sodium chloride is performed at a time after adding the pharmaceutical suspender.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 depicts an user spraying the antiviral composition into her nose.

[0037] FIG. 2 depicts a sagittal cross-section of the nasal cavity.

DETAILED DESCRIPTION OF EXAMPLE EMBODIMENTS

[0038] To assist in understanding the invention, reference is made to experimental examples to show specific embodiments in which the invention may be practiced. Experimental work was performed to make a nasal spray composition using anti-S1 subunit IgY antibodies. Polyclonal IgY antibodies can be obtained from antigen-vaccinated egg-laying hens. The eggs are collected and the IgY antibodies are extracted from the egg yolk. This process is described in the literature, such as Amro et al, “Production and purification of IgY antibodies from chicken egg yolk” (2018) Journal of Genetic Engineering & Biotechnology, 16(1):99-103. This article is incorporated by reference herein.

[0039] In this work, egg-laying hens were immunized with the SARS-CoV-2 viral spike protein to induce IgY antibody production. The eggs were collected and anti-S1 subunit IgY antibodies were extracted from the egg yolks. Three different formulations were made at different concentrations of the IgY antibody: 5 mg/ml, 10 mg/ml, and 20 mg/ml. Described below is the process for making the 10 mg/ml formulation.

[0040] Preparing sterile antibody. The anti-S1 subunit IgY antibodies were provided at a target stock concentration 40 mg/ml. This was diluted to 24.7% (v/v) with a density of 1.066 g/ml. A 266 gram batch of the diluted antibody was filtered through a sterile syringe filter (which was a 0.2 μm cellulose acetate membrane) into a tared beaker.

[0041] Preparing pharmaceutical vehicle. A top entry mixer was set with the mixing shaft and impeller centered in a beaker and positioned close to the bottom of the beaker without touching. Sterile water (USP) was added to the beaker. The mixer was started from speed zero and the speed was increased slowly to stir the water to form a vortex without drawing air into the liquid. The target mixer speed was 2,000 rpm, but was adjusted as needed.

[0042] While continually stirring, 20 grams of Vivapur® MCG 591 P (2% w/v) was added to the beaker and mixed for about 10 minutes until an aqueous suspension was formed. Vivapur® MCG 591 P is a powder blend of microcrystalline cellulose (86.2-91.7 wt %) and sodium carboxymethylcellulose (8.3-13.8 wt %). After initial “overhead” mixing, the suspension was homogenized with stirring speed of about 5,000 rpm for a duration of about 30 minutes until the suspension turned into a homogenous gel. The beaker was then covered with aluminum foil and placed in the autoclave at a temperature of about 120° C. for about 30 minutes duration to sterilize the gel.

[0043] After autoclaving, resume stirring with the mixer while allowing the gel to cool to room temperature (about 25° C.). Continue stirring at 500 rpm for about 10 minutes while adding more sterile water (USP) to make the gel composition at the desired concentration of ingredients.

[0044] Adding the antibody. While continually stirring further at 700 rpm for about 10 minutes, add the sterile IgY antibody preparation (as described above) to the gel. Then while continually mixing further for about 5 minutes, add 9 grams of granular sodium chloride (USP, solid form) to make the gel 0.9% w/v isotonic saline.

[0045] This yielded a sterile gel composition with 10 mg/ml concentration of the IgY antibody in 0.9% saline, with microcrystalline cellulose and sodium carboxymethylcellulose at 2 w/v % in aggregate. The gel composition was dispensed into 3 ml size nasal spray vials with 1.5 ml of the gel composition in each vial.

[0046] Additional formulations. Similar compositions at 5 mg/ml and 20 mg/ml of the IgY antibody were made by dilution of the 40 mg/ml stock to about 12.5% and about 50% (w/v), respectively.

[0047] Drawing Figures. FIG. 1 depicts an example of how this invention could be used. Shown here is a user 12 having a nasal sprayer 10, which contains an antiviral composition of this invention. She is spraying the antiviral composition into her nose. FIG. 2 depicts an example of how nasal spray droplets could be limited to deposition in the nasal cavity. Shown here is the nose 16 and a sagittal cross-section of the nasal cavity with its vestibule 14, atrium 22, internal turbinates 18, and nasopharynx 20. Deposition of the nasal spray droplets may be limited to the nasal cavity without traveling further down into the lungs.

Experimental Testing

[0048] The topical intranasal composition of this invention is designed to form a viscous layer within the nasal passage that traps the virus and prevents it advancing further into the airway. In order to achieve this, the composition must tolerate the high shear forces that are generated by the spray nozzle of nasal spray pumps. Shear forces may be problematic because they reduce the desired viscosity of the spray liquid. For experimental testing, we focused on blends of microcrystalline cellulose (MCC) and sodium carboxymethylcellulose (Na⋅CMC) for use as a pharmaceutical suspender. There are various grades of the product with different amounts of MCC and Na⋅CMC. Our experiments sought to find the mixture that was best suited for nasal spray of large biologic molecules, such as antibodies.

Making the Pharmaceutical Vehicle

[0049] An initial study was conducted to select the suspending agent for making a suitable pharmaceutical vehicle to serve as a carrier for the IgY antibodies. Because the objective of the composition is to trap the virus with antibodies, thereby preventing entry of the virus into mucosal cells, the viscous nature of the composition is important for therapeutic efficacy. Prolonging residence time in the nasal cavity would enhance therapeutic efficacy.

[0050] To achieve this, a blend of MCC and Na⋅CMC was used to create the suspension. Table 1 below gives the different blend options that were tested. Viscosity testing was performed on a rotational viscometer. For initial setup, the viscometer was tested in plain water at room temperature to give the following results: viscosity of 11.72 centipoise (cPs) at 200 rpm speed, giving a torque of 62.4%, with accuracy of ±0.30 cPs. The following commercial products were tested for viscosity at a concentration of 1.5 w/v % in water. The viscosity results were as follows:

TABLE-US-00001 TABLE 1 Materials & Viscosity Testing Product Viscosity Name Materials (cPs) Avicel microcrystalline cellulose and  18.60 CL-611 Na .Math. CMC (11.3-18.8 wt %) Avicel microcrystalline cellulose and 131.3  RC-591 Na .Math. CMC (8.3-13.8 wt %) Vivapur microcrystalline cellulose and  99.70 MCG 591P Na .Math. CMC (8.3-13.8 wt %) Methocel E5 microcrystalline cellulose Not tested

[0051] After evaluating the results, we decided that Vivapur MCG 591P was most suitable based on the viscosity results. Also, visual inspection showed that the Vivapur behaved like a gel when undisturbed and transformed to a viscous liquid when shaken.

[0052] Because the IgY antibodies were provided in watery-thin aqueous form, it must be added to a proportionally thicker liquid to obtain a 1.5 wt % pharmaceutical vehicle. Thus, the Vivapur suspension was made in purified water at a concentration thicker than 1.5 wt %. An appropriate amount of IgY antibody was pipetted into each 25 ml volumetric flask and diluted to volume with the thicker Vivapur suspension. With this, the final mixture contained 1.5 wt % Vivapur at each concentration of the antibody. Table 2 below shows the proportions used for each strength.

TABLE-US-00002 TABLE 2 Proportions of Vivapur Relative to IgY Control (0) 5 mg/mL 10 mg/mL 20 mg/mL Vivapur 1.5% 1.7% 2.0% 3.0% Concentration IgY N/A 40 mg/mL 40 mg/mL 40 mg/mL Concentration Amount of IgY N/A 3 mL 6 mL 12.5 mL q.s to volume N/A 25 mL 25 mL 25 mL

[0053] We performed HPLC (high-performance liquid chromatograph) on the samples for quality control testing (see more details below). Table 3 below shows the proportions used for each antibody strength, along with the recovery amount of each sample. These HPLC recovery results confirmed that the technique of adding the IgY antibody to a proportionally thicker Vivapur suspension produced an acceptable formulation.

TABLE-US-00003 TABLE 3 IgY Recovery by HPLC Control (0) 5 mg/mL 10 mg/mL 20 mg/mL Vivapur N/A  1.7%  2.0%  3.0% Concentration IgY N/A 40 mg/mL 40 mg/mL 40 mg/mL Concentration Amount of IgY N/A 6 mL 12.5 mL 25 mL q.s. to volume N/A 50 mL 50 mL 50 mL Recovery by N/A 94.1% 95.8% 98.1% HPLC

[0054] We then sought to make the pharmaceutical vehicle isotonic. Having the product be isotonic may reduce irritation to the nasal mucosa by avoiding inducement of osmotic flows. To create an isotonic suspension, sodium chloride was added to the homogenized Vivapur suspension. Sodium chloride 0.9% is a common fluid solution used in medical applications. To mimic this, we created a Vivapur suspension (without IgY) with 0.9% sodium chloride and studied the osmolality of the mixture. Table 4 below shows the osmolality of the resulting mixture (the target osmolality being 290 mOsm). These results indicate that a 1.5 wt % Vivapur suspension in 0.9% sodium chloride yielded the desired osmolality.

TABLE-US-00004 TABLE 4 Osmolality of Suspension with 0.9% Sodium Chloride (without IgY) Osmolality (mOsm) Trial 1 285 Trial 2 287 Trial 3 289 Average 287

[0055] We then studied how the IgY antibody alone (without saline) affected the solution osmolality. The testing results are shown in Table 5 below. These results indicate that the IgY antibody, at the different concentrations, has only minimal effect on overall osmolality. We believe that this minimal amount of solution osmolality is caused by the IgY antibody stock being provided in sodium acetate salt solution (as part of the protein reconstitution process performed by the supplier). Thus, we decided that the formulation could be made using 0.9% sodium chloride without the need to adjust the sodium chloride concentration according to the amount of IgY antibody. That is, the different strength antibody products could be made using the same sodium chloride addition technique.

TABLE-US-00005 TABLE 5 Osmolality (mOsm) without Sodium Chloride 5 mg/ml 10 mg/ml 20 mg/ml Trial 1 6 9 17 Trial 2 5 9 16 Trial 3 5 9 17 Average 5 9 17

[0056] We next performed visual observation of the formulation. This was done by dropping the liquid mixture onto a glass slide and making visual observations. Despite having satisfactory results on viscometer, HPLC, and osmolality, our visual observation indicated that more viscosity was needed. Thus, we decided to increase the Vivapur concentration to 2.0 wt % (up from 1.5 wt %). A comparison of the 1.5% versus the 2.0% formulation is shown in Table 6 below, using the 20 mg/mL strength for the IgY antibody, as an example. The IgY was calculated according to a presumed stock concentration of 40 mg/mL and density 1.066 g/ml. The q.s. was to 100 g of final suspension.

TABLE-US-00006 TABLE 6 Vivapur Amount Increased Raw Material Formulation #1 (1.5%) Formulation #2 (2.0%) Vivapur MCG 591P  1.5 g  2.0 g IgY Protein 53.3 g 53.3 g Sodium Chloride  0.9 g  0.9 g Purified Water 35.0 g 35.0 g Purified Water q.s. q.s.

[0057] Osmolality testing was conducted on Formulation #2 above to observe how increasing the concentration of Vivapur affected the osmolality. Table 7 below shows the results. As seen here, the osmolarity increased slightly compared to the about 290 mOsm expected for the 0.9% sodium chloride solution alone. This is because the IgY protein stock was provided in sodium acetate solution, which contributed slightly to the osmolality. This slight increase in osmolality is acceptable. We also measured density to modify the amount of added purified water at the end of the manufacturing process. The measured density of Formulation #2 was 1.077 g/mL and the acidity was pH 6.75.

TABLE-US-00007 TABLE 7 Osmolality Testing Trial Osmolality (mOsm) Trial 1 308 Trial 2 309 Trial 3 313 Average 310

[0058] Benefits of Homogenization. Originally, the Vivapur and purified water were only mixed with a top mixer. Although this achieved acceptable results, we found that homogenizing the suspension at high shear forces made it thicker more without having to add additional Vivapur. Table 8 below shows Formulations #1-3. Formulation #3 gave the best viscosity and gelling properties. The IgY was calculated according to a presumed stock concentration of 40 mg/ml and density 1.066 g/ml. For #1 and #2, the q.s. was to 100 grams of final suspension. For #3, the q.s. was to 107.7 g of final suspension. The amounts are expressed as gram units.

TABLE-US-00008 TABLE 8 Effect of Homogenization Raw Material Formulation #1 Formulation #2 Formulation #3 Vivapur MCG 591P 1.5 2.0 2.0 IgY Protein 53.3 53.3 53.3 Sodium Chloride 0.9 0.9 0.9 Purified Water 35.0 35.0 40.0 Purified Water q.s. q.s. q.s. Homogenization X X ✓

[0059] Tables 9-12 below show the final formulations for the control (no IgY), 5 mg/mL, 10 mg/mL, and 20 mg/mL strengths, respectively. Table 13 gives a summary of the cGMP (Current Good Manufacturing Practice) manufacturing process.

TABLE-US-00009 TABLE 9 Final Formulation, Placebo Control Ingredient w/w % Batch Weight (g) Vivapur MCG 591P 2.0 20.0 Sodium Chloride 0.9  9.0 Sterile Water q.s. Portion 1: 533 Portion 2: q.s Total 1077

TABLE-US-00010 TABLE 10 Final Formulation, IgY at 5 mg/mL Ingredient w/w % Batch Weight (g) Vivapur MCG 591P 2.0 20.0 Anti-S1 Protein IgY 12.4 133.5 Sodium Chloride 0.9 9.0 Sterile Water q.s. Portion 1: 533 Portion 2: q.s Total 1077

TABLE-US-00011 TABLE 11 Final Formulation, IgY at 10 mg/mL Ingredient w/w % Batch Weight (g) Vivapur MCG 591P 2.0 20.0 Anti-S1 Protein IgY 24.7 266.0 Sodium Chloride 0.9 9.0 Sterile Water q.s. Portion 1: 533 Portion 2: q.s Total 1077

TABLE-US-00012 TABLE 12 Final Formulation, IgY at 20 mg/mL Ingredient w/w % Batch Weight (g) Vivapur MCG 591P 2.0 20.0 Anti-S1 Protein IgY 49.4 533 Sodium Chloride 0.9 9.0 Sterile Water q.s. Portion 1: 500 Portion 2: q.s Total 1077

TABLE-US-00013 TABLE 13 Summary of cGMP Manufacturing Process Step # Procedure 1 All product contact materials are sterilized prior to batch start to minimize potential bioburden. 2 A calculation is performed based upon the purity of the anti-S1 protein IgY to determine if any adjustments to the theoretical water quantity are required. 3 The anti-S1 protein IgY is dispensed first. As the anti-S1 protein IgY is dispensed it is passed through a 0.22 μm filter to remove any bioburden. 4 All other ingredients are dispensed. 5 Water is added to a suitable container and mixing begins using a top entry mixer to form a vortex without introduction of air. 6 Vivapur MCG 591P is then added and mixed for at least 10 minutes to ensure homogeneity. 7 This suspension is then homogenized using high shear mixing for at least 30 minutes to form a more viscous suspension. 8 This suspension is then autoclaved to for at least 30 minutes at a minimum temperature of 120° C. to remove any bioburden. 9 The suspension is then cooled down to ≤25° C. 10 The resultant suspension is then weighed, and additional water is dispensed and added to make up for any evaporation losses during sterilization, etc., that have occurred to this point. The suspension is then mixed for at least 10 minutes to ensure homogeneity. 11 The batch yield is then calculated to ensure there are no irregularities prior to adding the anti-S1 protein IgY. 12 The anti-S1 protein IgY is then added and mixed at a slower speed for at least 10 minutes. 13 The sodium chloride is then added for isotonicity and then mixed for at least 20 minutes to ensure homogeneity. 14 Additional steps are then performed to calculate final yield and remove the appropriate samples. 15 The suspension is then packaged by adding 1.5 mL of suspension to a 3 mL dropper bottle w/control tip and placing the cap on the bottle. This process continues until the desired quantity of bottles have been filled.

[0060] Technical Observations: Sodium chloride was added to the mixture to make the suspension liquid isotonic. Having an isotonic liquid would avoid or reduce irritation to the nasal passageways. In the step where sodium chloride granules were added to the mixture, we observed that adding the sodium chloride after doing the high-temperature autoclaving was important. When the sodium chloride was added to the pharmaceutical vehicle initially and then autoclaved at high temperature, this caused the Vivapur blend to precipitate out of the mixture. This is an undesirable effect. Thus, adding sodium chloride in solid form (e.g. powder or granules) to the liquid composition after the IgY or the Vivapur may be critical to making a workable product. This is as opposed to adding IgY or Vivapur to an already-prepared saline sodium chloride aqueous solution.

[0061] Also, performing the high-temperature autoclave sterilization of the pharmaceutical vehicle before adding the IgY antibodies avoids the possibility of causing the antibodies to denature or degrade. Thus, pharmaceutical vehicle was autoclaved prior to adding the sodium chloride and separately from the sterilizing the antibody preparation.

[0062] In adding the IgY antibody to the pharmaceutical vehicle, we observed that the mixing should be performed slowly. Otherwise, it creates a frothy liquid instead of a gel. We believe that this is because the IgY antibody is an egg-based protein. Also, for storage, freezing the composition may be undesirable because the Vivapur excipients may separate out from the mixture during thawing.

Stability Testing

[0063] The following stability testing was performed at both refrigerated (2-4° C.) and room temperature. The duration of the stability testing was three months, and in some batches, up to six months.

[0064] pH Stability: The pH of the IgY stock was close to physiological. However, because the suspension formulation did not contain any buffers, unwanted changes in pH was a possibility. Thus, we tested for stability of pH and the results are shown in Tables 14 & 15. These results indicate that the pH of the suspension is stable in both temperature conditions.

TABLE-US-00014 TABLE 14 pH stability; refrigerated Batch Initial 2 wk. 1 mon. 2 mon. 3 mon. 6 mon. #1-Control (0) 5.6 5.6 5.7 5.6 5.7 N/A #2-5 mg/ml 6.5 6.4 6.5 6.5 6.5 #3-10 mg/ml 6.7 6.7 6.6 6.7 6.7 #4-20 mg/ml 6.8 6.8 6.7 6.8 6.8 6.1

TABLE-US-00015 TABLE 15 pH stability; room temperature Batch initial 2 wk. 1 mon. 2 mon. 3 mon. #1 - Control (0) 5.6 5.6 5.6 5.6 5.7 #2 - 5 mg/ml 6.5 6.5 6.5 6.5 6.5 #3 - 10 mg/ml 6.7 6.7 6.6 6.7 6.7 #4 - 20 mg/ml 6.8 6.8 6.8 6.8 6.7

[0065] Osmolality Stability: One of the objectives of the formulation design was isotonicity to avoid or reduce irritation to the nasal passageways. In the suspension, there are two factors affecting osmolality: sodium chloride concentration and IgY stock concentration. Because the IgY stock contained a small amount of sodium acetate, this could potentially affect the desired isotonic osmolality. Thus, we tested for stability of osmolality and the results are shown in Tables 16 & 17. These results indicate that the osmolality of the suspension is stable in both temperature conditions.

TABLE-US-00016 TABLE 16 Osmolality stability (mOsm); refrigerated Batch Initial 2 wk. 1 mon. 2 mon. 3 mon. 6 mon. #1-Control (0) 275 277 277 274 273 N/A #2-5 mg/ml 358 355 311 230 326 #3-10 mg/ml 288 284 286 289 288 #4-20 mg/ml 295 294 293 293 295 295

TABLE-US-00017 TABLE 17 Osmolality stability (mOsm); room temperature Batch Initial 2 wk. 1 mon. 2 mon. 3 mon. #1 - Control (0) 275 279 274 274 275 #2 - 5 mg/ml 358 233 241 320 310 #3 - 10 mg/ml 288 287 288 290 287 #4 - 20 mg/ml 295 295 292 292 311
For #2, the variance in osmolality was caused by inaccurate measurements from mixing problems.

[0066] Viscosity Stability: The viscosity of nasal spray compositions are known to change over time. As mentioned above, having sufficient viscosity is an important factor to the topical nasal composition. Thus, we tested for stability of viscosity and the results are shown in Tables 18 & 19. Viscosity here is measured as centipoise (cPs). These results indicate that the viscosity of the suspension is stable in both temperature conditions.

TABLE-US-00018 TABLE 18 Viscosity stability (cPs), refrigerated Batch Initial 2 wk. 1 mon. 2 mon. 3 mon. #1 - Contral (0) 48 45 48 48 50 #2 - 5 mg/ml 52 63 59 52 57 #3 - 10 mg/ml 51 56 55 57 60 #4 - 20 mg/ml 47 51 54 53 52

TABLE-US-00019 TABLE 19 Viscosity stability (cPs), room temperature Batch Initial 2 wk. 1 mon. 2 mon. 3 mon. #1 - Control (0) 48 43 50 51 51 #2 - 5 mg/ml 52 57 57 54 56 #3 - 10 mg/ml 51 63 53 * 61 #4 - 20 mg/ml 47 51 53 53 52 *Not performed.

[0067] IgY Stability: Being a biologic product, the IgY antibodies are vulnerable to degradation by a variety of factors. Thus, we developed an HPLC (high performance liquid chromatography) protocol to detect intact IgY for quality control purposes. In developing the HPLC protocol, we observed that slowing the flow rate from 0.5 to 0.4 ml/min enhanced the resolution between peaks for quantitative analysis. This HPLC protocol gave us the ability to accurately quantify the amount of intact IgY recovered. Tables 20 & 21 show the IgY amounts that were recovered.

TABLE-US-00020 TABLE 20 IgY stability (% recovery by HPLC), refrigerated Batch Initial 2 wk. 1 mon. 2 mon. 3 mon. 6 mon. #1-Control (0) N/A N/A N/A N/A N/A N/A #2-5 mg/ml 96% 96% 97% 95%  99% #3-10 mg/ml 97% 99% 98% 98%  99% #4-20 mg/ml 96% 98% 96% 96% 100% 102%

TABLE-US-00021 TABLE 21 IgY stability (% recovery by HPLC), room temperature Batch initial 2 wk. 1 mon. 2 mon. 3 mon. #1 - Control (0) N/A NA N/A N/A N/A #2 - 5 mg/ml 96% 100% 99%  97% 103% #3 - 10 mg/ml 97% 100% 99%  96% 104% #4 - 20 mg/ml 96%  99% 97% 100%  99%

[0068] Visual Observations: We also performed visual inspections of the batches through the three month testing duration. There were no irregularities in the refrigerated batches for the three month duration. However, there were irregularities in the room temperature batches. In a few of the samples, there was a color change to pale yellow, appearance of dark-colored foreign substances, or strong sulfuric odor. This happened in the 5 mg/ml batch at the three-month timepoint and thereon; in the 10 mg/ml batch at the one-month time point and thereon; and in the 20 mg/ml batch at the one-month timepoint and thereon.