THERAPIES FOR TREATING INFLAMMATORY DISORDERS

Abstract

The present invention is directed to an antibody composition for oral administration comprising intact blood-derived polyclonal antibodies that bind to a human tumour necrosis factor (TNF), and means for protecting the antibodies during gastrointestinal transit, as well as methods for manufacturing, kits, and therapeutic uses of the same.

Claims

1. An antibody composition for oral administration comprising intact blood-derived polyclonal antibodies that bind to a human tumour necrosis factor (TNF), and means for protecting the antibodies during gastrointestinal transit.

2. The antibody composition according to claim 1, wherein the blood-derived polyclonal antibodies are ovine blood-derived polyclonal antibodies.

3. The antibody composition according to claim 1, wherein the blood derived polyclonal antibodies are equine blood-derived polyclonal antibodies.

4. The antibody composition according to any one of the preceding claims, wherein the means for protecting the antibodies comprises at least a protease inhibitor.

5. The antibody composition according to claim 4, wherein the protease inhibitor is one or more selected from the Bowman-Birk inhibitor family of proteins.

6. The antibody composition according to any one of the preceding claims formulated as a liquid.

7. The antibody composition according to any one of the preceding claims, wherein the means for protecting the antibodies comprises a means obtainable from egg white.

8. The antibody composition according to any one of the preceding claims, wherein the means for protecting the antibodies comprises egg white, preferably powdered egg white.

9. The antibody composition according to any one of the preceding claims, wherein the means for protecting the antibodies comprises one or more antacid(s).

10. The antibody composition according to any one of the preceding claims, wherein the means for protecting the antibodies comprises one or more of: a. a polypeptide which binds specifically to and suppresses or inactivates the proteolytic activity of trypsin and/or chymotrypsin; and/or b. an antibody that binds to trypsin and/or chymotrypsin and suppresses or inactivates the protease activity of said trypsin and/or chymotrypsin; and/or c. an antacid.

11. The antibody composition according to any one of the preceding claims, wherein the composition comprises an antacid and: a. a polypeptide which binds specifically to and suppresses or inactivates the proteolytic activity of trypsin and/or chymotrypsin; and/or b. an antibody that binds to trypsin and/or chymotrypsin and inactivates the protease activity of said trypsin and/or chymotrypsin.

12. The antibody composition according to any one of the preceding claims, wherein the means comprises one or more of ovomucoid, ovostatin, ovomacroglobulin, or combinations thereof.

13. The antibody composition according to any one of the preceding claims, wherein at least 5% of the total antibodies present in the composition bind to TNF.

14. The antibody composition according to any one of the preceding claims, wherein at least 10% of the total antibodies present in the composition bind to TNF.

15. A method for manufacturing intact blood-derived polyclonal antibodies that bind to human tumour necrosis factor (TNF), said method comprising obtaining a blood sample from a non-human mammal that has been administered an immunogen comprising human TNF or a fragment thereof.

16. The method according to claim 15, wherein the non-human mammal is an ovine non-human mammal.

17. The method according to claim 15 or 16 further comprising obtaining serum from the blood sample.

18. The method according to claim 15, wherein the non-human mammal is an equine non-human mammal.

19. The method according to claim 15 or 18 further comprising obtaining plasma from the blood sample.

20. The method according to any one of claims 15-19 further comprising purifying antibodies using sodium sulphate precipitation or caprylic acid precipitation.

21. An antibody obtainable by the method of any one of claims 15-20.

22. An antibody composition according to any one of claims 1-14 for use in treating an inflammatory disorder, wherein the antibody composition is orally administered to a subject.

23. Use of an antibody composition according to any one of claims 1-14 in the manufacture of a medicament for treating an inflammatory disorder, wherein the antibody composition is orally administered to a subject.

24. A method of treating an inflammatory disorder comprising orally administering an antibody composition according to any one of claims 1-14.

25. The antibody composition for use, use or method according to any one of claims 22-24, wherein the antibodies and the means for protecting the antibodies are administered simultaneously.

26. The antibody composition for use, use or method according to any one of claims 22-24, wherein the antibodies and the means for protecting the antibodies are administered separately.

27. The antibody composition for use, use or method according to any one of claims 22-26, wherein the inflammatory disorder is septic shock.

28. The antibody composition for use, use or method according to any one of claims 22-27, wherein the inflammatory disorder is a gastrointestinal disorder, preferably an intestinal disorder.

29. The antibody composition for use, use or method according to any one of claims 22-28, wherein the inflammatory disorder is an inflammatory bowel disease, an intestinal infection, a graft vs. host disorder, a disorder caused by: non-steroidal anti-inflammatory drugs, stress, alcohol, bowel surgery, ischaemia and reperfusion, food allergy, or combinations thereof.

30. The antibody composition for use, use or method according to any one of claims 22-29, wherein the inflammatory disorder is an inflammatory bowel disease selected from ulcerative colitis, Crohn's disease, or combinations thereof.

31. The antibody composition for use, use or method according to any one of claims 22-30, wherein the antibody composition is administered to a subject at a dose of 2 g or less daily.

32. The antibody composition for use, use or method according to any one of claims 22-31, wherein the antibody composition is administered to a subject at a dose of 1 g or less daily.

33. A kit comprising an antibody composition according to any one of claims 1-14, and instructions for use of same.

34. A foodstuff comprising an antibody composition according to any one of claims 1-14.

35. The foodstuff according to claim 34, wherein the foodstuff is a dairy product, preferably a dairy product selected from yogurt, cheese, or milk.

36. The foodstuff according to claim 34 or 35 further comprising a probiotic, and optionally a prebiotic.

Description

FIGURES

[0231] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying Figures, in which:

[0232] FIG. 1 shows inhibition of bovine trypsin by Intact Anti-TNF purified with either caprylic acid (.box-tangle-solidup.) or sodium sulphate (.square-solid.). TNF antisera (.circle-solid.) was used as a control.

[0233] FIG. 2 shows results of a direct ELISA for detection of anti-TNF IgG in antisera (.circle-solid.) and IgG purified by way of caprylic acid precipitation (.box-tangle-solidup.).

[0234] FIG. 3 shows results of an immunocytotoxicity assay (IOTA) testing the neutralising activity of Anti-TNF Fragment (.square-solid.), Intact Anti-TNF (.box-tangle-solidup.) and TNF antisera (.circle-solid.) using L929 cells. Starting concentration: Anti-TNF Fragment 50 mg/ml; Intact Anti-TNF 210 mg/ml; TNF antisera protein concentration 86 mg/ml.

[0235] FIG. 4 shows the results of an ELISA comparing binding to murine TNF by ovine blood-derived intact PcAbs (anti-human TNF IgG) and monoclonal antibody Infliximab. Anti-human TNF IgG (.circle-solid.), Infliximab (.square-solid.), and negative control (PCB) (.box-tangle-solidup.).

EXAMPLES

Example 1

Preparation of Ovine Antisera to Human TNF

[0236] Mature human TNF (hTNF) (UniProtKB Accession No.: P01375) was obtained from R&D Systems, Boehringer. The amino acid sequence is shown as SEQ ID No. 1.

[0237] The immunogen for the primary immunisation of merino wether sheep comprised Freund's complete adjuvant and 100 g of hTNF per sheep. The protein:adjuvant mixture was injected subcutaneously and equally into 6 injection sites chosen for their proximity to the axillary, inguinal, and prescapular drainage lymph glands. Each sheep was reimmunized at 28-day intervals with 100 g of hTNF and Freund's incomplete adjuvant, and blood samples were collected 14 days later at approximately 4 weekly intervals at processing facilities at Turretfield Research Centre (Rosedale, South Australia, Australia) according to strict state and national ethical guidelines for animal welfare. The animals were not terminally bled. A total of 10 mL of blood per kg of body weight can be collected from the external jugular vein without detriment to the animal.

[0238] Ovine antisera was subsequently stored at 20 C.

Example 2

Purification of Polyclonal Ovine Antibodies to Human TNF (Intact Anti-TNF)

[0239] Two different methods for purification of ovine PcAb were used in order to determine which method would co-isolate to a larger extent the efficient inhibitor of human trypsin, 1-anti-trypsin. Either caprylic acid precipitation, which precipitates albumin and keeps the IgG in solution, or the sodium sulphate precipitation that precipitates IgG were used. The purified IgG was filtered and stored at 20 C. ready for inclusion in the proposed formulation for oral administration or for further characterisation.

[0240] The presence of protease inhibitors in antisera and the two IgG fractions purified using either caprylic acid or sodium sulphate was assayed against trypsin and chymotrypsin by way of a colorimetric assay. The assay was based on the methods of Kakade M L et al. Determination of trypsin inhibitor activity of soy products: a collaborative analysis of an improved procedure. Cereal Chem 51: 376-381, 1974 (which is incorporated herein by reference) and measured cleavage of Na-Benzoyl-DL-Arginine-p-Nitroanilide Solution (L-BAPNA) (colourless) to p-nitroanaline (a yellow substrate) by trypsin.

[0241] Two-fold dilutions of inhibitor sample were diluted in 50 mM Tris-buffer, pH 8.2, containing 20 mM CaCl.sub.2 (100 l final volume) across a 96-well plate (Grenier UV-Star). To each well, 100 l of trypsin (0.2 mg/ml diluted in 1 mM HCL) or chymotrypsin (1 mg/ml in WFI) solution was added followed by 100 l of either L-BAPNA or NSLPN (2 mM in DMSO) for trypsin or chymotrypsin, respectively. Reactions were terminated after 5 minutes for the trypsin assay and after 60 minutes for the chymotrypsin assay with the addition of 50 l stop solution (30% v/v acetic acid) and the absorbance measured spectrophotometrically at 410 nm using Omega PolarStar. Blank samples were prepared by adding the stop solution prior to the substrate solution. The average absorbance value was then plotted against the dilution, resulting in a dose response curve. Representative curves are shown in FIG. 1, which demonstrate that the sodium sulphate purification retains the al-anti-trypsin inhibitor much better that the caprylic acid precipitation procedure.

[0242] These two methods can also be used to assess the survival of protease inhibitors in the oral formulation after incubation in simulated gastric and intestinal fluids.

Example 3

Enzyme-Linked Immunosorbent Assay for Characterising Antibody Binding

[0243] The specific antibodies produced bound to multiple epitopes on the surface of recombinant human TNF (rhTNF) but not to recombinant rodent TNF. The avidity of binding was extremely high.

[0244] A direct ELISA assay was developed for detection of anti-TNF IgG in the ovine antisera and in the purified fraction of IgG (purified by way of caprylic acid precipitation) from this antisera (Intact Anti-TNF). Immulon 4HBx microtiter plates were coated with 1 g/mL hTNF. Plates were washed with 3 changes of phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBST) and blocked for 1 hour at 37 C. with blocking buffer (2.5% fetal calf serum diluted in PBS). Plates were washed and incubated for 1 hour at 37 C. with antisera at initial dilutions of 1:1000, followed by 1:2 serial dilutions; washed with PBST; and incubated with a donkey anti-ovine IgG horseradish peroxidase conjugate for 1 hour at 37 C. After further washing, 3,3,5,5-Tetramethylbenzidine (TMB) liquid substrate solution was added, and the reaction was stopped after approximately 10 minutes by the addition of 1.0 M HCL before reading the optical density at 450 nm.

[0245] The developed assay showed very low background which was tested using pre-immune ovine serum (FIG. 2).

Example 4

Immunocytotoxicity Assay for Characterising Antibody Neutralisation

[0246] The L929 mouse fibrosarcoma cell line (commercially available from Sigma-Aldrich, The Old Brickyard, New Road, Gillingham, Dorset, SP8 4XT, UK) was used to test the cytotoxic effects of TNF as well as the neutralising ability of antibodies to TNF. An assay was therefore developed to test neutralisation of the cytotoxic effect of rhTNF by the ovine PcAb in the antisera, and by purified IgG from the antisera (Intact Anti-TNF), and by fragments thereof (Anti-TNF Fragment).

[0247] Anti-TNF Fragment was prepared by subjecting a portion of the stock of ovine antisera of Example 1 to papain digestion. The Fab were present at a concentration of 10 g/L and about 10% of the total Fab were specific for TNF. An affinity chromatography step was not included in its manufacture. In the presence of excess of Fab, about 12 molecules of Fab become attached to each TNF trimer.

[0248] As a challenging dose we used the 1090 hTNF concentration of 13 ng/ml determined from a cytotoxicity assay (data not shown). Briefly, L929 cells containing twice the necessary challenging dose in DMEM were co-incubated with an equal volume of various dilutions of hTNF antisera or Anti-TNF Fragment or Intact Anti-TNF for 24 h. As a positive control (maximum killing), 2.5 g/ml hTNF was used. Antibody toxin neutralisation titres were estimated by colorimetric assays based on cell staining with neutral red.

[0249] Antibody toxin neutralisation titres were estimated by colorimetric assays based on cell staining with neutral red (representative curves shown in FIG. 3).

[0250] The specific antibody concentration was calculated as follow:

Specific Ab conc [g/L]=[CCD(g/L)LC50(g/L)][MW Ab/(MW AgBS)]EC5010.sup.6 [0251] CCD (g/L)challenging dose=13 g/L (LC90 determined from the TNF cytotoxicity on L929 cells) [0252] LC50 (g/L)=0.3 g/L (determined from the TNF cytotoxicity on L929 cells) [0253] BSbinding sites=2 for whole IgG [0254] MW Ab=160 000 Da [0255] MW Ag (TNF)=51 000 Da

[0256] Taking the above into account, the specific PcAb concentration in the antisera was calculated at 2.9 g/L.

Example 5

Formulation of Intact Anti-TNF

[0257] The choice of protease inhibitors to protect the PcAb of the invention (Intact Anti-TNF) from digestion as well as the inhibitor's survival in gastric and intestinal fluids was assessed against trypsin and chymotrypsin using colourimetric assay, based on the method of Kakade M L et al. Determination of trypsin inhibitor activity of soy products: a collaborative analysis of an improved procedure. Cereal Chem 51: 376-381, 1974. The trypsin assay is described above. The chymotrypsin assay utilised a colourless NSLPN substrate producing a yellow colour.

[0258] The two antacids in Table 1, magnesium hydroxide and aluminium hydroxide gel, were added to neutralise the low gastric pH, and thereby prevent pepsin in the stomach from degrading the active components of Intact Anti-TNFthe ovine IgG against TNF and the egg white trypsin and chymotrypsin inhibitors. Other constituents were antimicrobials methylparaben and propylparaben which are believed to maintain a low bioburden. The antifoaming agent simethicone was added to prevent protein denaturation when the suspension is mixed prior to use. Sweeteners in the form of sodium saccharin and mannitol reduce the bitterness of the two antacids. A flavouring agent, peppermint oil, was used to improve the taste. A suspending agent, xanthan gum, was added to keep all the above components in suspension.

[0259] It was experimentally determined that adding 200 mM Glycine to the formulation significantly improved stability of the suspension.

TABLE-US-00008 TABLE 1 Composition of Intact Anti-TNF oral formulation. Category Material name Concentration Active IgG Intact Anti-TNF 50 g/l components Protease Egg white dried (EWD) 60 g/l Excipients inhibitor Antacid Magnesium hydroxide 23.4 g/l Dried aluminium 26.4 g/l hydroxide gel Antimicrobial Methylparaben (E218) 2 g/l Propylparaben (E216) 0.6 g/l Suspension Glycine (200 mM) 15 g/l stability Antifoaming Simethicone 16.89 g/l agent Sweeteners Sodium saccharin 0.4 g/l Mannitol 21 g/l Flavour Peppermint oil 0.2 g/l Suspending Xanthan gum 4 g/l agent

Example 6

Physicochemical Characterisation of the Composition Formulated for Oral Delivery

[0260] The physical appearance of the suspension and its purity were ensured by carefully performing visual inspections. Sedimentation volume of the suspension was determined by pouring 50 ml of the formulation into a 100 ml measuring cylinder and the sedimentation volume was monitored and recorded at different time intervals.

[0261] Duplicate results were obtained and the sedimentation volume was calculated according to the equation:

F=Vu/Vo

[0262] Where F is sedimentation volume, Vuultimate height of sediment and Voinitial height of total suspension.

[0263] Within 48 hours of the beginning of the sedimentation experiment, no sedimentation was observed (Vu=0). Therefore, the pharmaceutical suspension remained very stable within 48 hours without any separation. Furthermore, after 48 hours the cylinder with the suspension was turned upside-down and none of the layers were observed to be disposed at the bottom of the cylinder. This demonstrates that there were no layers forming within the suspension. In other word, there was no flocculation observed within the prepared suspension during the 48-hour period of the experiment.

Example 7

Case Study

[0264] A 27 year old white male on an emergency admission to hospital is diagnosed as suffering from acute, severe ulcerative colitis which is confirmed by colonoscopy and biopsy. The subject is immediately administered an intravenous course of hydrocortisone but fails to respond over the next six days. There is concern that he might require a total colectomy and, consequently, he is orally administered a formulation of the invention (e.g. as per Example 5). For the first two weeks he receives 50 ml twice daily and, thereafter, 50 ml daily for a further 12 weeks.

[0265] The patient makes a quick and excellent recovery based on his answers to the UK bowel disease questionnaire (UK-IBDQ) and various clinical parameters. In addition measured parameters such as C-reactive protein return to normal values, and he experiences no serious adverse effects. At the end of the study there is significant mucosal healing as assessed by a second colonoscopy and biopsy.

Example 8

Synergistic Effects of the Compositions Formulated for Oral Delivery

[0266] The following formulations are provided: [0267] Formulation A: Intact Anti-TNF; [0268] Formulation B: Egg White Dried Protease Inhibitor; [0269] Formulation C: Bowman-Birk Inhibitor (soybean); [0270] Formulation D: Intact Anti-TNF+Egg White Dried Protease Inhibitor; and [0271] Formulation E: Intact Anti-TNF+Bowman-Birk Inhibitor (soybean).

[0272] 25 patients with ulcerative colitis and 25 patients with Crohn's Disease provide consent to be involved in a study to test the efficacy of Formulations A-E. The patients are split into groups of 5 and administered one of Formulations A-E (i.e. 5 patients with ulcerative colitis are administered Formulation A and 5 patients with Crohn's Disease are administered Formulation A, 5 patients with ulcerative colitis are administered Formulation B and 5 patients with Crohn's Disease are administered Formulation B, etc.). The dosage regime is 20 ml (equivalent to 1 g of Intact Anti-TNF) twice daily for 4 weeks, and once daily thereafter.

[0273] Physicians determine that patients in both disease groups administered Formulations D and E show a greater improvement and reduced symptoms when compared with patients administered Formulations A-C. Colonoscopies reveal an improvement in the surface layers of the intestinal tract post-treatment. The improvement in patients administered Formulations D and E is much greater than the improvement in patients administered Formulations A-C, and much greater than the expected combined improvement of Formulations A+B and Formulations A+C (as determined by way of colonoscopy). Thus, the combination of PcAbs and protease inhibitors (e.g. EWD protease inhibitors and/or Bowman-Birk Inhibitors) yields unexpected synergistic effects.

Comparative Example 9

[0274] Comparative Analysis of Specific Antibody Titres from Sheep Serum (Ovine) & Hen Eggs

[0275] A study was undertaken to assess the concentration and avidities of specific IgY obtained from hen eggs in comparison with specific antibody concentrations from ovine antisera.

[0276] A group of 10 chickens and 5 sheep were immunised with human interleukin-6 (hIL-6, a pro-inflammatory cytokine like TNF) and the titres and avidities of the resultant specific PcAb was compared. The average avidity constants were 1.310.sup.10 L/mol for chicken IgY vs 3.110.sup.10 L/mol for the ovine antibodies. However, the levels of specific PcAb attained in the sheep (with an average titre of 1:200,000) were more than ten times the titres found in egg yolk (1:20,000). This tenfold or more difference in the concentration of specific PcAb was also apparent when sheep and hens were immunised with a number of other immunogens.

[0277] The above experiment shows the advantages of antibodies derived from blood, and from an ovine source in particular.

Example 10

[0278] Comparative Analysis of Specific Antibody Titres Sourced from Sheep Serum (Ovine) & Cow's Milk (Bovine)

[0279] A study was undertaken to assess the potential of colostrum and milk from suitably immunised cows as a source of PcAb.

[0280] Cows were immunised with human TNF and the titres of the resulting specific PcAb determined first in the colostrum and then in serial samples of milk. The maximum titre obtained in colostrum was 1:275,000 (as compared with 1:800,000 in ovine antisera) and, after the first milking, levels rapidly fell to approximately 1:27,500.

[0281] Thus, blood-derived sources were shown to yield higher concentrations of antibodies that bind to human TNF when compared to milk/colostrum-derived sources.

Example 11

Stability of Oral Antibody Formulations

[0282] The oral formulation of Example 5 was tested for antibody binding and neutralising activity following storage for approximately 12 months. It was shown that there was no deterioration in protease inhibitor activity, and no change in the physical stability of the formulation, which retained antibody binding and neutralising activity.

Example 12

Efficacy of Antimicrobials of the Oral Antibody Formulations

[0283] The antimicrobial agents in the oral formulation of Example 5Methylparaben (E218) (concentration 2 g/L) and Propylparaben (E216) (0.6 g/L)were subjected to external antimicrobial testing to European Pharmacopeia standards for such organisms as S. aureus, P. aeruginosa, E. coli, C. albicans and A. brasiliensis. Complete sterility was shown.

Example 13

[0284] Comparison of Blood-Derived Polyclonal Antibodies with Monoclonal Antibodies

[0285] A comparative antigen-binding assay was performed using blood-derived (ovine) polyclonal antibodies that bind to human TNF and Infliximab (Schering-Plough Ltd), a monoclonal antibody that binds to human TNF.

[0286] FIG. 4 shows that the monoclonal antibody, Infliximab, does not bind to murine TNF, whereas the ovine derived polyclonal antibodies raised towards human TNF do.

[0287] The blood-derived polyclonal antibodies of the invention were shown to bind to and neutralise murine TNF albeit at a concentration approximately 100-fold higher than that needed to neutralise human TNF. No neutralisation of murine TNF was observed by Infliximab, indicative of an overall reduced neutralisation capability when compared to antibodies of the invention.

TABLE-US-00009 SEQUENCES SEQIDNo.1 VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQ LVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLL SAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDY LDFAESGQVYFGIIAL SEQIDNo.2 MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTL FCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVA NPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFK GQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAK PWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL SEQIDNo.3 MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTL FCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVA NPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFK GQGCP SEQIDNo.4 VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQ LVVPSEGLYLIYSQVLFKGQGCP SEQIDNo.5 VRSSSRTP SEQIDNo.6 HVVANPQAEGQLQWLNRR SEQIDNo.7 NGVELR SEQIDNo.8 VPSEG SEQIDNo.9 CPSTHVL SEQIDNo.10 ISRIAVSYQTK SEQIDNo.11 PCQRETPEGAEAK SEQIDNo.12 DRLSAEINRPDYLDFA SEQIDNo.13(VariantHumanTNF P84L) MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTL FCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTLSDKPVAHVVA NPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFK GQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAK PWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

[0288] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.