Stabilizing compositions for immobilized biomolecules

Abstract

The present invention relates to the use of a composition comprising (a) at least three different amino acids, (b) at least two different amino acids and a saponin or (c) at least one dipeptide or tripeptide for stabilizing biomolecules immobilized on a solid carrier. The invention furthermore relates to a method for producing stabilized biomolecules, comprising embedding the biomolecules in the composition according to the invention and a method of producing a solid carrier having biomolecules attached thereto. The invention furthermore relates to a solid carrier producible or produced by the method of the invention and a method of diagnosing a disease using the carrier of the invention.

Claims

1. A method for producing stabilized biomolecules, comprising (a) reversibly attaching the biomolecules to a solid carrier; and (b) embedding the biomolecules in a composition selected from: (i) a composition comprising at least three different amino acids, or (ii) a composition comprising at least two different amino acids and a saponin, wherein the composition of (i) does not contain dipeptides or tripeptides.

2. The method of claim 1, wherein stabilizing biomolecules includes stabilizing the structure and/or activity of biomolecules, enhancing the shelf-life of biomolecules and/or protecting biomolecules against stress-mediated damage.

3. The method of claim 1, wherein the composition comprising at least 3 different amino acids comprises at least 4 or at least 5 different amino acids.

4. The method of claim 3, wherein the composition comprising at least 5 different amino acids comprises at least one amino acid of each group of, (a) an amino acid with non polar, aliphatic R groups; (b) an amino acid with polar, uncharged R groups; (c) an amino acid with positively charged R groups; (d) an amino acid with negatively charged R groups; and (e) an amino acid with aromatic R groups.

5. The method of claim 3, wherein the composition comprising at least 5 different amino acids comprises amino acids selected from (a) alanine, glutamate, lysine, threonine and tryptophan; (b) aspartate, arginine, phenylalanine, serine and valine; (c) proline, serine, asparagine, aspartate, threonine, and phenylalanine; (d) tyrosine, isoleucine, leucine, threonine, and valine; or (e) arginine, glycine, histidine, alanine, glutamate, lysine, and tryptophan.

6. The method of claim 1, wherein the composition comprising at least three different amino acids, or the composition comprising at least two different amino acids and a saponin, comprises less than 1% by dry weight cysteine.

7. The method of claim 1, wherein the composition comprising at least three different amino acids, or the composition comprising at least two different amino acids and a saponin, further comprises less than 1% Tween.

8. The method of claim 1, wherein the saponin is glycyrrhizic acid or a derivative thereof.

9. A method of producing a solid carrier having biomolecules attached thereto, comprising the steps of (a) reversibly attaching the biomolecules to the solid carrier; and (b) incubating the carrier of step (a) in a composition selected from: (i) a composition comprising at least three different amino acids, or (ii) a composition comprising at least two different amino acids and a saponin, wherein the composition of (i) does not contain dipeptides or tripeptides.

10. The method according to claim 1 or 9, further comprising sterilizing the solid carrier after step (b).

11. The method according to claim 10 wherein the sterilization of the carrier is effected by ethylene oxide, beta radiation, gamma radiation, X-ray, heat inactivation, autoclaving or plasma sterilization.

12. The method of claim 1, wherein the biomolecules are reversibly attached to the solid carrier via a cleavable linker, and wherein the biomolecule can be released from the solid carrier by using a means for cleaving the linker.

13. The method of claim 12, wherein the cleavable linker is selected from the group consisting of ethylene glycol bis(succinimidylsuccinate), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, dithiobis(succinimidylpropionate), disuccinimidyl tartarate, succinimidyl 2-([4,4′-azipentanamido]ethyl)-1,3′-dithiproprionate, and sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-proprionate.

14. The method according to claim 1 or 9 further comprising (c) subjecting the solid carrier to drying.

15. A solid carrier produced by the method of claim 9.

16. The solid carrier of claim 15, wherein the biomolecules are proteins, peptides, nucleic acids, carbohydrates, lipids, fatty acids, polyalcohols and combinations or modifications thereof, wherein the proteins preferably are antibodies, enzymes, receptors, cytokines, hormones, membrane proteins, growth factors, albumins, globulins, transport proteins or blood coagulation factors.

17. The solid carrier of claim 15, wherein the biomolecules specifically bind to a marker protein indicative for a disease, a non-cellular pathogen, a cell or a toxin.

18. A method of preparing a medical device comprising the solid carrier of claim 15, wherein the medical device is selected from the group consisting of an implant, a tubing, a catheter, a stent, a tubing, a wound dressing and a medical device used in extracorporeal circulation.

19. A method for diagnosing a disease comprising the steps of: (a) contacting a sample obtained from a patient with a solid carrier according to claim 16 under suitable conditions to allow specific binding of the biomolecules attached to the carrier to said marker protein indicative for a disease, said non-cellular pathogen, said cell or said toxin; and (b) detecting whether said marker protein indicative for the disease, said non-cellular pathogen, said cell or said toxin has been bound to the biomolecules.

20. A method for producing stabilized biomolecules, comprising (a) adsorbing biomolecules directly to the surface of a solid carrier, and (b) covering the biomolecules in a composition selected from: (i) a composition comprising at least three different amino acids, or (ii) a composition comprising at least two different amino acids and a saponin, wherein the composition of (i) does not contain dipeptides or tripeptides, so that the biomolecules are partially or completely covered by the composition comprising at least three different amino acids, or the composition comprising at least two different amino acids and a saponin, wherein the covered biomolecules can be released from the carrier by adding a liquid to solubilize/dissolve the covered biomolecules.

21. The method of claim 20, further comprising: sterilizing the covered biomolecules; and releasing the sterilized covered biomolecules from the carrier.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The figures show:

(2) FIG. 1: A biomolecule, e.g. an antibody is attached to a solid carrier, preferably by use of spacer or linker molecules. The antibody is embedded by the postcoating solution and thereby protected of stress influences like irradiation.

(3) FIGS. 2 and 3: The protective effect of amino acid postcoatings increases with the number of different amino acids used (independent of the amino acid). The amino acids were selected as representatives of the following groups: amino acids with aromatic/positively charged/negatively charged/polar non-charged/non polar aliphatic side-chain.

(4) With postcoatings consisting of 5 different amino acids after irradiation with 25 kGy approx. 65% of the antigen binding ability compared to the not irradiated control is maintained; after accelerated aging procedure (7 days at 45° C.) approx. 85% of the antigen binding ability compared to the untreated control is maintained.

(5) FIGS. 4 and 5: The protective effect of amino acid postcoatings cannot be further enhanced by increasing the number of amino acids above 5. After irradiation with 25 kGy about 70% antigen binding ability compared to untreated control is maintained with all postcoatings containing more than 5 amino acids; after accelerated aging (7 days at 45° C.) about 85% is maintained.

(6) FIGS. 6 and 7: The combination of amino acids shows a protective effect of 65% (4 amino acids) respectively 85% (8 amino acids), whereas the average effect of the single amino acids is 22% (4 amino acids) respectively 33% (8 amino acids) antigen binding ability compared to the unsterilized control. The maximal effect of single amino acids is at 28% (4 amino acids) respectively 55% (8 amino acids).

(7) FIG. 8: The addition of 1 mM Glycyrrhizic acid to amino acid postcoatings enhances the protective effect of all postcoatings to a maximum value of 70-80% (antigen binding ability compared to not irradiated control).

(8) FIG. 9: An amino acid postcoating consisting of five different amino acids (200 mM) and 1 mM glycyrrhizic acid shows a protective effect of 80% antigen binding ability compared to the untreated control after irradiation (25 kGy) and 85% after accelerated aging procedure, respectively. An postcoating consisting of albumin and mannitol shows only 65% remaining activity of after irradiation (25 kGy). After accelerated aging for 7 days at 45° C. there is almost no activity remaining with albumin and mannitol, similar to the controls without any postcoating.

(9) FIG. 10: A mixture of 18 amino acids was used to block unspecific binding to an ELISA plate without inhibiting specific antigen-antibody interactions. The blocking efficiency is comparable to standard albumin blockings.

(10) FIG. 11: An amino acid postcoating consisting of 18 different amino acids (20 g/L) and 0.25 mM glycyrrhizic acid shows a protective effect of 83% DNAse activity compared to the untreated control after irradiation (25 kGy). An postcoating consisting of albumin and mannitol shows 95% remaining activity of after irradiation (25 kGy).

(11) FIG. 12: An amino acid postcoating consisting of 18 different amino acids (20 g/L in PBS) shows a protective effect of 85% antigen binding ability compared to the untreated control after irradiation (25 kGy) and 90% after accelerated aging procedure, respectively. With the addition of 1 mM glycyrrhizic acid to the amino acid postcoating the protective effect is 92% antigen binding ability compared to the untreated control after irradiation (25 kGy) and 95% after accelerated aging procedure, respectively.

(12) FIG. 13: Amino acid postcoatings consisting of 18 different amino acids (20 g/L in PBS) show a protective effect of 55-60%. Without postcoating, the amount of intact ds-DNA is reduced to 20%, with albumin and mannitol 85% are preserved.

(13) FIG. 14: anti-Hepatitis A test (functional ELISA) of an amino acid composition used for protecting anti Hepatitis antibodies after lyophilisation and sterilization.

(14) FIG. 15: The structural class of saponins has the potential to enhance the protective effect of amino acid combinations.

(15) FIG. 16: Combinations of at least 3 amino acids and combinations of 2 amino acids with the addition of glycyrrhizic acid provide a maximal protection of an immobilized antibody when sterilized with 50 kGy beta irradiation.

(16) FIG. 17: The amino acid composition provides protection under different stress conditions. The best protection is provided for beta irradiation with different doses and artificial aging under elevated temperature. The protection for gamma irradiation or ethylene oxide sterilisation is less but still relevant.

(17) FIGS. 18 and 19: Amino acid postcoatings containing at least 5 amino acids provide protection during long time storage. For the non sterilized samples, after 62 days at 45° C. about 80% of the antigen binding ability is preserved. Sterilized samples (beta, 25 kGy) maintain about 70% of their antigen binding ability after 62 days at 45° C. Amino acid postcoatings containing only 2 amino acids maintain only about 40% antigen binding ability during the storage process, regardless of sterilization. Without postcoating only 20-30% activity is preserved.

(18) FIGS. 20 and 21: The addition of 1 mM glycyrrhizic acid to the postcoating solutions enforces the protecting effect. Amino acid postcoatings containing at least 5 amino acids and glycyrrhizic acid provide protection during long time storage. For the non sterilized samples, after 62 days at 45° C. about 90% of the antigen binding ability is preserved. Sterilized samples (beta, 25 kGy) maintain about 80% of their antigen binding ability after 62 days at 45° C. Amino acid postcoatings containing 2 amino acids and glycyrrhizic acid maintain about 70% antigen binding ability during the storage process, regardless of sterilization. Without postcoating only 20-30% activity is preserved.

(19) FIG. 22: Samples sterilized with gamma irradiation maintain about 85% activity when protected with an amino acid postcoating containing 18 amino acids; this effect is not further enhanced with glycyrrhizic acid; with 5 amino acids the remaining activity is 75%; with 2 amino acids only 40% are maintained. The protection with 2 amino acids is improved by the addition of glycyrrhizic acid, here the remaining activity is 65%. Samples sterilized with ETO maintain about 85% activity when protected with an amino acid postcoating containing 18 amino acids; this effect is not further enhanced with glycyrrhizic acid. Postcoatings containing 5 or 2 amino acids have only little protecting effect; the addition of glycyrrhizic acid enhances the protection marginally.

(20) FIG. 23: Amino acid postcoating of amino acids, dipeptides or mixtures thereof, optionally together with glycyrrhizic acid.

(21) FIG. 24

(22) An example is shown, where the vial itself is the carrier. First, the biomolecule was attached to the carrier by drying. Subsequently, the stabilizer solution was added and also dried. The biomolecule (here interleukin 8=IL8) loses most of its biological function during subsequent sterilisation (25 kGy irradiation) if no stabilizer is added. In contrast, a stabilizer solution with different amino acids protected the biomolecule. Shown is the chemotactic activity of IL8 on human neutrophil granulocytes.

(23) FIG. 25

(24) An example is shown, where the vial itself is the carrier. First, the biomolecule was attached to the carrier by drying. Subsequently, the stabilizer solution was added and also dried. The biomolecule (here interleukin 8=IL8) loses most of its biological function during accelerated storage (45° C.) if no stabilizer is added. In contrast, a stabilizer solution with different amino acids protected the biomolecule. Shown is the chemotactic activity of IL8 on human neutrophil granulocytes.

(25) FIG. 26

(26) An example is shown, where the vial itself is the carrier. First, the biomolecule was attached to the carrier by drying. Subsequently, the stabilizer solution was added and also dried. The biomolecule (here ds-DNA) loses part of its biological function during subsequent sterilisation (25 kGy irradiation) if no stabilizer is added. In contrast, a stabilizer solution with different amino acids protected the biomolecule. Shown is the detectable DNA amount in percent of starting value.

(27) FIG. 27

(28) An example is shown, where the stabilizer itself is the carrier. The biomolecule and the stabilizer solution were added and dried together. The biomolecule (here interleukin 8=IL8) loses most of its biological function during subsequent sterilisation (25 kGy irradiation) if no stabilizer is added. In contrast, a stabilizer solution with different amino acids protected the biomolecule. Shown is the chemotactic activity of IL8 on human neutrophil granulocytes.

(29) FIG. 28

(30) An example is shown, where the stabilizer itself is the carrier. The biomolecule and the stabilizer solution were added and dried together. The biomolecule (here an anti-mouse IgG antibody) loses most of its biological function during subsequent storage and or sterilisation (25 kGy irradiation) if no stabilizer is added. In contrast, a stabilizer solution with different amino acids protected the biomolecule. Shown is the specific binding to the antigen.

(31) FIG. 29

(32) Influence of different desorption solutions on the biological activity of a biomolecule (here an anti-mouse IgG antibody). With the exception of 0.5 M H2SO4 none of the other desorption solutions tested in this experiment had a significant influence on the biological activity of the biomolecule. Shown is the specific binding to the antigen.

(33) FIG. 30

(34) The desorption of a biomolecule (here an anti-mouse IgG antibody) is tested after the biomolecule was attached to an open porous polyurethane foam (supplier A, large pores). While citrate buffer pH 4.75 and 1 M NaCl only desorbed small amounts of the biomolecule, significantly more biomolecule could be desorbed with 1M NaCl+0.02 M imidazole and phosphate buffered saline, respectively. Shown is the specific binding to the antigen.

(35) FIG. 31

(36) The desorption of a biomolecule (here an anti-mouse IgG antibody) is tested after the biomolecule was attached to an open porous polyurethane foam (supplier B, smaller pores). Citrate buffer pH 4.75 and phosphate buffered saline desorbed a little bit better than 1 M NaCl with or without 0.02 M imidazole. Shown is the specific binding to the antigen.

(37) FIG. 32

(38) The desorption of a biomolecule (here an anti-mouse IgG antibody) is tested after the biomolecule was attached to an open porous polyurethane foam (supplier Smith&Nephews, small pores). The biomolecule (here an anti-mouse IgG antibody) loses most of its biological function during subsequent storage and or sterilisation (25 kGy irradiation) if no stabilizer is added. In contrast a stabilizer solution with different amino acids protected the biomolecule. The recovery of the antibody is almost 100% (5 μg/mL). Shown is the specific binding to the antigen.

(39) FIG. 33

(40) The desorption of a biomolecule (here an anti-mouse IgG antibody) is tested after the biomolecule was attached to an PVA-hydrogel. The biomolecule loses most of its biological function during subsequent storage and or sterilisation (25 kGy irradiation) if no stabilizer is added. In contrast, a stabilizer solution with different amino acids protected the biomolecule. The recovery of the eluted antibody is very high. Shown is the specific binding to the antigen.

(41) FIG. 34

(42) Chemical Structures of examples for cleavable linkers

EXAMPLES

(43) The examples illustrate the invention.

(44) Materials & Methods

(45) All experiments were based on the same basic ELISA assay design.

(46) Adsorption of LO-MM-3 to an ELISA Plate and Application of Postcoatings

(47) A monoclonal antibody LO-MM-3 (anti-mouse-IgM, Acris, SM1495P) was adsorbed to the surface of a 96 well ELISA plate (Greiner Bio-one, 655061). LO-MM-3 was diluted in PBS (without Ca.sup.2+/Mg.sup.2+, PAA, H15-002) to a concentration of 2 μg/mL. 100 μL of the antibody solution was pipetted to each well and incubated over night at 5° C. The plate was washed 2 times with washing buffer (25× concentrate, Invitrogen, WB02).

(48) 200 μL of each assayed stabilizing composition (post-coating) were pipetted per well. At least 4 wells were treated identically with the same postcoating to calculate means and standard deviations in the following analysis. The plate was incubated with the postcoating solutions for 1 hour at ambient temperature. The postcoatings were discarded and the plate was dried at ambient temperature for at least 1 hour.

(49) Amino acids used for postcoatings: L-alanine (Sigma-Aldrich, A7627), L-arginine (Sigma-Aldrich, A5131), L-asparagine (Sigma-Aldrich, A0884), L-aspartate (Sigma-Aldrich, A9256), L-cysteine (Sigma-Aldrich, 1276), L-glutamine (AppliChem, A1420), L-glutamate (Sigma-Aldrich, G1251), glycine (Merck, 1042010), L-histidine (Sigma-Aldrich, H8125), L-isoleucine (Sigma-Aldrich, I2752), L-leucine (Sigma-Aldrich, L8000), L-lysine (Sigma-Aldrich, L5626), L-methionine (Sigma-Aldrich, M9625), L-phenylalanine (Sigma-Aldrich, P2126), L-proline (Sigma-Aldrich, P0380), L-serine (Sigma-Aldrich, S4500), L-threonine (Sigma-Aldrich, T8625), L-tryptophan (Sigma-Aldrich, T0254), L-tyrosine (Sigma-Aldrich, T3754), L-valine (Sigma-Aldrich, V0500)

(50) Stress Exposure of the Coated Surface

(51) Three identically treated plates were exposed to different environmental conditions. One plate was sterilized by irradiation (beta, 25 kGy). The irradiation was conducted at Beta-Gamma-Service, Bruchsal, Germany. One plate was treated with an accelerated aging procedure (7 days at 45° C.). Based on a simplification of the Arrhenius equation it is assumed that by increasing the storage temperature by 10° C., the speed of reaction, i.e. aging, is doubled (Hemmerich, 1998: General Aging Theory and Simplified Protocol for Accelerated Aging of Medical Devices). This implies that an accelerated aging for 7 days at 45° C. equals a real time aging of 16 weeks of cooled storage (5° C.).

(52) An identical plate without stress exposition served as a control in each experiment. The protective effect was calculated as the remaining antibody functionality after stress conditions compared to the untreated control.

(53) $\mathrm{functionality}\left(\%\right)=\frac{\mathrm{stressed}\mathrm{plate}.Math.100}{\mathrm{control}\mathrm{plate}}$
ELISA Detection of LO-MM-3 Functionality

(54) The dried postcoatings were removed from the wells by washing the plate 3 times. The ELISA plate was blocked by pipetting 300 μL blocking solution (10 g/L albumin in PBS) to each well and incubating for 1 hour at ambient temperature. The plate was washed 3 times.

(55) The antigen to LO-MM-3, CH11 (mouse IgM, MBL, SY001), was diluted to 125 ng/mL and 200 μL of the antigen solution was pipetted to each well. The plate was incubated for 1 hour at ambient temperature and washed 3 times.

(56) The bound antigen was detected by a detection antibody LO-MM-9 (biotinylated anti-mouse-IgM, AbD serotec, MCA199B), which was diluted with PBS to 50 ng/mL. To each well 200 μL were added and incubated for 1 hour at ambient temperature. The plate was washed 3 times.

(57) Streptavidin (Horseradish peroxidase (HRP) labeled, Pierce, 21126) was diluted with PBS to 100 ng/mL. 200 μL were added to each well and incubated for 1 hour at ambient temperature. The plate was washed 3 times.

(58) A ready-to-use ELISA substrate solution for HRP (TMB=tetramethylbenzidine, Invitrogen, 00-2023) was diluted 1:2 with aqua dest. and 200 μL were pipetted to each well. The plate was incubated for 20 minutes at ambient temperature and protected from light. To stop the colour reaction, to each well 50 μL of H.sub.2SO.sub.4 (diluted 1:5 with aqua dest., Merck, 1007311000) were added. The resulting yellow color was detected by measuring the absorption at a wavelength of 450 nm (Fusion Photometer A153601, PerkinElmer).

Example 1: Postcoatings Consisting of at Least 5 Amino Acids Show a Maximal Protection Against Stress

(59) Experiment:

(60) The amino acids were dissolved either in 0.5 M NaOH (Merck, 106482) or 0.5 M HCl (Merck, 100319) to obtain stock solutions with a maximal concentration. The amino acid stock solutions were mixed together to get a total amino acid concentration of 200 mM in the postcoating solution. The amino acids were used in equimolar ratio (2 amino acids: 2×100 mM; 3 amino acids: 3×67 mM; 4 amino acids: 4×50 mM, 5×40 mM; etc.)

(61) The pH of the amino acid mixtures was set to approx. 7.0; and the mixtures were further diluted in PBS to get the final concentration of 200 mM.

(62) Adsorption of LO-MM-3 to the plate and application of the postcoating; sterilization and accelerated aging; as well as the general ELISA procedure were conducted as described in the Materials & Methods section.

(63) Results:

(64) As shown in FIGS. 2 and 3, the protective effect of amino acid postcoatings increases with the number of different amino acids used. The amino acids were selected as representatives of the following groups: amino acids with aromatic/positively charged/negatively charged/polar non-charged/non polar aliphatic side-chain. It is not essential to use particular amino acids; they can be substituted with a different amino acid from the same group, or with similar characteristics respectively (Taylor, 1985: The Classification of Amino Acid Conservation).

(65) With postcoatings consisting of 5 different amino acids after irradiation with 25 kGy approx. 65% of the antigen binding ability compared to the non-irradiated control is maintained (FIG. 1); after accelerated aging procedure (7 days at 45° C.) approx. 85% of the antigen binding ability compared to the untreated control is maintained (FIG. 2).

(66) The protective effect of amino acid postcoatings cannot be further enhanced by increasing the number of amino acids above 5. As shown in FIGS. 4 and 5, postcoatings with 8, 10 or even 18 amino acids have virtually the same effect.

Example 2: The Combination of Amino Acids Reveals a Protective Effect not Attributed to One Particular Single Amino Acid

(67) Experiment:

(68) The amino acids were dissolved either in 0.5 M NaOH (Merck, 106482) or 0.5 M HCl (Merck, 100319) to obtain stock solutions with a maximal concentration. The amino acid stock solutions were mixed together to get a total amino acid concentration of 200 mM in the postcoating solution. The amino acids were used in equimolar ratio (4 amino acids: 4×50 mM; 8 amino acids: 8×25 mM). For the single amino acid solutions the amino acids were dissolved in aqua dest. at the maximal soluble concentration, if possible 400 mM. The pH of all postcoating solutions was set to approx. 7.0. Concentrations of the single amino acids: Ala 400 mM, Glu 100 mM, Lys 400 mM, Thr 400 mM, Asn 200 mM, Asp 100 mM, His 300 mM, Pro 400 mM, Ser 400 mM, Trp 60 mM, Val 400 mM.

(69) Adsorption of LO-MM-3 to the plate and application of the postcoating; sterilization and accelerated aging; as well as the general ELISA procedure were conducted as described in the Materials & Methods section.

(70) Result:

(71) As can be seen in FIGS. 6 and 7, the protective effect of an amino acid mixture cannot be attributed to a protective effect of one single amino acid of the mixture.

(72) The combination of amino acids shows a protective effect of 65% (4 amino acids) and 85% (8 amino acids), whereas the average effect of the single amino acids of those mixtures is 22% (4 amino acids) and 33% (8 amino acids) antigen binding as ability compared to the unsterilized control. The maximal effect of single amino acids is at 28% (4 amino acids) and 55% (8 amino acids).

Example 3: The Addition of Glycyrrhizic Acid to the Solution Further Increases the Protective Effect of Amino Acid Postcoatings

(73) Experiment:

(74) The amino acids were dissolved either in 0.5 M NaOH (Merck, 106482) or 0.5 M HCl (Merck, 100319) to obtain stock solutions with a maximal concentration. The amino acid stock solutions were mixed together to get a total amino acid concentration of 200 mM in the postcoating solution. The amino acids were used in equimolar ratio (2 amino acids: 2×100 mM; 3 amino acids: 3×67 mM; 4 amino acids: 4×50 mM; etc.). Glycyrrhizic acid (ammonium salt, Fluka, 50531) was dissolved in 50% ethanol (absolute, Sigma-Aldrich, 32205) to obtain a 25 mM stock solution. Glycyrrhizic acid was diluted 1:25 to obtain a concentration of 1 mM in the postcoating solutions.

(75) The pH of the amino acid mixtures was set to approx. 7.0; and the mixtures were further diluted in PBS to get the final amino acid concentration of 200 mM.

(76) Adsorption of LO-MM-3 to the plate and application of the postcoating; sterilization and accelerated aging; as well as the general ELISA procedure were conducted as described in the Materials & Methods section.

(77) Results:

(78) The addition of glycyrrhizic acid to different amino acid postcoatings enhances the protective effect of all postcoatings to a maximum value of 75-80% (antigen binding ability compared to unirradiated control) as shown in FIG. 8. Glyzyrrhizic acid alone does not have a protective effect as a postcoating.

Example 4: Amino Acid Postcoatings Provide a Protection Against Stress Conditions at Least Comparable to Postcoatings Containing Conventional Protecting Substances Like Albumin and Mannitol

(79) Experiment:

(80) The amino acid postcoating was prepared as described in example 3. Human albumin was used at a concentration of 20 g/L and mannitol at 10 g/L, diluted in PBS.

(81) Adsorption of LO-MM-3 to the plate and application of the postcoating; sterilization and accelerated aging; as well as the general ELISA procedure were conducted as described in the Materials & Methods section.

(82) Results:

(83) As depicted in FIG. 9, amino acid postcoatings provide a protective effect comparable to or even better than postcoatings based on albumin and mannitol, depending on the form of stress applied.

(84) An amino acid postcoating consisting of five different amino acids (200 mM) and 1 mM glycyrrhizic acid shows a protective effect of 80% antigen binding ability compared to the untreated control after irradiation (25 kGy) and 85% after accelerated aging procedure, respectively. The postcoating consisting of albumin and mannitol shows 65% activity of the untreated control after irradiation (25 kGy). After accelerated aging for 7 days at 45° C. there is almost no activity remaining with albumin and mannitol, similar to the controls without any postcoating.

Example 5: Amino Acid Mixtures Block Unspecific Binding, e.g. to ELISA Plates

(85) Experiment:

(86) LO-MM-3 was adsorbed to an ELISA plate as described in material & methods. The plate surface was blocked by incubating the wells with 300 μL blocking solution for 1 h at ambient temperature. As blocking either 10 g/L human albumin in PBS or 20 g/L of a mixture of 18 amino acids in PBS were used. The following ELISA procedure was conducted as described in the Materials & Methods section.

(87) Results:

(88) Amino acid mixtures can also block unspecific binding to surfaces, as shown in FIG. 10. A mixture of 18 amino acids blocks unspecific binding to an ELISA plate without inhibiting specific antigen-antibody interactions. The blocking efficiency is comparable to standard albumin blockings.

Example 6: Amino Acid Mixtures Provide Protection Against Stress for Enzymes Like DNAse

(89) Experiment:

(90) Adsorption of DNAse to an ELISA Plate and Application of Postcoatings

(91) The enzyme DNAse (Sigma Aldrich, DN25) was adsorbed to the surface of a 96 well ELISA plate (Greiner Bio-one, 655061). DNAse was diluted in PBS (without Ca.sup.2+/Mg.sup.2+, PAA, H15-002) to a concentration of 1 mg/mL. 100 μL of the enzyme solution was pipetted to each well and incubated over night at 5° C. The plate was washed 2 times with PBS.sup.−/−.

(92) Four wells were treated identically to calculate means and standard deviations in the following analysis. Unspecific binding to the plate was blocked with 200 μL blocking solution per well and incubated for 1 hour at ambient temperature. The plate was washed 2 times with PBS.sup.−/−. 200 μL of each postcoating were pipetted per well. The plate was incubated with the postcoating solutions for 1 hour at ambient temperature. The postcoatings were discarded and the plate was dried at ambient temperature.

(93) Blocking and Postcoating Solutions:

(94) Blocking: 20 g/L human albumin (Biotest Pharma) in PBS.sup.−/−

(95) Postcoating: 20 g/L human albumin+10 g/L mannitol (Serag Wiesner, 219675) in PBS.sup.−/−

(96) Blocking: 20 g/L amino acids (Ala, Asp, Arg, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val) in PBS.sup.−/−

(97) Postcoating: 20 g/L amino acids (Ala, Asp, Arg, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val)+0.25 mM glycyrrhizic acid (ammonium salt, Fluke, 50531) in PBS.sup.−/−
Stress Exposure of the Coated Surface

(98) One plate was sterilized by irradiation (beta, 25 kGy). The irradiation was conducted at Beta-Gamma-Service, Bruchsal, Germany.

(99) An identical plate without stress exposure served as a control. The protective effect was calculated as the remaining DNAse functionality after stress conditions compared to the untreated control.

(100) $\mathrm{functionality}\left(\%\right)=\frac{\mathrm{stressed}\mathrm{plate}.Math.100}{\mathrm{control}\mathrm{plate}}$
Detection of DNAse Functionality

(101) The plate was washed 3 times with PBS.sup.−/−. Ds-DNA Standard (Sigma Aldrich, D1501) was diluted to 1 μg/mL in PBS (with Ca.sup.2+/Mg.sup.2+, Hyclone, SH3026401). To each well 50 μL of the DNA-solution were added and incubated for 1 hour at 37° C. The fluorescent DNA-dye Picogreen (Molecular Probes, P7581) was diluted 1:1000 (PBS) and 150 μL of the dye were added to the DNA-solution in each well.

(102) The picogreen fluorescence was measured, the excitation filter was set to 485 nm and the emission was detected at 530 nm (Fusion Photometer A153601, PerkinElmer). The fluorescence signal correlates to the DNA concentration. DNAse activity was determined as the reduction of DNA, i.e. fluorescence signal.

(103) Results:

(104) As depicted in FIG. 11, amino acid postcoatings provide a protective effect against stress (irradiation) also for enzymes like DNAse. The protection is comparable to that of poscoatings based on albumin and mannitol

(105) An amino acid postcoating consisting of 18 different amino acids (20 g/L) and 0.25 mM glycyrrhizic acid shows a protective effect of 83% DNAse activity compared to the untreated control after irradiation (25 kGy). The postcoating consisting of albumin and mannitol shows 95% activity of the untreated control after irradiation (25 kGy).

Example 7: Amino Acid Postcoatings Provide a Protection Against Stress Conditions for Therapeutic Antibodies Like IgG Infliximab

(106) Experiment:

(107) The therapeutic antibody Infliximab (humanized IgG, anti-Human-TNF-α, Centocor, DD7701504) was adsorbed to the ELISA plate surface. Infliximab was diluted with PBS−/− to a concentration of 1 μg/mL and 100 μL of the solution was pipetted to each well. The plate was incubated over night at 5° C. and washed 2× with washing buffer.

(108) Application of the postcoating; sterilization and accelerated aging were conducted as described in the Materials & Methods section.

(109) ELISA Detection of Infliximab Functionality

(110) The dried postcoatings were removed from the wells by washing the plate 3 times. The ELISA plate was blocked by pipetting 300 μL blocking solution (10 g/L albumin in PBS) to each well and incubating for 1 hour at ambient temperature. The plate was washed 3 times.

(111) The antigen to Infliximab, TNF-α (recombinant human, R&D, Cat 210-TA), was diluted to 1 ng/mL and 200 μL of the antigen solution was pipetted to each well. The plate was incubated for 1 hour at ambient temperature and washed 3 times.

(112) The bound antigen was detected by a detection antibody (HRP labelled anti-human-TNF-α, R&D, Cat DTA00C). The ready-to-use-solution of the detection antibody was diluted 1:2 with PBS. To each well 200 μL were added and incubated for 1 hour at ambient temperature. The plate was washed 3 times.

(113) 200 μL of a ready-to-use ELISA substrate solution for HRP (TMB=tetramethylbenzidine) were pipetted to each well. The plate was incubated for 20 minutes at ambient temperature and protected from light. To stop the color reaction, to each well 50 μL of H.sub.2SO.sub.4 (diluted 1:5 with aqua dest.) were added. The resulting yellow colour was detected by measuring the absorption at a wavelength of 450 nm.

(114) Results:

(115) As depicted in FIG. 12, amino acid postcoatings with or without glycyrrhizic acid provide a protective effect comparable to or even better than poscoatings based on albumin and mannitol, depending on the form of stress applied.

(116) An amino acid postcoating consisting of 18 different amino acids (20 g/L in PBS) shows a protective effect of 85% antigen binding ability compared to the untreated control after irradiation (25 kGy) and 90% after accelerated aging procedure, respectively. With the addition of 1 mM glycyrrhizic acid to the amino acid postcoating the protective effect is 92% antigen binding ability compared to the untreated control after irradiation (25 kGy) and 95% after accelerated aging procedure, respectively. The postcoating consisting of albumin and mannitol shows 72% activity of the untreated control after irradiation (25 kGy). After accelerated aging for 6 days at 45° C. there is only 15% activity remaining with albumin and mannitol, similar to the controls without any postcoating.

Example 8: Amino Acid Postcoatings Provide a Protection Against Stress Conditions for Nucleic Acids (dsDNA)

(117) Experiment:

(118) Ds-DNA (Sigma, D1501) was adsorbed to the ELISA plate surface. The DNA was dissolved in PBS.sup.−/−. (1 mg/mL) with 1 mM EDTA (Fluka, 50531) to inhibit possible DNAse activity. The DNA was diluted with PBS 1:64 to 15 μg/mL and 100 μL of the solution was pipetted to each well. The plate was incubated over night at 5° C. and washed 2× with PBS.sup.−/−.

(119) Application of the postcoating and sterilization were conducted as described in the Materials & Methods section.

(120) Detection of Intact Ds-DNA with Picogreen

(121) The dried postcoatings were removed from the wells by washing the plate 3 times. To each well 100 μL Picogreen dye (diluted 1:1000 in PBS.sup.−/−, Molecular Probes, P7581) were added. The picogreen fluorescence was measured immediately, the excitation filter was set to 485 nm and the emission was detected at 530 nm (Fusion Photometer A153601, PerkinElmer). The fluorescence signal correlates to the concentration of intact ds-DNA.

(122) Results:

(123) As depicted in FIG. 13, amino acid postcoatings with provide a protective effect of 55-60%, independent of the addition of glyzyrrhizic acid or mannitol. Without postcoating, the amount of intact ds-DNA is reduced to 20% when irradiated with 25 kGy. Conventional stabilizing substances like albumin and mannitol provide a protection of about 85%.

Example 9: Protective Effect of a Specific Amino Acid Composition

(124) 20 mg human anti-Hepatitis A antibody (Beriglobin (human IgG, AK), CSL Behring) and 40 mg of a protective composition were dissolved in water to a total volume of 525 μL per sample and lyophilized. Afterwards, the samples were dissolved in 1 mL water and tested for functionality using an HAV (Hepatitis A virus) IgG ELISA.

(125) Composition:

(126) 20 g Arginine

(127) 20 g Histidine

(128) 20 g Lysine

(129) 3 g Glutamic acid

(130) 2 g Tryptophane

(131) 20 g Glycine

(132) 15 g Alanine

(133) 0.2 g Tween 80

(134) 1 g Glycyrrhizic acid ammonium salt

(135) The pH was adjusted to about 7.2 using NaOH and/or NaCl. Afterwards, the solutions were subjected to sterile filtration.

(136) 400 μL of solution (corresponding to 40 mg solid compounds) were mixed with 125 μL Beriglobin (corresponding to 20 mg antibody)

(137) Lyophilisation:

(138) Lyophilisation was carried out as follows:

(139) initial freezing temperature −40° C.;

(140) start of vacuum of 0.1 mBar after 3 h freezing time;

(141) temperature rise of about 1.5° C./h for 23 h;

(142) 6 h drying over night at 6° C. and 0.004 mBar.

(143) Sterilisation:

(144) The lyophilized samples were irradiated with 25 kGy and one with 50 kGy Beta-radiation.

(145) Results:

(146) The results of the HAV-ELISA are depicted in FIG. 14.

(147) After drying and sterilization white, non-odorant, inherently stable cakes were obtained. 1 mL water was added to each sample and the dissolution behaviour was monitored. Dissolution took place within less than 30 seconds and was free of aggregates. Even after 24 h neither macroscopic nor microscopic aggregation was detectable.

(148) Conclusion:

(149) Application of the composition resulted in only little loss of the Hepatitis A antibody portion of Beriglobin as compared to the not irradiated control.

Example 10: Test of Different Saponins as Stabilizing Compounds. The Structural Class of Saponins has a Stabilizing Effect on Antibodies

(150) Materials & Methods

(151) All experiments were based on the same basic ELISA assay design. (see above)

(152) Adsorption of LO-MM-3 to an ELISA plate and application of postcoatings

(153) Stress exposure of the coated surface

(154) ELISA detection of LO-MM-3 functionality

(155) Experiment:

(156) Adsorption of LO-MM-3 to the plate and application of the postcoating and sterilization; as well as the general ELISA procedure were conducted as described in the Materials & Methods section. The irradiation dose (electron beam) was 50 kGy.

(157) Results:

(158) The results of the experiment are depicted in FIG. 15. The structural class of saponins has the potential to enhance the protective effect of amino acid combinations. Preferred is the use of the saponin glycyrrhizic acid.

Example 11: Stabilizing Compositions Comprising at Least 3 Different Amino Acids or at Least Two Different Amino Acids and a Saponin Such Glycyrrhizic Acid Provide Very Good Protection

(159) Materials & Methods

(160) All experiments were based on the same basic ELISA assay design. (see above)

(161) Adsorption of LO-MM-3 to an ELISA plate and application of postcoatings

(162) Stress exposure of the coated surface

(163) ELISA detection of LO-MM-3 functionality

(164) Experiment:

(165) The amino acids were dissolved either in 0.5 M NaOH (Merck, 106482) or 0.5 M HCl (Merck, 100319) to obtain stock solutions with a maximal concentration. The amino acid stock solutions were mixed together in different combinations to get total amino acid concentrations of 20 g/L in the postcoating solutions.

(166) The pH of the amino acid mixtures was set to approx. 7.0.

(167) Adsorption of LO-MM-3 to the plate and application of the postcoating and sterilization; as well as the general ELISA procedure were conducted as described in the Materials & Methods section. The irradiation dose (electron beam) was 50 kGy.

(168) Results:

(169) The results of the experiment are depicted in FIG. 16. Combinations of at least 3 amino acids and combinations of 2 amino acids with the addition of glycyrrhizic acid provide a maximal protection (more than 75%) of an immobilized antibody when sterilized with 50 kGy beta irradiation.

Example 12: A Preferred Amino Acid Composition Provides Protection Under Different Stress Conditions

(170) Materials & Methods

(171) All experiments were based on the same basic ELISA assay design. (see above)

(172) Adsorption of LO-MM-3 to an ELISA Plate and Application of Postcoatings

(173) Stress Exposure of the Coated Surface

(174) ELISA Detection of LO-MM-3 Functionality

(175) Experiment:

(176) Compositions Used:

(177) Protective Composition A (Compound Per Liter)

(178) 20 g Arginine

(179) 20 g Histidine

(180) 20 g Lysine

(181) 3 g Glutamine

(182) 2 g Tryptophan

(183) 20 g Glycine

(184) 15 g Alanine

(185) 0.2 g Tween 80

(186) 1 g Glycyrrhizic acid ammonium salt

(187) The pH was adjusted to about 7.2 using NaOH and/or HCl. Afterwards, the solutions were subjected to sterile filtration.

(188) Adsorption of LO-MM-3 to the plate and application of the postcoating and sterilization; as well as the general ELISA procedure were conducted as described in the Materials &

(189) Methods section.

(190) Results:

(191) The results of the experiment are depicted in FIG. 17. The amino acid composition provides protection under different stress conditions. The best protection is provided for beta irradiation with different doses and artificial aging under elevated temperature. The protection for gamma irradiation or ethylene oxide sterilisation is less but still relevant.

Example 13: Amino Acid Postcoatings Provide Protection During Long-Time Storage

(192) Experiment:

(193) The amino acids were dissolved either in 0.5 M NaOH (Merck, 106482) or 0.5 M HCl (Merck, 100319) to obtain stock solutions with a maximal concentration. The amino acid stock solutions were mixed together to get a total amino acid concentration of 200 mM in the postcoating solution. The amino acids were used in equimolar ratio. 18 amino acids: Ala, Arg, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val 5 amino acids (1): Asp, Arg, Phe, Ser, Val 5 amino acids (2): Ala, Glu, Lys, Thr, Trp 2 amino acids (1): Asp, Val 2 amino acids (2): Ala, Glu

(194) The pH of the amino acid mixtures was set to approx. 7.0; and the mixtures were further diluted in PBS to get the final concentration of 200 mM.

(195) Adsorption of LO-MM-3 to the plate and application of the postcoating and sterilization; as well as the general ELISA procedure were conducted as described in the Materials & Methods section. Long-time storage was simulated by an accelerated aging procedure. The plates were stored at 45° C. and antibody activity was determined after 0, 10, 25, 41 and 62 days of storage. This equals real time aging at 5° C. of 0, 6, 12, 24 and 36 months.

(196) Results:

(197) Amino acid postcoatings containing at least 5 amino acids provide protection during long time storage. For the non-sterilized samples, after 62 days at 45° C. about 80% of the antigen binding ability is persevered. Sterilized samples (beta, 25 kGy) maintain about 70% of their antigen binding ability after 62 days at 45° C. Amino acid postcoatings containing only 2 amino acids maintain only about 40% antigen binding ability during the storage process, regardless of sterilization. The addition of 1 mM glycyrrhizic acid to the postcoating solutions enforces the protecting effect: For the non-sterilized samples containing at least 5 amino acids and glycyrrhizic acid, after 62 days at 45° C. about 90% of the antigen binding ability is preserved. Sterilized samples (beta, 25 kGy) maintain about 80% of their antigen binding ability after 62 days at 45° C. Amino acid postcoatings containing 2 amino acids and glycyrrhizic acid maintain about 70% antigen binding ability during the storage process.

Example 14: Amino Acid Postcoatings Provide Protection During Different Sterilization Processes

(198) Experiment:

(199) The amino acids were dissolved either in 0.5 M NaOH (Merck, 106482) or 0.5 M HCl (Merck, 100319) to obtain stock solutions with a maximal concentration. The amino acid stock solutions were mixed together to get a total amino acid concentration of 200 mM in the postcoating solution. The amino acids were used in equimolar ratio. 18 amino acids: Ala, Arg, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val 5 amino acids: Asp, Arg, Phe, Ser, Val 2 amino acids: Asp, Val

(200) The pH of the amino acid mixtures was set to approx. 7.0; and the mixtures were further diluted in PBS to get the final concentration of 200 mM.

(201) Adsorption of LO-MM-3 to the plate and application of the postcoating and sterilization; as well as the general ELISA procedure were conducted as described in the Materials & Methods section. One plate was irradiated (gamma, 25 kGy). The irradiation was conducted at Beta-Gamma-Service, Bruchsal, Germany. Another plate was sterilized by EO (ETO BO1 cycle); the sterilization was conducted at Rose GmbH, Trier, Germany.

(202) Results:

(203) Samples sterilized with gamma irradiation maintain about 85% activity when protected with an amino acid postcoating containing 18 amino acids; this effect is not further enhanced with glycyrrhizic acid; with 5 amino acids the remaining activity is 75%; with 2 amino acids only 40% are maintained. The protection with 2 amino acids is improved by the addition of glyccyrhizic acid; here the remaining activity is 65%. Samples sterilized with ETO maintain about 85% activity when protected with an amino acid postcoating containing 18 amino acids; this effect is not further enhanced with glycyrrhizic acid. Postcoatings containing 5 or 2 amino acids have only little protecting effect; the addition of glycyrrhizic acid enhances the protection marginally.

Example 15: Postcoatings Consisting of Amino Acids and Dipeptides Provide Protection Against High Irradiation Doses

(204) Materials & Methods

(205) All experiments were based on the same basic ELISA assay design. (see above) Adsorption of LO-MM-3 to an ELISA plate and application of postcoatings Stress exposure of the coated surface ELISA detection of LO-MM-3 functionality
Experiment:

(206) The amino acids were dissolved either in 0.5 M NaOH (Merck, 106482) or 0.5 M HCl (Merck, 100319) to obtain stock solutions with a maximum concentration. The amino acid stock solutions were mixed together to get a total amino acid concentration of 20 g/L in the postcoating solution. The dipeptides alone were used with a concentration of 10 g/L and in combination with amino acids with 2 g/L.

(207) 7 amino acids: Arg, His, Lys, Glu, Trp, Gly, Ala

(208) 2 dipeptides: Gly-Tyr, Gly-Gln

(209) The pH of the amino acid mixtures was set to approx. 7.0.

(210) Adsorption of LO-MM-3 to the plate and application of the postcoating and sterilization; as well as the general ELISA procedure were conducted as described in the Materials & Methods section. The irradiation dose (electron beam) was 50 kGy.

(211) Results:

(212) Samples sterilized with 50 kGy beta irradiation maintain about 60% activity when protected with an amino acid postcoating containing only 7 amino acids; this effect is further enhanced with the addition of dipeptides such as Gly-Tyr or dipeptide combinations. Glycyrrhizic Acid does not enhance the protective effect of the amino acid dipeptide combination further.

Example 16: Interleukin-8 in Glass-Vials was Sterilized, the Vial Itself is the Carrier

(213) Experiment:

(214) Interleukin-8 (IL-8, R&D, 208-IL) was diluted in PBS (without Ca.sup.2+/Mg.sup.2+, PAA, H15-002) to 10 μg/mL. 5 μL of the solution (50 ng IL-8) were added to glass vials and rotated for 4 hours until dried. 25 μL of a stabilizing solution (20 g/L amino acid mixture and 1 mM glyzyrrhizic acid (ammonium salt, Fluke, 50531)) were added and rotated/dried overnight.

(215) The vials were sterilized with 25 kGy (beta irradiation). Unsterilized controls were stored under cool conditions.

(216) Assay:

(217) Neutrophile granulocytes were isolated from 10% ACDA whole blood. 20 mL ACDA blood (10%) were sedimented with 2 mL HES (Grifols 662650). The supernatant was pipetted to 7 mL Percoll (L6143) and centrifuged 20 min at 2000×g. The isolated granulocytes were resuspended in 1% autologous serum and set to a cell count of 0.5×10.sup.6/mL.

(218) As positive controls 5 μL IL-8-solution (50 ng) were dissolved in 25 μL of the stabilizing solution (A and B). To each sterile vial 1 mL PBS (with Ca.sup.2+/Mg.sup.2+, Hyclone, SH3026401) (with 1% autologous serum) were added to dissolve the dried film.

(219) To detect the chemotactic activity of the samples, the complete IL-8 solutions from the sterile and non-sterile vials and the controls were pipetted into 12-well-plates. Migration filters (3 μm, Corning, 3462) were inserted and 500 μL of the granulocyte suspension was pipetted into the filters. The plates were incubated for 30 min at 37° C. The number of migrated cells was detected by counting the cells in each well via FACS and counting beads (Invitrogen, C36950).

(220) Results:

(221) see FIG. 26

(222) The biomolecule (here interleukin 8=IL8) loses most of its biological function during subsequent sterilisation (25 kGy irradiation) if no stabilizer is added. In contrast, a with different amino acids protected the biomolecule. Shown is the chemotactic activity of IL8 on human neutrophil granulocytes.

Example 17: Interleukin-8 in Glass-Vials was Sterilized, the Stabilizer Itself is the Carrier

(223) Experiment:

(224) Interleukin-8 (IL-8) was diluted in PBS (without Ca.sup.2+/Mg.sup.2+, PAA, H15-002) to 10 μg/mL. 5 μL of the solution (50 ng IL-8) and 25 μL of a stabilizing solution (20 g/L amino acid mixture and 1 mM glyzyrrhizic acid (ammonium salt, Fluke, 50531)) were mixed and pipetted into glass vials. The vials were rotated/dried over night.

(225) The vials were sterilized by irradiation with 25 kGy. Unsterilized controls were stored under cool conditions.

(226) Assay:

(227) Neutrophile granulocytes were isolated from 10% ACDA whole blood. 20 mL ACDA blood (10%) were sedimented with 2 mL HES (Grifols 662650). The supernatant was pipetted to 7 mL Percoll (L6143) and centrifuged 20 min at 2000×g. The isolated granulocytes were resuspended in 1% autologous serum and set to a cell count of 0.5×10.sup.6/mL.

(228) As positive controls 5 μL IL-8-solution (50 ng) were dissolved in 25 μL of a stabilizing solution (A and B). To each sterile vial 1 mL PBS (with Ca.sup.2+/Mg.sup.2+, Hyclone, SH3026401) (with 1% autologous serum) were added to dissolve the dried film.

(229) To detect the chemotactic activity of the samples, the complete IL-8-solutions from the sterile and non-sterile vials and the controls were pipetted into 12-well-plates. Migration filters (3 μm) were inserted and 500 μL of the granulocyte suspension was pipetted into the filters. The plates were incubated 30 min at 37° C. The number of migrated cells was detected by counting the cells in each well (via FACS and counting beads).

(230) Results:

(231) see FIG. 29

(232) The biomolecule (here interleukin 8=IL8) loses most of its biological function during subsequent sterilisation (25 kGy irradiation) if no stabilizer is added. In contrast, a stabilizer solution with different amino acids protected the biomolecule. Shown is the chemotactic activity of IL8 on human neutrophil granulocytes.

Example 18: Anti-Mouse-IgG in Glass-Vials was Sterilized, the Stabilizer Itself is the Carrier

(233) Experiment:

(234) Anti-Mouse-IgG (biotinylated, Jackson ImmunoResearch, 115-065-003) was diluted in PBS (without Ca.sup.2+/Mg.sup.2+, PAA, H15-002) to 4 μg/mL. 25 μL (100 ng) of the antibody solution and 25 μL of a 2× concentrated stabilizing solution (20 g/L amino acid mixture and 1 mM glyzyrrhizic acid (ammonium salt, Fluke, 50531)) were mixed and pipetted into glass vials. The vials were rotated/dried overnight.

(235) The vials were sterilized by irradiation with 25 kGy. Unsterilized controls were stored under cool conditions.

(236) Assay:

(237) An ELISA plate (Greiner Bio-one, 655061) was coated with the antigen (mouse IgG, Innovativ Research, Ir-Ms-Gf): the antigen was diluted to 1 μg/mL, 100 μL were pipetted to each well and incubated over night at 4° C. The plate was washed twice with washing buffer (25× concentrate, Invitrogen, W802). The plate was blocked with Albumin (5%) and washed again 3 times.

(238) To all sample vials 200 μL PBS were added to dissolve the dried film (theoretically 5 μg/mL). The samples were diluted to 10 ng/mL with PBS. To calculate the antibody concentration a serial dilution of fresh antibody was prepared.

(239) The samples and standard were pipetted to the ELISA plate (2×200 μL each) an incubated 1 h at ambient temperature. The plate was washed 3×. To each well 200 μL Streptavidin solution (Horseradish peroxidase (HRP) labeled, Pierce, 21126, diluted to 0.1 μg/mL in PBS) were added and incubated 1 h at ambient temperature. The plate was washed 3×. HRP chromogenic substrate TMB (TMB=tetramethylbenzidine, Invitrogen, 00-2023) was diluted 1:2 in H2O and 200 μL were added to each well. The plate was incubated 15 min at ambient temperature and was protected from light. To stop the color reaction 50 μL diluted H2SO4 (diluted 1:5 with aqua dest., Merck, 1007311000) were added. The absorption of the plate was detected at 450 nm (Fusion Photometer A153601, PerkinElmer).

(240) Results:

(241) see FIG. 30

(242) The biomolecule (here an anti-mouse IgG antibody) loses most of its biological function during subsequent storage and or sterilisation (25 kGy irradiation) if no stabilizer is added. In contrast, a stabilizer solution with different amino acids protected the biomolecule. Shown is the specific binding to the antigen.

Example 19: Anti-Mouse-IgG was Sterilized, the Carrier is an Polyurethane Foam

(243) Experiment:

(244) From a fine porous polyurethan (PU) foam (Smith&Nephew, 66012608) samples with a defined diameter (1 cm) were punched. Anti-Mouse-IgG (biotinylated, Jackson ImmunoResearch, 115-065-003) was attached to the samples: the antibody was diluted to 5 μg/mL either in PBS (without Ca.sup.2+/Mg.sup.2+, PAA, H15-002) or in a stabilizing solution (20 g/L amino acid mixture and 1 mM glyzyrrhizic acid (ammonium salt, Fluke, 50531)) and the PU samples were covered with antibody solutions. The samples were incubated 1 h at 37° C.

(245) The antibody solution was removed and the PU samples were air dried for 2 h. The samples were sterilized via beta irradiation (25 kGy) and unsterile controls were stored under cool conditions.

(246) Assay:

(247) An ELISA plate (Greiner Bio-one, 655061) was coated with the antigen (mouse IgG, Innovativ Research, Ir-Ms-Gf): the antigen was diluted to 1 μg/mL, 100 μL were pipetted to each well and incubated over night at 4° C. The plate was washed 2× with washing buffer (25× concentrate, Invitrogen, W802). The plate was blocked with Albumin (5%) and washed again 3×.

(248) The PU samples were covered with PBS and incubated 1 h at ambient temperature. The sample solutions were collected and diluted 1:20 and further serial diluted 1:4 with PBS. To calculate the antibody concentration of the samples a serial dilution of fresh antibody was prepared.

(249) The samples and standard were pipetted to the ELISA plate (2×200 μL each) an incubated 1 h at ambient temperature. The plate was washed 3×. To each well 200 μL Streptavidin solution (Horseradish peroxidase (HRP) labeled, Pierce, 21126, diluted to 0.1 μg/mL in PBS) were added and incubated 1 h at ambient temperature. The plate was washed 3×. HRP cromogenic substrate TMB (TMB=tetramethylbenzidine, Invitrogen, 00-2023) was diluted 1:2 in H2O and 200 μL were added to each well. The plate was incubated 15 min at ambient temperature and was protected from light. To stop the color reaction 50 μL diluted H2SO4 (diluted 1:5 with aqua dest., Merck, 1007311000) were added. The absorption of the plate was detected at 450 nm (Fusion Photometer A153601, PerkinElmer).

(250) Results:

(251) The biomolecule (here an anti-mouse IgG antibody) loses most of its biological function during subsequent storage and or sterilisation (25 kGy irradiation) if no stabilizer is added. In contrast a stabilizer solution protected the biomolecule. The recovery of the antibody is almost 100% (5 μg/mL). Shown is the specific binding to the antigen.

Example 20: Anti-Mouse-IgG was Sterilized, the Carrier is an PVA Hydrogel

(252) Experiment:

(253) A 7% (m/v) solution of polyvinylalcohol (PVA, Sigma, 341584-25G) in water (heated to 85° C.) was prepared. The solution was cooled down to ambient temperature. Anti mouse IgG (biotinylated, Jackson ImmunoResearch, 115-065-003) was diluted to 200 μg/mL in PBS.

(254) The hydrogel mixture was composed as follows: 6.75 mL PVA solution (7%) 4.5 μL anti mouse IgG (200 μg/mL) 2.25 either PBS or stabilizing solution ((20 g/L amino acid mixture and 1 mM glyzyrrhizic acid (ammonium salt, Fluke, 50531)) in PBS)

(255) PVA hydrogels were poured into small petri dishes (diameter 35 mm, 2 mL solution). The hydrogel films were air dried for 48 h. The samples were sterilized via beta irradiation (25 kGy) and unsterile controls were stored under cool conditions.

(256) Assay:

(257) An ELISA plate (Greiner Bio-one, 655061) was coated with the antigen (mouse IgG, Innovativ Research, Ir-Ms-Gf): the antigen was diluted to 1 μg/mL, 100 μL were pipetted to each well and incubated over night at 4° C. The plate was washed 2× with washing buffer (25× concentrate, Invitrogen, W802). The plate was blocked with Albumin (5%) and washed again 3×.

(258) The PVA hydrogels were placed in 6 well plates and covered with 2 mL PBS (without Ca.sup.2+/Mg.sup.2+, PAA, H15-002). After 30 min, 1 h and 2 h the PBS was collected and replaced with fresh PBS.

(259) Serial dilutions of the samples and the standard were pipetted into the ELISA plate (2×200 μL each) and incubated for 1 h at ambient temperature. The plate was washed 3×. To each well 200 μL Streptavidin solution (Horseradish peroxidase (HRP) labeled, Pierce, 21126, diluted to 0.1 μg/mL in PBS) were added and incubated 1 h at ambient temperature. The plate was washed 3×. Chromogenic substrate TMB (TMB=tetramethylbenzidine, Invitrogen, 00-2023) was diluted 1:2 in H2O and 200 μL were added to each well. The plate was incubated 15 min at ambient temperature and was protected from light. To stop the color reaction 50 μL diluted H2SO4 (diluted 1:5 with aqua dest., Merck, 1007311000) were added. The absorption of the plate was detected at 450 nm (Fusion Photometer A153601, PerkinElmer).

(260) Results:

(261) The biomolecule (here an anti-mouse IgG antibody) loses most of its biological function during subsequent storage and or sterilisation (25 kGy irradiation) if no stabilizer is added. In contrast a stabilizer solution protected the biomolecule. The recovery of the eluted antibody is very high. Shown is the specific binding to the antigen.