Means and methods of sterilization of biofunctional compositions

Abstract

The present invention, inter alia, relates to a closed sterilized container comprising at least one carrier which is a stabilizer; and at least one biomolecule reversibly attached to the carrier, wherein said carrier partially or completely covers the attached biomolecules and wherein said at least one carrier is selected from the group consisting of (poly)peptides such as dipeptides or tripeptides, amino acids, polyalcohols, polyethyleneglycols, ionic liquids, compatible solutes, saponins and a mixture thereof. The invention also relates to methods for producing sterilized containers according to the invention and uses thereof.

Claims

1. A sterile container selected from vials, ampullas, cryocontainers, phials, flasks, bottles and bags, comprising a stabilizer composition acting as a carrier, at least one biomolecule selected from the group consisting of an antibody, enzyme, receptor, membrane protein, transport protein, blood coagulation factor, hormone, cytokine and function fragments of any of the foregoing, wherein the at least one biomolecule is reversibly attached to the carrier, and optionally a lid, wherein the stabilizer composition partially or completely covers the attached biomolecules, and comprises at least three different naturally occurring amino acids, and wherein the at least three different naturally occurring amino acids are not in the form of peptides.

2. The container according to claim 1, wherein the at least one biomolecule is attached to the carrier such that it can be released from the carrier prior to use.

3. The container according to claim 1, wherein the carrier is solid.

4. The container according to claim 1, wherein the carrier is semi-solid.

5. The container according to claim 1, wherein the carrier is solubilizable.

6. The container according to claim 3, wherein the solid carrier is porous.

7. The container according to claim 5, wherein the solubilizable carrier comprises proteinaceous and/or carbohydrate structures that dissolve in aqueous, optionally buffered solutions.

8. The container according to claim 1, wherein the at least one stabilizer is comprised in a buffered solution.

9. The container according to claim 1, wherein the at least one stabilizer is a mixture of at least four different naturally occurring amino acids.

10. The container according to claim 1, wherein said mixture comprises at least one amino acid of each group of (a) an amino acid with nonpolar, 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.

11. The container according to claim 9, wherein the amino acids comprised in said mixture are alanine, glutamic acid, lysine, threonine and tryptophan.

12. The container according to claim 9, wherein the amino acids comprised in said mixture are aspartate, arginine, phenylalanine, serine and valine.

13. The container of claim 1, wherein the container is a closable sterile container.

Description

(1) The figures show:

(2) FIG. 1

(3) A vial is shown comprising a carrier with the reversibly attached biomolecule and the protective coating. The biomolecule (e.g. cytokine) is embedded by the stabilizer solution and thereby protected against stress influences like dehydration during storage or irradiation during sterilization.

(4) FIG. 2

(5) Different carriers are shown: a) The carrier is a non dissolvable gel or a dissolvable gel b) The carrier is a non woven fibre network c) The carrier is a mesh or woven network d) The protective coating itself is the carrier and covers the biomolecule or the vial itself is the carrier e) The carrier has an open porous (like a sponge) or sintered structure f) The carrier comprises of nano-, micro- or macro-particle g) The vial itself is the carrier and has micro or macro structures that increase the surface (e.g. needle like structures).

(6) FIG. 3

(7) 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 both stabilizer solution A (Albumin and Mannitol) and B (solution with different amino acids) protected the biomolecule. Shown is the chemotactic activity of IL8 on human neutrophil granulocytes.

(8) FIG. 4

(9) 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 both stabilizer solution A (Albumin and Mannitol) and B (solution with different amino acids) protected the biomolecule. Shown is the chemotactic activity of IL8 on human neutrophil granulocytes.

(10) FIG. 5

(11) 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 both stabilizer solution A (Albumin and Mannitol) and B (solution with different amino acids) protected the biomolecule. Shown is the detectable DNA amount in percent of staring value.

(12) FIG. 6

(13) 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 both stabilizer solution A (Albumin and Mannitol) and B (solution with different amino acids) protected the biomolecule. Shown is the chemotactic activity of IL8 on human neutrophil granulocytes.

(14) FIG. 7

(15) 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 both stabilizer solution A (Albumin and Mannitol) and B (solution with different amino acids) protected the biomolecule. Shown is the specific binding to the antigen.

(16) FIG. 8

(17) 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 H.sub.2SO.sub.4 none of the other desorption solution tested in this experiment had a significant influence on the biological activity of the biomolecule. Shown is the specific binding to the antigen.

(18) FIG. 9

(19) 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 1 M NaCl+0.02 M imidazole and phosphate buffered saline, respectively. Shown is the specific binding to the antigen.

(20) FIG. 10

(21) 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.

(22) FIG. 11

(23) 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 (Albumin and Mannitol) protected the biomolecule. The recovery of the antibody is almost 100% (5 μg/ml). Shown is the specific binding to the antigen.

(24) FIG. 12

(25) The desorption of a biomolecule (here an anti-mouse IgG antibody) is tested after the biomolecule was attached to a 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 A (Albumin and Mannitol) and B (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.

(26) FIG. 13

(27) Chemical Structures of examples for cleavable linkers

(28) FIG. 14

(29) Anti-Hepatitis A test (functional ELISA) of an amino acid composition used for protecting anti Hepatitis antibodies after lyophilisation and sterilization.

(30) FIG. 15

(31) 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.

(32) FIG. 16

(33) 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.

(34) FIG. 17

(35) 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.

(36) FIGS. 18 and 19

(37) Amino acid postcoatings containing at least 5 amino acids provide protection during longtime 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.

(38) FIGS. 20 and 21

(39) 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 longtime 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.

(40) FIG. 22

(41) 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.

(42) FIG. 23

(43) Amino acid postcoating of amino acids, dipeptides or mixtures thereof, optionally together with glycyrrhizic acid.

(44) The examples illustrate the invention.

EXAMPLE 1

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

(45) Experiment:

(46) 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 (A=20 g/l albumin (Biotest Pharma) and 10 g/l mannitol (Serag Wiesner, 219675), B=20 g/l amino acid mixture and 1 mM glyzyrrhizic acid (ammonium salt, Fluka, 50531)) were added and rotated/dried over night.

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

(48) Assay:

(49) Neutrophil 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.

(50) 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.

(51) 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).

(52) Results:

(53) See FIG. 3

(54) 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 both stabilizer solution A (Albumin and Mannitol) and B (solution with different amino acids) protected the biomolecule. Shown is the chemotactic activity of IL8 on human neutrophil granulocytes.

EXAMPLE 2

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

(55) Experiment:

(56) 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 an stabilizing solution (A=20 g/l albumin (Biotest Pharma) and 10 g/l mannitol (Serag Wiesner, 219675), B=20 g/l amino acid mixture and 1 mM glyzyrrhizic acid (ammonium salt, Fluka, 50531)) were mixed and pipetted into glass vials. The vials were rotated/dried over night.

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

(58) Assay:

(59) Neutrophil 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.

(60) 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.

(61) 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).

(62) Results:

(63) See FIG. 6

(64) 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 both stabilizer solution A (Albumin and Mannitol) and B (solution with different amino acids) protected the biomolecule. Shown is the chemotactic activity of IL8 on human neutrophil granulocytes.

EXAMPLE 3

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

(65) Experiment:

(66) 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 an 2×concentrated stabilizing solution (A=20 g/l albumin (Biotest Pharma) and 10 g/l mannitol (Serag Wiesner, 219675), B=20 g/l amino acid mixture and 1 mM glyzyrrhizic acid (ammonium salt, Fluka, 50531)) were mixed and pipetted into glass vials. The vials were rotated/dried over night.

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

(68) Assay:

(69) 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, WB02). The plate was blocked with Albumin (5%) and washed again 3 times.

(70) 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.

(71) 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) labelled, 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).

(72) Results:

(73) See FIG. 7

(74) 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 both stabilizer solution A (Albumin and Mannitol) and B (solution with different amino acids) protected the biomolecule. Shown is the specific binding to the antigen.

EXAMPLE 4

Anti-Mouse-IgG was Sterilized, the Carrier is a Polyurethane Foam

(75) Experiment:

(76) From a fine porous polyurethane (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 albumin (Biotest Pharma) and 10 g/l mannitol (Serag Wiesner, 219675) in PBS) and the PU samples were covered with antibody solutions. The samples were incubated for 1 h at 37° C.

(77) 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.

(78) Assay:

(79) 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, WB02). The plate was blocked with Albumin (5%) and washed again 3×.

(80) 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.

(81) The samples and standard were pipetted to the ELISA plate (2×200 μl each) and 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 H.sub.2O 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).

(82) Results:

(83) 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 (Albumin and Mannitol) protected the biomolecule. The recovery of the antibody is almost 100% (5 μg/ml). Shown is the specific binding to the antigen.

EXAMPLE 5

Anti-Mouse-IgG was Sterilized, the Carrier is a PVA Hydrogel

(84) Experiment:

(85) 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.

(86) 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 albumin (Biotest Pharma) and 10 g/l mannitol (Serag Wiesner, 219675) in PBS)

(87) 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.

(88) Assay:

(89) 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, WB02). The plate was blocked with Albumin (5%) and washed again 3×.

(90) 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.

(91) 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 H.sub.2O 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 H.sub.2SO.sub.4 (diluted 1:5 with aqua dest., Merck, 1007311000) were added. The absorption of the plate was detected at 450 nm (Fusion Photometer A153601, PerkinElmer).

(92) Results:

(93) 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 (Albumin and Mannitol) protected the biomolecule. The recovery of the eluted antibody is very high. Shown is the specific binding to the antigen.

EXAMPLE 6

Protective Effect of a Specific Amino Acid Composition

(94) 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.

(95) Composition:

(96) 20 g Arginine

(97) 20 g Histidine

(98) 20 g Lysine

(99) 3 g Glutamic acid

(100) 2 g Tryptophan

(101) 20 g Glycine

(102) 15 g Alanine

(103) 0.2 g Tween 80

(104) 1 g Glycyrrhizic acid ammonium salt

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

(106) 400 μl of solution (corresponding to 40 mg solid compounds) were mixed with 125 μl Beriglobin (corresponding to 20 mg antibody)

(107) Lyophilisation:

(108) Lyophilisation was carried out as follows:

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

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

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

(112) 6 h drying over night at 6° C. and 0.004 mBar.

(113) Sterilisation:

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

(115) Results:

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

(117) 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.

(118) Conclusion:

(119) 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 7

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

(120) Materials & Methods

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

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

(123) Stress exposure of the coated surface

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

(125) Experiment:

(126) 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.

(127) Results:

(128) 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 8

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

(129) Materials & Methods

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

(131) Adsorption of LO-MM-3 to an EL ISA plate and application of postcoatings

(132) Stress exposure of the coated surface

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

(134) Experiment:

(135) 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.

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

(137) 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.

(138) Results:

(139) 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 9

A Preferred Amino Acid Composition Provides Protection Under Different Stress Conditions

(140) Materials & Methods

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

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

(143) Stress exposure of the coated surface

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

(145) Experiment:

(146) Compositions used:

(147) protective composition A (compound per liter)

(148) 20 g Arginine

(149) 20 g Histidine

(150) 20 g Lysine

(151) 3 g Glutamine

(152) 2 g Tryptophan

(153) 20 g Glycine

(154) 15 g Alanine

(155) 0.2 g Tween 80

(156) 1 g Glycyrrhizic acid ammonium salt

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

(158) 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.

(159) Results: 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 10

Amino Acid Postcoatings Provide Protection During Long-Time Storage

(160) Experiment:

(161) 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.

(162) 18 amino acids: Ala, Arg, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val

(163) 5 amino acids (1): Asp, Arg, Phe, Ser, Val

(164) 5 amino acids (2): Ala, Glu, Lys, Thr, Trp

(165) 2 amino acids (1): Asp, Val

(166) 2 amino acids (2): Ala, Glu

(167) 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.

(168) 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.

(169) Results:

(170) Amino acid postcoatings containing at least 5 amino acids provide protection during longtime 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. 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 11

Amino Acid Postcoatings Provide Protection During Different Sterilization Processes

(171) Experiment:

(172) 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.

(173) 18 amino acids: Ala, Arg, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val

(174) 5 amino acids: Asp, Arg, Phe, Ser, Val

(175) 2 amino acids: Asp, Val

(176) 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.

(177) 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.

(178) Results:

(179) 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 12

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

(180) Materials & Methods 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

(181) Experiment:

(182) 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.

(183) 7 amino acids: Arg, His, Lys, Glu, Trp, Gly, Ala

(184) 2 dipeptides: Gly-Tyr, Gly-Gln

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

(186) 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.

(187) Results:

(188) 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.