|HAO WANATA MARIAUS009797895B2 DEL MALA MAN UN CONT E (12 ) United States Patent ( 10 ) Patent No. : US 9 , 797 ,895 B2 Margraf et al. (45 ) Date of Patent: Oct . 24 , 2017

( 54 ) STABILIZING COMPOSITIONS FOR 2001/ 0049361 A1* 12 /2001 Yamashita ...... A61K 9/ 0019 514 /54 IMMOBILIZED BIOMOLECULES 2001/ 0055617 Al* 12/ 2001 Mattern A61K 38 / 1816 424 /489 (75 ) Inventors : Stefan Margraf, Frankfurt (DE ); Anja 2003/ 0031584 Al * 2 / 2003 Burgess A61L 2 /0011 Breuer , Babenhausen (DE ) ; Martin 422/ 22 Scholz , Oberursel (DE ); Jens 2003 /0118598 A1* 6 /2003 Hunt ...... A61K 9 /0019 424 / 184 . 1 Altrichter, Kavelstorf (DE ) 2003/ 0138402 A1 * 7 /2003 Yamashita ...... A61K 9 / 146 424 / 85 . 4 ( 73 ) Assignee : Leukocare AG , Munich (DE ) 2003 /0138437 A1 * 7 /2003 Hunt ...... A61K 8 /02 424 / 184 . 1 ( * ) Notice : Subject to any disclaimer, the term of this 2003 /0138460 Al * 7 /2003 Hunt 424 /239 . 1 patent is extended or adjusted under 35 2003/ 0215575 A1 * 11 / 2003 Martin et al...... 427 /407 . 1 U . S . C . 154 (b ) by 1194 days . 2003 /0232198 Al * 12 / 2003 Lamberti ...... A61L 27/ 26 428 /423 . 1 2004 /0022792 A1 * 2 /2004 Klinke et al ...... 424 / 178 . 1 ( 21 ) Appl. No. : 13 /262 ,542 2004 / 0033243 A1 * 2 / 2004 Metcalfe ...... A61K 38 / 2207 424 / 400 (22 ) PCT Filed : Mar. 31, 2010 ( Continued ) ( 86 ) PCT No. : PCT/ EP2010 /054391 $ 371 ( c ) ( 1 ), FOREIGN PATENT DOCUMENTS (2 ), ( 4 ) Date : Dec . 8 , 2011 CN 1409758 A 4 / 2003 CN 1522155 A 8 /2004 CN 101910202 A 12 /2010 ( 87 ) PCT Pub . No. : W02010 /115835 EP 1852443 * 11 /2007 CO7K 17 / 02 PCT Pub . Date : Oct . 14 , 2010 EP 1852443 A 11 / 2007 EP 2058335 * 5 / 2009 CO7K 17/ 02 EP 2058335 A 5 /2009 (65 ) Prior Publication Data JP S62194459 A 8 / 1987 JP 2003517153 A 5 / 2003 US 2012 /0107829 A1 May 3 , 2012 WO 2005 /083433 A 9 /2005 ( 30 ) Foreign Application Priority Data WO 2007128550 A1 11 /2007 Mar . 31 , 2009 (EP ) 09004734 OTHER PUBLICATIONS (51 ) Int. Ci. Cleland et al. , “ A specific molar ratio of stabilizer to protein is GOIN 33 /53 ( 2006 .01 ) required for storage stability of a lyophilized monoclonal anibody, " GOIN 33 /543 ( 2006 .01 ) J . Pharm . Sci ., Am . Pharm . Assoc. , Mar . 1 , 2001, pp . 310 -321 , vol. U . S . CI. 90 , No . 3 . (52 ) Polifke et al. , “ Langzeit - Stabilisierung von Assay -Komponenten , ” CPC ...... GOIN 33 / 54393 (2013 .01 ) Laborwelt , Nov. 1, 2007 , pp . 35 -39 , vol. 8 , No . 6 . ( 58 ) Field of Classification Search Vincken et al. , “ Saponins, classification and occurrence in the plant None kingdom ,” Phytochemistry , Pergamon Press , GB , pp . 275 - 297 , vol. See application file for complete search history . 68, No . 3 , 2007 . Durand - Fleith , Odette , International Search Report, PCT/ EP2010 / (56 ) References Cited 054391 , dated Oct. 13 , 2010 , European Patent Office . U . S. PATENT DOCUMENTS Primary Examiner — Ann Lam 4 ,340 ,589 A * 7 / 1982 Uemura ...... A61K 47 /02 ( 74 ) Attorney , Agent, or Firm — Gavrilovich , Dodd & 424 / 94 .63 Lindsey LLP 5 ,494 ,807 A * 2 / 1996 Paoletti ...... C07K 14 /005 424 / 199 . 1 5 ,547 , 794 A * 8/ 1996 Demizu ...... GO3G 9 /0804 (57 ) ABSTRACT 430 / 108 . 1 The present invention relates to the use of a composition 5, 730 ,933 A 3/ 1998 Peterson 5 ,780 , 295 A * 7 / 1998 Livesey et al ...... 435 / 307 . 1 comprising (a ) at least three different amino acids , ( b ) at 5 ,863 , 542 A * 1 / 1999 Paoletti ...... C07K 14 / 005 least two different amino acids and a saponin or ( c ) at least 424 / 188 . 1 one dipeptide or tripeptide for stabilizing biomolecules 6 , 197 ,291 B1 * 3 /2001 Terao ...... A61K 38 / 2053 immobilized on a solid carrier . The invention furthermore 424 /85 . 2 relates to a method for producing stabilized biomolecules , 6 ,309 ,647 B1 * 10 /2001 Paoletti C07K 14 /005 comprising embedding the biomolecules in the composition 424 / 186 . 1 according to the invention and a method of producing a solid 6 ,737 ,405 B2 * 5 /2004 Roemisch et al...... 424 / 130 . 1 6 ,770 ,729 B2 * 8 / 2004 Van Antwerp ...... A61K 31 /436 carrier having biomolecules attached thereto . The invention 424 /422 furthermore relates to a solid carrier producible or produced 7 ,052 , 875 B1 * 5 /2006 Terada ...... A61K 48 /0008 by the method of the invention and a method of diagnosing 435 /91 . 1 a disease using the carrier of the invention . 8 ,685 ,459 B2 * 4/ 2014 Altrichter ...... C12Q 1/ 22 422 / 22 21 Claims, 30 Drawing Sheets US 9 ,797 ,895 B2 Page 2

( 56 ) References Cited 2008 / 0161400 A1 * 7 / 2008 Virsik ...... A61K 31/ 05 514 /567 U . S . PATENT DOCUMENTS 2009/ 0093047 A1* 4 /2009 Klapproth et al ...... 435 / 288 . 7 2009/ 0130756 A1 * 5 /2009 Klann ...... A01N 1/ 02 2004 /0131620 Al * 7 / 2004 Maeda ...... CO7K 16 /06 435 /374 424 / 159 . 1 2009 /0226530 A1 * 9 /2009 Lassner ...... A61K 9 / 1605 2005 / 0136542 A1 * 6 / 2005 Todtleben et al. . 436 / 15 514 / 1. 1 2005 / 0169886 A1 * 8 / 2005 Brody ...... A61K 9 /0024 2009 /0269778 Al * 10 /2009 Margraf ...... CO7K 17 / 14 424 / 85 . 6 435 / 7 . 1 2006 / 0067950 A1 * 3 / 2006 Taylor ...... 424 / 239. 1 2009/ 0304628 A1* 12 /2009 Bello Rivero ...... A61K 9 / 19 2006 /0110379 A1 5 / 2006 Green et al. 424 /85 . 5 2006 /0160101 A1. * 77 // 2006 PouletPoulet ...... A61K 39 /118 435 / 6 . 16 2010 / 0233671 A1 * 9 / 2010 Bakaltcheva ...... A61K 9 / 0019 2007 / 0141641 A1 * 6 / 2007 Fang et al...... 435 / 7 . 2 435 / 2 2008/ 0031855 A1 * 2 / 2008 Okano ...... A61K 35 /768 2010/ 0291665 Al * 11/ 2010 Margraf ...... CO7K 17 / 14 424 / 93 . 6 435 /287 . 1 2008/ 0032962 A1* 2/ 2008 Heubes ...... A61K 9/ 0019 2012 /0093803 A1 * 4 /2012 Altrichter et al ...... 424 / 130 . 1 514 /202 2014 /0134699 A1* 5 /2014 Scholz C12N 7 / 00 2008 /0044928 A1 * 2 /2008 Char et al ...... 436 /524 435 / 174 2008 /0102524 A1 * 5 / 2008 Ueda ...... A61K 9 / 19 435 / 456 456 * cited by examiner atent Oct. 24 , 2017 Sheet 1 of 30 US 9, 797 , 895 B2

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STABILIZING COMPOSITIONS FOR proteins ( e . g . albumin ) in order to protect bio -molecules IMMOBILIZED BIOMOLECULES from degradation (Polifke T . & Rauch P , Laborwelt ( 2007 ) Vol. 6 , pp 1 - 4 ) . CROSS REFERENCE TO RELATED Stabilizers that contain serum - derived proteins have the APPLICATION 5 advantage that , in addition to their stabilizing effect , they also function as blocking agents to prevent unspecific bind This application is a U . S . National Stage Application filed ing . However, as albumins and the like are of human or under 35 U . S . C . $ 371 based upon International Application animal origin , they may contain pathogens like for example No. PCT/ EP2010 /054391 , filed Mar. 31, 2010 , which appli - viruses or prions . Therefore, complex cost and time inten cation claims priority to European Application No. 10 sive purification methods have to be applied in order to 09004734 . 1 , filed Mar. 31 , 2009 , the disclosures of which remove these pathogens . are incorporated herein by reference in their entirety . Another disadvantage of conventional methods for pre venting damage to immobilized biomolecules during stress SUMMARY like sterilization or long -term storage is that these methods 15 require that said biomolecules be frozen ( U . S . Pat . No. The present invention relates to the use of a composition 5 ,730 ,933 ; Cleland J. L . et al. , Journal of Pharmaceutical comprising ( a ) at least three different amino acids, ( b ) at Sciences (2001 ) Vol. 90 , No. 3 , pp . 310 - 321) . For example , least two different amino acids and a saponin or ( c ) at least U . S . Pat. No . 5 , 730 , 933 discloses a method for sterilizing one dipeptide or tripeptide for stabilizing biomolecules antibodies , by which the activity of these antibodies can be immobilized on a solid carrier. The invention furthermore 20 retained by freezing them during sterilization . However , relates to a method for producing stabilized biomolecules , freezing can lead to conformational changes of the biomol comprising embedding the biomolecules in the composition ecules that can affect their biofunctionality and constitutes a according to the invention and a method of producing a solid further step in the preparation of immobilized biomolecules carrier having biomolecules attached thereto . The invention causing additional costs . Furthermore , the stabilizing com furthermore relates to a solid carrier producible or produced 25 position contains serum proteins like albumin which are of by the method of the invention and a method of diagnosing animal origin and therefore bear the risk of contaminating a disease using the carrier of the invention . the biomolecules which are incubated therein . In this specification , a number of documents including Therefore , a need exists for improved methods and means patent applications and manufacturer ' s manuals are cited for stabilizing and protecting immobilized biomolecules that The disclosure of these documents , while not considered 30 are used in therapy and diagnosis . Thus , the object of the relevant for the patentability of this invention , is herewith invention is the provision of methods and means for the incorporated by reference in its entirety . More specifically , stabilization of biomolecules that avoid the disadvantages of all referenced documents are incorporated by reference to the state of the art. the same extent as if each individual document was specific cally and individually indicated to be incorporated by ref- 35 DETAILED DESCRIPTION erence . The present invention relates to the use of a composition BACKGROUND comprising at least two different amino acids for stabilizing biomolecules immobilized on a solid carrier. One major challenge when working with immobilized 40 In particular , the present invention relates to the use of a biomolecules as for example proteins like antibodies in composition comprising ( a ) at least three different amino diagnostic and therapeutic applications is to maintain the acids, ( b ) at least two different amino acids and a saponin activity of the biomolecules . However, since the chemical, and / or ( c ) at least one dipeptide for stabilizing biomolecules physical or physiological properties of bio - functional mol- on a solid carrier . ecules are often significantly altered by variations in the 45 A composition used according to the invention can be compounds ' surrounding environment, this is a difficult task . liquid or solid , depending on the intended use or state . A For example , changes in pH , ionic strength , temperature or composition covering and /or embedding the biomolecules storage can result in reversible or irreversible changes in the attached to a carrier according to the invention as described character of compounds. Especially biomolecules which are below is generally solid , whereas in the method of producing subjected to stress like e . g . long - term storage, transport or 50 a carrier of the invention as also described below , the sterilization procedures have to be stabilized in order to composition is usually liquid . After removal of the liquid prevent a loss of activity . part of the composition and / or drying , the composition For clinical immunodiagnostics , a sufficient stabilization covering and / or embedding the biomolecules attached to a of immobilized biomolecules that are used in diagnostic carrier according to the invention as described below is assays is required for the market entry of products according 55 again solid . If the composition is liquid , it is preferably to the ‘ EU In Vitro Diagnostics Guideline ' (IVDD 98 /79 / aqueous. EC ). Amino acids are defined as organic molecules that have a Implantable medical devices containing immobilized carboxylic and an amino functional group . They are the biomolecules , for example , are exposed to a wide variety of essential building blocks of proteins . In connection with the biological agents present in the tissues of the body, e .g . 60 present invention , the term “ amino acid ” refers to free amino acids , bases , ions and the like , depending on the location of acids which are not bound to each other to form oligo - or the implant in the body . Some of these agents can degrade polymers such as dipeptides , tripeptides , oligopeptides or the bio -molecules of the device leading to damage or even proteins. device failure . The amino acids that are contained in the stabilizing Commercially available carriers or devices containing 65 composition can be selected from naturally occurring amino immobilized biomolecule preparations therefore contain sta - acids as well as artificial amino acids or derivatives thereof. bilizers like sugars (e .g . trehalose ) and /or serum -derived Naturally occurring amino acids are e. g. the 20 proteino US 9 ,797 , 895 B2 genic amino acids glycine, proline , arginine , alanine , aspara - useful and applicable as pharmaceutically active agent( s ) , gine , aspartic acid , glutamic acid ( in connection with the diagnostic agent ( s ) and / or research tool( S ) . present invention , the terms aspartic acid and glutamic acid For the person skilled in the art , it is understood that the also include the salts of these amino acids ), glutamine , term “ biomolecules ” as applied in the present invention also cysteine, phenylalanine , lysine , leucine , isoleucine , histi- 5 comprises biomolecules as described above which are com dine , methionine , serine , valine , tyrosine , threonine and prised in or on structures such as eukaryotic or prokaryotic tryptophan . Other naturally occurring amino acids are e . g . cells , tissues , viruses as well as fragments thereof ( e . g . carnitine , ornithine , hydroxyproline , homocysteine , citrul organelles, membranes or capsids ) . In this embodiment of line, hydroxylysine or beta -alanine . the present invention , the biomolecules can be attached to Derivates of amino acids are e . g . n -acetyl - tryptophan , 10 the solid carrier in isolated form or comprised in or on said phosphonoserine , phosphonothreonine, phosphonotyrosine , eukaryotic or prokaryotic cells , tissues , viruses or fragments melanin , argininosuccinic acid and salts thereof or DOPA . thereof. Alternatively, the structures comprising the biomol Artificial amino acids are amino acids that have a different ecules , preferably on their surface , may serve as carriers side chain length and / or side chain structure and /or have the according to the present invention . amino group at a site different from the alpha - C - atom . 15 The term “ polypeptide ” interchangeably used with the The term “ stabilizing ” in the context of the present term “ protein ” as used herein describes a group ofmolecules invention relates to any effect of the composition used which comprises the group of polypeptides , consisting of according to the present invention resulting in the stabiliza - more than 30 amino acids . In contrast thereto , a molecule tion of the structure and / or activity of a ( immobilized ) consisting of up to 30 amino acids is designated “ peptide ” . biomolecule , the elongation of the shelf - life of a biomol- 20 Also in line with the definition the term “ peptide” describes ecule and /or the protection of a biomolecule against stress . fragments of proteins of a length of 30 amino acids or less . This results in a biological activity of the biomolecule which Polypeptides or peptides may further form dimers , trimers is retained to a significant degree. and higher oligomers , i. e . consisting of more than one The term “ stress ” as used in connection with the present polypeptide or peptide molecule . Polypeptide or peptide invention and as interchangeably used with the term " stress - 25 molecules forming such dimers , trimers etc . may be identi mediated damage” relates to any environmental influence cal or non - identical . The corresponding higher order struc resulting in a loss of activity of a biomolecule . Exemplary t ures are , consequently , termed homo - or heterodimers , stress factors are heat, drought or radiation , such as radiation homo - or heterotrimers etc . The terms " polypeptide ” , " pro induced by sterilization methods described below . Another tein ” and “ peptide ” also refer to naturally modified poly stress factor is the chemical environment. For example gases 30 peptides / proteins and peptides , wherein the modification is such as ethylene oxide used in sterilization can contribute to effected e . g . by glycosylation , acetylation , phosphorylation the loss of activity of a biomolecule . and the like . Such modifications are well known in the art . The term “ biological activity ” relates to a naturally occur The term “ nucleic acid ” or “ nucleic acid molecule ” , in ring activity of a biomolecule . Biological activities depend accordance with the present invention , includes DNA , such on the specific biomolecule and include binding affinity to 35 as cDNA or genomic DNA , and RNA such as antisense other (bio )molecules or catalytic activities. The biological RNA or siRNA . Further included are nucleic acid mimick activity of an antibody, for example , requires the specific ing molecules known in the art such as synthetic or semi binding of its antigen . The biological activity of a nucleic synthetic derivatives of DNA or RNA and mixed polymers . acid probe for example requires its ability to specifically Such nucleic acid mimicking molecules or nucleic acid hybridize to the nucleic acid target to which it is comple - 40 derivatives include phosphorothioate nucleic acid , phospho mentary . In this regard the term " retained to a significant ramidate nucleic acid , 2 - 0 -methoxyethyl ribonucleic acid , degree ” denotes a remaining biological activity of ( immo - morpholino nucleic acid , hexitol nucleic acid (HNA ) , locked bilized ) biomolecules having been exposed to stress as nucleic acid (LNA ) and peptide nucleic acid (PNA ) (see compared to immobilized ) biomolecules not exposed to Braasch and Corey , Chem Biol 2001, 8 : 1 ) . LNA is an RNA stress of at least 50 % , more preferably at least 60 % , more 45 derivative in which the ribose ring is constrained by a preferably at least 70 % and most preferably at least 80 % . methylene linkage between the 2 - oxygen and the 4 ' -carbon . The term " biomolecule ” describes any organic molecule Nucleic acid molecules may contain additional non - natural that can be or is produced by a living organism , including , or derivative nucleotide bases, as will be readily appreciated preferably biodegradable , polymeric molecules such as pro - by those skilled in the art . Nucleic acid molecules further teins or peptides , carbohydrates and nucleic acids as well as 50 include ribozymes , aptamers , plasmids and chromosomes . small molecules such as primary metabolites , secondary Nucleic acid molecules may be used in accordance with the metabolites, and natural products . The term “ biomolecule " present invention in isolated form or in complex with other not only comprises native molecules as they can be isolated biomolecules such as proteins , e . g . histone proteins or from a living organism but also naturally occurring or proteins of the ribosome. artificial molecules which are produced synthetically, semi- 55 The term " carbohydrate ” relates to an organic compound synthetically or recombinantly . Artificial molecules are e . g . that is an aldehyde or ketone with a number of hydroxyl those derived from naturally occurring molecules with groups added , usually one on each carbon atom that is not alterations introduced . Biodegradable polymeric molecules part of the aldehyde or ketone functional group . Depending other than those belonging to the above -mentioned classes on the length of the molecule, carbohydrates are termed are lignin and polyhydroxylalcanoates ( natural polymers ) 60 mono - , oligo - or polysaccharides . As soon as carbohydrates and polyalkylene esters , polylactic acid and its copolymers , are bound to non -carbohydrate molecules , the resulting polyamide esters , polyvinyl esters , polyvinyl alcohols and molecules are termed glycosides . Modified carbohydrates polyanhydrides ( artificial polymers ). Preferred biomolecules have for example N -acetylester , carboxyl - or sulfate - side exert properties which are relevant for pharmaceutical , diag - chains and may contain glucuronic acid , iduronic acid , nostic and /or scientific applications. In other words, the 65 galactosamine , glucosamine . biomolecules applicable in the present invention preferably Preferred biomolecules are proteins, peptides, nucleic exert a biological activity which makes them preferably acids and their derivatives , carbohydrates and their deriva US 9 ,797 ,895 B2 tives as well as lipids and fatty acids, polyalcohols and The antibody may be of any class of antibody . It is most combinations or modifications thereof. Examples of proteins preferred that the antibody is monoclonal and of the IgG , are antibodies or fragments thereof which retain their bind - IgM or IgY class . IgY antibodies represent the analogs of ing specificity , enzymes , receptors , membrane proteins (op - IgG antibodies in chicken . tionally without their transmembrane domain ), growth fac - 5 The term “ immobilized ” or “ immobilizing” as inter tors, albumins, globulins, cytokines, transport proteins, changeably used with the term " attached ” or “ attaching” blood coagulation factors and protein hormones . Exemplary relates to the fixation of the biomolecules on the carrier . Said carbohydrates are amylopectin , glycogen , starch , alpha - and immobilization or attachment or fixation may be irreversible beta - glucan , dextran , and glycosaminoglycans like or reversible . Fixation may be due to covalent or non hyaluronic acid , heparin , heparan sulfate , chondroitin sulfate covalent bonds formed between the material of the carrier and derivatives thereof such as glycosides . and the biomolecules. Exemplary non -covalent interactions Especially preferred proteins are antibodies or fragments include those leading to adsorption . The fixation of the thereof which retain their binding specificity . Antibodies biomolecule ( s ) to the carrier can be performed by any applicable in the present invention can be , for example , 15 technique known in the state of the art ( see for example , polyclonal or monoclonal. The term “ antibody ” also com - Hermanson , G . T ., Bioconjugate Technique (2008 ), 2na prises derivatives of antibodies which still retain their bind - edition ): ing specificity . Fragments of antibodies comprise , inter alia , The fixation may for example be effected via a direct Fab fragments , F ( ab ') 2 or Fv fragments . Techniques for the binding of the biomolecules to the solid carrier. Alterna production of antibodies and fragments thereof are well 20 tively , the fixation may be effected via an indirect binding known in the art and described , e . g . in Harlow and Lane via a third compound such as spacer and linker molecules , “ Antibodies , A Laboratory Manual ” , Cold Spring Harbor e . g . silanes or proteins such as biotin , avidin or streptavidin , Laboratory Press , 1988 and Harlow and Lane “ Using Anti- covering the solid carrier. The term " covering” as used bodies : A Laboratory Manual” Cold Spring Harbor Labo - throughout the present invention comprises full coverage as ratory Press , 1998 . These antibodies can be used , for 25 well as partial coverage of the solid carrier. In both alter example, for immunoprecipitation of molecules of interest natives, the binding can be a binding via a covalent or a or for removing unwanted molecules from body liquids of a non -covalent bond . A covalent bond can be achieved e . g . by patient. a chemical reaction between the bio -molecules and a carrier material or between the spacer / linker molecule and the The term “ antibody ” also includes embodiments such esas 30 biomolecules . Examples for non - covalent binding comprise synthetic , chimeric , single chain and humanized antibodies weak bonds such as van - der- Waal' s bonds or other polar or derivatives or fragments thereof which still retain their bonds . Such non -covalent bonds occur e . g . between poly binding specificity . Various procedures are known in the art peptides or peptides and a solid carrier with a polyethylene and may be used for the production of such antibodies and / or surface . fragments . Further , techniques described for the productionproduction 35, In a preferred embodiment , the biomolecules are revers of single chain antibodiesles can bebe adapted to produceproduce singlesingle ibly attached on said solid carrier. chain antibodies specifically binding to a molecule of inter The term “ reversibly attached " defines that the biomol est or fragments thereof . Also , transgenic animals may be ecules , when attached to the carrier , can be released from used to express humanized antibodies . Most preferably, the said carrier with suitable means. Depending on the kind of antibody is a monoclonal antibody . For the preparation of 40 attachment, e . g . of whether the attachment is covalent or monoclonal antibodies, any technique that provides antibod non -covalent , differentmeans of releasing the biomolecules ies produced by continuous cell line cultures can be used . are applicable . Examples are cleavage by proteases in the Examples for such techniques include the hybridoma tech - case of proteins as biomolecules, a pH change or a change nique (Kohler and Milstein Nature 256 ( 1975 ), 495 -497 ) , the in temperature . The biomolecules are attached to the carrier trioma technique , the human B - cell hybridoma technique 45 until needed and only then released from the carrier . (Kozbor , Immunology Today 4 ( 1983 ) , 72 ) and the EBV . The techniques mentioned above are only examples for hybridoma technique to produce human monoclonal anti - immobilizing or reversibly attaching biomolecules on solid bodies ( Cole et al ., Monoclonal Antibodies and Cancer carriers . It is emphasized that the present invention is not Therapy, Alan R . Liss, Inc . ( 1985 ) , 77 - 96 ) . Surface plasmon limited to these examples. Instead , any conventional method e used 50 known in the state of the art can be applied to immobilize or resonance as employed in the BIAcore system can be used 30 reversibly attach the biomolecule (s ) on a solid carrier . to increase the efficiency of phage antibodies which bind to The reversible attachment of the biomolecule is chosen in an epitope of a biomolecule of interest ( Schier, Human such a way that the biomolecule can be quickly released Antibodies Hybridomas 7 ( 1996 ), 97 - 105 ; Malmborg , J . from the carrier. In this regard , the term " quickly ” means Immunol. Methods 183 (1995 ) , 7 - 13 ) . It is also envisaged in 55 that more than 50 % of said biomolecule , such as 60 % , 70 % the context of this invention that the term “ antibody ” com or 80 % , can be released within 2 hours or less , such as 1 prises antibody constructs that may be expressed in cells , hour, 30 minutes or 20 minutes, in any combination such as e . g . antibody constructs which may be transfected and/ or 60 % in 30 minutes or 20 minutes , 70 % in 30 minutes or 20 transduced via , amongst others, viruses or plasmid vectors . minutes or 80 % in 30 minutes or 20 minutes, preferably Once the antibody or fragment thereof has been obtained , 60 more than 85 % within 10 minutes or less and most prefer the antibody itself or the DNA encoding it can be sequenced ably more than 98 % within 1 minute . This can be achieved providing for the information to recombinantly produce the e. g . by applying one of the methods described below . antibody or fragment thereof in small or large scale . Meth . Furthermore , the reversible attachment of the biomolecules ods of the production of a recombinant antibody are known is preferably chosen in such a way that the biomolecules are to the person skilled in the art . 65 released immediately before the clinical application . The antibody or derivative or fragment thereof can be Reversible attachment , like irreversible attachment, can further chemically modified as is well known in the art . be covalent or non -covalent . US 9 ,797 ,895 B2

Preferably, the non - covalent bonds are non -covalent the latter that are derived from humans or animals, the amino bonds with high affinity and specificity . Examples for such acids of the stabilizing composition used according to the non -covalent bonds are those formed by the streptavidin - present invention can be produced synthetically . Therefore , biotin or the avidin -biotin system . In this example , strepta - the risk of contamination by pathogens like viruses or prions vidin /avidin is coupled covalently to a suitable carrier. The 5 is reduced or avoided . biotinylated biomolecule then is bound non - covalently but Besides its protective effect on biomolecules , the stabi with high affinity to the streptavidin / avidin . By adding lizing composition also was found to have the additional excess biotin , the binding is competitively suppressed and effect of blocking free binding sites on the solid carrier the biotinylated biomolecule is released . having biomolecules attached thereto . The biomolecule may be attached via a linker , preferably 10 Furthermore, as confirmed in the appended examples, the a cleavable linker . Suitable linkers can be selected , but are stabilizing composition used according to the invention is not limited to universally applicable : it is suitable for the protection of a ) linkers with disulfide- bridges like SDAD (NHS - SS - Di- different molecules such as antibodies (e .g . IgM , IgG ), azirine ), SulfoSAND , DSP that can easily be cleaved by the enzymes ( e . g. DNAse ) or nucleic acids. addition of reagents with SH groups like thiols ,mercap - 15 The method for stabilizing biomolecules of the present tanes, cysteine, mecaptoethanol or dithiothreitol. invention has the further advantage that, unlike conventional b ) linkers with peptide bonds which can be cleaved with a stabilization methods , it does not require the freezing of the specific protease , preferably a human enzyme biomolecules . Thus , conformational changes that can occur c ) linkers which are cleavable via ultrasound during the freezing process can be avoided . Therefore, the d ) linkers with ester bonds like EGS that can be cleaved by 20 stabilization method of the present invention is especially e . g . hydroxylamine suitable for instable or labile biomolecules, like e . g . anti e ) linkers with Sulfons like BSOCOES that can be cleaved bodies, especially the complex structure of IgM molecules. at higher ph ( e. g . pH 11. 6 ) Further surprisingly , it was found that by incubating d ) linkers with cis - Diols like DST that can be cleaved by biomolecules such as antibodies in the stabilizing composi sodium -meta - periodate . 25 tion of the present invention , the ability of these biomol Alternatively , the biomolecules may be reversibly ecules to exert their biological activity , e . g . in the case of attached by drying as indicated above and as described in antibodies to bind their antigen , can be retained to at least detail further below in connection with the methods of the 65 % when being sterilized by irradiation with 25 kGy as invention . In this embodiment, reversible attachment is compared to untreated controls . achieved in that the biomolecules and the molecules used as 30 That is , with the stabilizing composition of the present a stabilizer ( i. e . amino acids, optionally in connection with invention , a significant increase of retention of functionality a saponin and /or optionally in connection with at least one of the embedded biomolecules can be achieved compared to di- and /or tripeptide as described ) and the solid carrier stick conventional stabilizers as e . g . disclosed in U . S . Pat . No . together after drying . Release of the biomolecules takes 5 , 730 , 9333 when being irradiated with 25 kGy. place upon addition of a liquid to the above compounds thus 35 The composition used according to the present invention dissolving / solubilising the biomolecules and the stabilizing preferably does not contain proteins or fragments of proteins molecules from the carrier. which are not amino acids, dipeptides and / or tripeptides. The term “ solid carrier" defines in the context of the Accordingly , in this preferred embodiment of the present invention a carrier of solid material. The material of the invention , the composition does not contain proteins or carrier may be either of compact or porous structure . As 40 fragments thereof consisting ofmore than three amino acids , described herein below , it is preferred that the carrier is of a nor hydrolysed proteins that are of human or animal origin . material selected from the group consisting of glass, medical Such a composition has the advantage that there is no risk of grade stainless steel, metal alloys ( e . g . chrome cobalt contaminating the bio -molecules embedded therein . It is molybdenum , titan nitride oxide ) , hydroxyapatite , silicone , furthermore a cost effective alternative to known stabilizing polystyrene , poly - L - lactic acid ; polyurethane , polyester , 45 compositions. polysulfone , polyethylene , polypropylene , polyacryl, poly In a preferred embodiment of the present invention , the acrylnitrile , polyamide, PMMA , fleece wadding , open stabilizing composition comprises between 2 and 18 differ porous foam plastic or glass and reticular plastic or glass and ent amino acids, more preferably 2 to 10 , even more structures derived from marine sponges ( porifera ) . preferably 2 to 8 and most preferably 2 to 5 or 5 to 8 different In the course of the present invention , it was surprisingly 50 amino acids. Alternatively , the composition comprises at found that biomolecules immobilized on a solid carrier can least 2 , at least 3 , at least 4 , at least 5 , at least 6 , at least 7 be stabilized by applying a composition comprising ( a ) at or at least 8 different amino acids or more such as at least 9 , least three different amino acids , ( b ) at least two different at least 10 , at least 11 , at least 12 , at least 13 , at least 14 , at amino acids and a saponin or ( c ) at least one dipeptide . This least 15 , at least 16 , at least 17 , at least 18 , at least 19 or at composition covers or embeds the biomolecules and shields 55 least 20 different amino acids . The number of different them from adverse influences . Analogous to the term “ Cov - amino acids comprised in the composition preferably does ering ” , the term “ embedding ” as used throughout the present not exceed 18 . invention comprises partial or complete embedding of the As apparent from the appended examples, the composi biomolecules by the composition used according to the tion according to the invention when used for stabilizing invention . As shown in the appended examples , immobi - 60 immobilized biomolecules exerts its advantageous effects lized biomolecules covered by or embedded in the compo - already when two or three different amino acids are present. sition used according to the invention show a marked The effect then gradually increases and the maximum effect increase in resistance to stress such as ageing or sterilization is reached when five to eight different amino acids are while essentially retaining their biological activity. present in the stabilizing composition . Depending on the A major advantage of the stabilizing composition of the 65 procedure applied for testing the stabilizing effect , the present invention is that amino acids and not serum - derived maximum effect is reached when 5 different amino acids are proteins like e . g . albumins, are used as stabilizers . Unlike present (such as for enhanced ageing) or when eight different US 9 ,797 , 895 B2 10 amino acids are present ( such as for sterilization ). The amino acids than alanine , glutamic acid , lysine , threonine advantageous effects do not or do not essentially decrease and tryptophan are comprised in the composition . with the addition of further different amino acids to the In an alternative preferred embodiment, the amino acids stabilizing composition used according to the invention but comprised in the composition are aspartic acid , arginine , are preferably retained . Accordingly , also compositions 5 phenylalanine , serine , valine . Similarly , to the above , comprising at least 9 , at least 10 , at least 11 such as at least whereas in certain embodiments of the present invention , the 12 , at least 13 , at least 14 , at least 15 , at least 16 , at least 17 , composition according to the invention comprisesmore than at least 18 , at least 19 or at least 20 different amino acids can the above 5 amino acids, in a further preferred embodiment be used for stabilizing immobilized biomolecules . no other amino acids than aspartate , arginine , phenylalanine , In a preferred embodiment, the composition comprises at 10 serine , valine are comprised in the composition . least 4 or at least 5 amino acids . In another preferred embodiment, the amino acids com As apparent from the examples , the composition accord prised in the composition are proline, serine , asparagine , ing to the invention , when comprising at least 4 or at least aspartic acid , threonine and phenylalanine ; or tyrosine , 5 amino acids has an even more advantageous effect on the isoleucine , leucine , threonine and valine ; or arginine, gly stability of biomolecules immobilized on a solid carrier. 15 cine , histidine , alanine, glutamic acid , lysine and tryptophan . Depending on the combination of amino acids , the residual The last combination of amino acids , i . e . arginine , glycine , biological activity after stress exposure amounts to up to histidine , alanine , glutamic acid , lysine and tryptophan , has 87 % . been shown to be especially advantageous with regard to its In a further preferred embodiment, the composition com properties after sterilization , in particular after irradiation . prises at least one amino acid of each group of ( a ) an amino 20 Said combination of amino acids was shown not to exert any acid with non polar, aliphatic R groups; (b ) an amino acid displeasing odor or discoloration after irradiation . with polar , uncharged R groups; ( c ) an amino acid with Again , whereas in certain embodiments of the present positively charged R groups; ( d ) an amino acid with nega invention , the composition according to the invention com tively charged R groups; and ( e ) an amino acid with aro - prises more than 5 or 6 or 7 amino acids, in a further matic R groups. 25 preferred embodiment no other amino acids than those of the The naturally occurring amino acids can be classified into combinations listed in this preferred embodiment are com the above characteristic groups (Nelson D . L . & Cox M . M ., prised in the composition . ‘ Lehninger Biochemie ’ ( 2005 ) , pp . 122 - 127 ) from each of In another preferred embodiment , the composition further which at least one amino acid is selected for the composition comprises less than 1 % , more preferably less than 0 . 3 % according to the invention . Also other than naturally occur - 30 Tween , preferably Tween 80 . ring amino acids such as artificial amino acids can be Tween is a generic term for polysorbates which are a class classified accordingly . Whereas more than one amino acid of of emulsifiers and surfactants used in some pharmaceuticals each group such as at least two or at least three can be and food preparations. They are often used to solubilize oily comprised in the composition according to the invention , it ingredients into water- based products . Polysorbates are oily is presently preferred that only one amino acid is selected 35 liquids derived from PEG -ylated sorbitan ( a derivative of from each group. The skilled person further understands that sorbitol) esterified with fatty acids . Examples for polysor not the same number of amino acids of each group has to be bates are polysorbate 20 ( Tween 20 or polyoxyethylene (20 ) present in the composition used according to the invention . sorbitan monolaurate ) , polysorbate 40 ( Tween 40 or poly Rather, any combination of amino acids can be chosen as oxyethylene (20 ) sorbitan monopalmitate ) , polysorbate 60 long as at least one amino acids of each group is present. 40 ( Tween 60 or polyoxyethylene (20 ) sorbitan monostearate ), In a preferred embodiment, the composition comprises and polysorbate 80 ( Tween 80 or polyoxyethylene (20 ) less than 1 % , preferably less than 0 . 5 % by dry weight sorbitan monooleate ) . Tween 80 is most preferred in the cysteine within the mixture of at least two or at least three compositions used in the present invention . amino acids . This also applies to compositions comprising at In the course of the present invention , it has been found least 4 , at least 5 , at least 6 , at least 7 , at least 8 , at least 9 45 that the addition of less than 1 Tween ( preferably related to or at least 10 amino acids or even more such as at least 11 , the dry mass of biomolecules and amino acids ) avoids at least 12, at least 13 , at least 14 , at least 15 , at least 16 , at foaming during handling of the liquid composition accord least 17 , at least 18 , at least 19 or at least 20 different amino ing to the invention . acids . As indicated above , the present invention also relates to Due to the SH group comprised in cysteine , compositions 50 the use of a composition comprising at least one di- and /or comprising cysteine may be subjected to oxidation taking tripeptide for stabilizing biomolecules immobilized on a place e . g . at the SH groups and resulting in displeasing solid carrier. The definitions given above and the preferred odors and / or possible the composition changing color to embodiment described above , where applicable , mutatis shades of brown . In order to minimize these undesired mutandis apply to this embodiment of the present invention . effects , in particular when the composition is used in con - 55 Accordingly , the composition may be comprised of at least nection with medical devices, the amount of cysteine com - two, at least three , at least four, at least five , at least six , at prised in the composition should be reduced as described least seven , at least eight, at least nine or at least ten different above . However, even if these effects are undesired , they do di - and / or tripeptides . Exemplary dipeptides are glycylglu not affect the suitability of compositions of the invention tamine (Gly -Gln , displaying an enhanced stability as com comprising a higher percentage of cysteines or otherwise 60 pared to glutamine alone ) , glycyltyrosine (Gly - Tyr ), alanyl exerting brownish colors or a displeasing odor. glutamine (Ala -Gln , the latter two displaying an increased In an especially preferred embodiment, the amino acids solubility in water as compared to tyrosine alone ) and comprised in the composition are alanine , glutamic acid , glycylglycine . Further naturally occurring dipeptides are lysine , threonine and tryptophan . Whereas in certain Carnosine (( beta -alanyl - L -histidine ) , Anserine (beta -alanyl embodiments of the present invention , the composition 65 N -methyl histidine ) , Homoanserine ( N - ( 4 - Aminobutyryl) according to the invention comprises more than the above 5 L -histidine ), Kyotorphin ( L - tyrosyl- L -arginine ), Balenine amino acids, in a further preferred embodiment no other (or ophidine ) (beta -alanyl - N tau -methyl histidine ), Glorin US 9 ,797 , 895 B2 ( N - propionyl - y - L - glutamyl- L -ornithine - 8 - lac ethyl ester ) acid with two amino acid residues and a free 30 - COOH and Barettin ( cyclo - [ (6 - bromo - 8 - en -tryptophan )- arginine ]) . function and conjugates of at least one residue of amino acid Further artificial dipeptides are Aspartame ( N - L - a - aspartyl alkyl esters in the carbohydrate part of the glycyrrhizic acid L -phenylalanine 1 -methyl ester ) and pseudoproline. Exem molecule . Examples of specific derivatives can be found e . plary tripeptides are Glutathione ( Y - glutamyl- cysteinyl- gly - 5 g . in Kondratenko et al. (Russian Journal of Bioorganic cine ) and its analogues Ophthalmic acid ( L - y - glutamyl- L - Chemistry , Vol 30 ( 2 ), (2004 ) , pp . 148 - 153 ) . - aminobutyryl- glycine ) and Norophthalmic acid In the course of the present invention , it has surprisingly ( y - glutamyl- alanyl- glycine ) . Further tripeptides are Isoleu cine- Proline -Proline ( IPP ), Glypromate (gly - pro -glu ), Thy been found that upon addition of glycyrrhizic acid to the rotropin -releasing hormone ( TRH , thyroliberin or protirelin ) 10 above described composition , biomolecules retain even ( L -pyroglutamyl - L -histidinyl - L -prolinamide ) , Melanostatin more of their biological activity than by using compositions ( prolyl- leucyl - glycinamide ) , Leupeptin ( N - acetyl- L - leucyl comprising only amino acids as stabilizing agents . In this L - leucyl- L - argininal) and Eisenin ( pGlu -Gln - Ala - OH ) . It is regard , it is particularly remarkable that glycyrrhizic acid preferred that the at least one tripeptide and more preferred alone does not exert any stabilizing or protective effect on all tripeptides used as a stabilizer , when used in connection 15 immobilized biomolecules . with medical applications according to the invention ( see In another preferred embodiment, the stabilizing compo below ) do not exert any pharmacologicalological propertiesproperties. The sition further comprises a polyalcohol, preferably a sugar or composition according to this embodiment of the invention sugar alcohol which is selected from xylose , mannose , preferably does not contain proteins or fragments of proteins glucose , fructose , lactose , maltose , sucrose , trehalose , which are not amino acids, dipeptides or tripeptides . Accord - 20 raffinose , dextran , mannitol, sorbitol or inositol. ingly , in this preferred embodiment of the present invention , In an additional preferred embodiment, the liquid embodi the composition does not contain proteins or fragments ment of the composition is a preferably buffered , preferably thereof consisting of more than three amino acids. Instead , aqueous solution but can also be water or a non buffered , the composition according to this embodiment preferably preferably aqueous , salt solution . The buffer to be used further comprises at least one amino acid , preferably at least 25 depends, inter alia , on the biomolecule which is to be two , more preferably at least three , even more preferably at covered/ embedded . Buffers generally suitable for the con least four , at least five , at least six , at least seven , at least tact with biomolecules are e . g . phosphate , citrate , acetate , eight, at least nine , at least ten different amino acids or more borate , carbonate , lactate , ammonium , glycine , barbiturate , such as at least eleven , at least 12 , at least 13 , at least 14 , at HEPES , MOPS , MES , TRIS . Exemplary buffers suitable for least 15 , at least 16 , at least 17 , at least 18 , at least 19 or at 30 antibodies are described further below . least 20 different amino acids. In another embodiment, the present invention relates to a In a preferred embodiment, the composition used in method for stabilizing biomolecules or for producing stabi accordance with the invention comprises a saponin . lized biomolecules , comprising (a ) attaching the biomol Saponins are a class of chemical compounds forming ecules to a solid carrier and ( b ) embedding the biomolecules secondary metabolites which are found in natural sources, 35 in the composition according to the invention . Possible ways derive from natural sources or can be chemically synthe - of immobilizing have been described above and can be sized . Saponins are found in particular abundance in various applied in the present method . Preferably , in step b ), the plant species . Saponins are amphipathic glycosides grouped biomolecules are covered or embedded partially or com phenomenologically by the soap - like foaming they produce pletely in the composition . when shaken in aqueous solutions, and structurally by their 40 The detailed procedure of embedding is described further composition of one or more hydrophilic glycoside moieties below in connection with the method of the invention of combined with a lipophilic triterpene derivative . Examples producing a solid carrier, in particular step ( b ) thereof, but of saponins are glycyrrhicic acid , glycyrrhetinic acid , glu - optionally also steps ( c ) , ( d ) and /or ( e ) . curonic acid , escin , hederacoside and digitonin . It is pre - The present invention furthermore relates to a method of ferred that the saponin used as a stabilizer , when used in 45 producing a solid carrier having biomolecules attached connection with medical applications according to the thereto , comprising the steps of invention ( see below ) do not exert any pharmacological ( a ) attaching the biomolecules to the solid carrier ; and properties . (b ) incubating the carrier of step ( a ) in the composition In a more preferred embodiment, a saponin is comprised described according to the invention . in a composition comprising any number of different amino 50 The terms " attaching ” and “ immobilizing ” in connection acids comprised in the above list of minimum numbers of with the biomolecules have been defined and the details of amino acids, such as in a composition comprising at least 3 , the procedure have been described above . Said definition at least 4 , at least 5 , at least 6 , at least 7 , at least 8 , at least and description equally apply to the methods of the present 9 or at least 10 different amino acids or even more , such as invention and other embodiments described below . In par at least eleven , at least 12 , at least 13 , at least 14 , at least 15 , 55 ticular, the attachment of the biomolecules can be reversible at least 16 , at least 17 , at least 18 , at least 19 or at least 20 or irreversible as defined above . different amino acids . The term “ incubating ” refers to an incubation under In a more preferred embodiment, the saponin is glycyr - conditions that allow the compounds recited in step ( a ) to rhizic acid (also : glycyrrhicic acid ) , also known as glycyr - attach to the carrier and the biomolecules attached to the rhizin or glycyrrhizinic acid or a derivative thereof. 60 solid carrier in step ( a ) to be embedded in the stabilizing Derivatives of glycyrrhizic acid are well -known in the art composition recited in step (b ). In this embodiment, the and include those produced by transformation of glycyr - composition to be used for incubation with the solid carrier rhizic acid on carboxyl and hydroxyl groups, by conjugation is liquid and preferably an aqueous solution . The composi of amino acid residues into the carbohydrate part or the tion may have been originally a solid composition . In this introduction of 2 - acetamido - B - D - glucopyranosylamine into 65 embodiment, the originally solid composition is dissolved in the glycoside chain of glycyrrhizic acid . Other derivatives a preferably aqueous medium and thus is comprised in a are amides of glycyrrhizic acid , conjugates of glycyrrhizic preferably aqueous solution . US 9 ,797 , 895 B2 13 14 Thereby a postcoating layer is formed above and /or (c ) subjecting the solid carrier to drying . around the biomolecules which stabilizes the biomolecules , Drying can be effected using well -known techniques such inter alia , by reducing their accessible surface and by as air -drying , spray -drying , lyophilisation and precipitation . associating with complementary areas of their tertiary struc - Further suitable techniques comprise crystallization and ture . Whereas the composition is liquid upon application to 5 microcrystallization . In the case of beads or nanoparticles as the biomolecules , its is later dehydrated resulting in a solid a carrier, drying can be effected by lyophilisation whereas composition embedding or covering the biomolecules. The for other carriers other drying techniques are preferred . biomolecules can be embedded partially or completely in the In an alternative preferred embodiment of the invention , composition in step b ). The skilled person understands that the method further comprises a step ( c ) after step (b ): incubation conditions need to be adapted to the biomol- 10 ( c ) drying the carrier until the residual liquid content is ecules attached to the solid carrier. Incubation conditions include those where incubations are < 20 % ( w / w ) . effected from 20 minutes to 12 hours depending on the step In accordance with this preferred embodiment of the and the temperature . Antibodies, for example, can be incu invention the residual liquid content may be calculated in a bated for 1 hour at 37° C ., which is the maximum tempera - 15 manner as known for the determination of the water content ture where IgM antibodies are stable . IgG antibodies can be of wood . In the case of wood , the percentage of water incubated to up to 50° C . The temperature range may be content of wood ( x ) is determined by the calculation of the from 4° C . to 50° C . , preferably 20° C . to 37° C ., depending ratio between the mass of the water in the sample ( m ) and on the step and the incubation time. the mass of the water containing wood sample ( m ,, ), mul In a preferred embodiment, incubation in step (b ) is 20 tiplied with 100 . The mass of a water in the sample ( m ) can effected for 1 hour at room temperature . It is understood that be determined by subtraction of the mass of the sample after in order to expedite the procedure or to improve the results , its desiccation from the mass of the water containing wood the incubation times and temperatures can be varied accord sample ( m .) . Accordingly , the percentage of water content ing to the substances used in the incubation . The skilled of wood is calculated by the following formula : artisan is aware that the term “ room temperature ” can imply 25 different temperatures depending on the location and the outside temperature. It usually ranges between 17° C . and X ( % ) = ** * * * 100 23° C . mu Preferably , the liquid composition in step ( b ) is buffered and more preferably of low salt content. A low salt content 30 according to the present invention is defined as a salt The residual water contentof a preparation of carriers can concentration of 0 . 9 % ( w / w ) or less , preferably less than be determined in analogy, wherein m ,, , is the mass of the 0 . 2 % ( w / w ) . Generally , but not exclusively , for antibodies water in the sample of carriers and m ,, is the mass of the such as IgG and IgY antibodies, a more alkaline buffer, e . g . sample of carriers after the complete desiccation of the made of 15 mM sodium carbonate and 35 mM sodium 35 sample . In case of a spongiform carrier, my is determined hydrogencarbonate in water ( pH 9 ), is useful whereas for the after squeezing the excess water out of the pores. attachment of IgM a more neutral buffer (pH 7 . 0 to 7 . 4 ) , e . g . In another preferred embodiment of the invention , the a phosphate buffer like PBS, is favorable . method further comprises a step ( a ') after step ( a ) : A solid carrier resulting from step ( a ), that is a solid carrier (a ') drying the carrier until the residual liquid content of the onto which biomolecules are attached , is also termed 40 composition is less than 10 % , preferably less than 5 % , “ coated ' carrier in the context of the present invention . more preferably less than 2 % such as less than 1 % , 0 . 5 % A solid coated carrier resulting from step ( b ) , that is a or 0 . 2 of the originally applied composition . Preferred solid carrier which is incubated with the composition drying methods are described above and include also according to the invention covering and / or embedding the methods which are used for removing the composition . It attached biomolecules , is also termed ' postcoated ' carrier in 45 is most preferred that drying is effected by air - drying , the context of the present invention . spray -drying , lyophilisation , precipitation , crystallization In a preferred embodiment, the method further comprises or microcrystallization . a step (c ): The method of the invention may further comprise a step ( c) removing the composition applied in step (b ) . ( a " ) subsequent to step ( a ) and prior to step ( b ) and option Steps (a ), ( b ) and (c ) are carried out in the above described 50 ally after step (a ') : order. (a " ) incubating the carrier in a buffered aqueous solution The term “ removing ” refers to the qualitative or quanti comprising a blocking agent and removing the aqueous tative removal of the liquid composition after step (b ) . Thus , solution . the composition is removed at least to a degree where the blocking agent may be used in the method for the remaining amount of the composition does not significantly 55 production of a solid carrier in order to prevent unspecific change the quality of a solution used in a subsequent step of binding ofmaterial added in subsequent steps of the method . the method of the invention . Quantitative removal results in This blocking may also have a positive effect on the con drying of the carrier. The removal may be effected e . g . by formation stability of the biomolecules before the incubation suction , e . g . by using a conventional pump to which a of the carrier in the liquid composition according to the pipette may be attached , and the like , compression or 60 invention in step ( b ) . blowing . Optionally , such steps are combined and may be In this regard , the composition according to the invention further combined with air drying . Preferably , the solution is can be used as blocking agent. Other blocking agents in line removed in volume to at least 95 % such as at least 98 % or with the present invention comprise but are not limited to at least 99 % , 99 . 5 % or 99 .8 % . human serum and proteins of such sera ( e . g . albumin ) , milk , Accordingly , in a more preferred embodiment, where 65 egg proteins, plant derived proteins ( e . g . soya , wheat ) quantitative removal is effected , themethod of the invention including hydrolysates of said proteins ( e . g . gelatin , colla comprises step ( c ): gen ). US 9 ,797 ,895 B2 15 present invention have been described herein above in the invention further comprises a step ( e ) of sterilizing the solid context of the characterization of the method of the inven carrier after step ( b ), ( c ) or ( d ) . The ability to sterilize the tion . solid coated carrier produced by the method of the invention According to a preferred embodiment of the solid post allows, inter alia , the production of the solid coated carriers 5 coated carrier of the invention , the biomolecules that are under non - sterile or semi- sterile conditions, wherein the so attached to the solid carrier are e . g . proteins , peptides , produced carriers may still be used in the method of the nucleic acids , carbohydrates , lipids, fatty acids , polyalcohols invention described below which involves the contact of the and combinations or modifications thereof as described solid coated carrier with a body liquid in vivo , ex vivo or in above . Proteins are preferably antibodies , more preferably vitro . This represents a further superior feature of the 10 antibodies of the IgG , IgY or IgM class . Other preferred method and the so produced carrier of the invention since , proteins are cytokines , proteinaceous agonists or antagonists due to that feature, the costs for the production of the solid other than antibodies such as enzymes , growth factors , carrier can be reduced compared to costs for the production albumins or globulins , receptors , hormones , membrane pro of carriers which may not be sterilized . This is because , in teins, albumins, globulins , transport proteins or blood the production process , sterile conditions are not required . 15 coagulation factors . As it is well known, conventionally prepared carriers are not in another more preferred embodiment, the biomolecules suitable for sterilization and must be produced under com - specifically bind to a marker protein indicative for a disease , pletely sterile conditions . Moreover, a cost contributing a pathogen , preferably a non - cellular pathogen , a cell or a feature is that said carriers may be recycled after their use by toxin . The term " specifically binds” , interchangeably used sterilization . The recycled carriers may subsequently be 20 with “ specifically interacts with ” , in accordance with the used in a further treatment of the same or a different patient present invention means that the biomolecule does not or or in a further or the same method for diagnosing . Prefer - essentially does not cross -react with other structures or ably , the sterilization or recycling of the carrier is effected by epitopes than those of the pathogen or marker protein ethylene oxide ( EO ) , beta radiation , gamma radiation , X -ray , indicative for a disease . Preferred biomolecules of this heat inactivation , autoclaving or plasma sterilization 25 embodiment are antibodies as described above . Cross - reac depending on the carrier material. It is most preferred that tivity of a panel of antibodies under investigation may be the carrier is sterilized by B - or y - radiation . Suitable in this tested , for example , by assessing binding of said panel of embodiment is B -radiation with a dose of 25 kgray using an antibodies under conventional conditions to the structure / electron accelerator with 10 MeV . To a certain extent ster - epitope of interest as well as to a number of more or less ilization with ethylene oxide can be applied to the carrier of 30 ( structurally and/ or functionally ) closely related structures! the invention . In general, the method of sterilization has to epitopes . Only those antibodies that bind to the structurel be chosen in order not to harm the desired activity of the epitope of interest in its relevant context ( e . g . a specific biological material attached to / immobilized on the solid motif in the structure of a protein indicative for a disease ) carrier . This can be effected with the biomolecules as defined but do not or do not essentially bind to any of the other above , in isolated form , in complex with each other or other 35 structures/ epitopes are considered specific for the structurel biomolecules or present on higher order structures such as epitope of interest and thus to be antibodies to be used in cells or fragments of cells . For living cells of any kind , accordance with this invention . Corresponding methods are suitable means of sterilization are not known to date . Thus, described e . g . in Harlow and Lane , 1988 and 1999 , loc cit . embodiments where living cells are used as the biological Examples for diseases for which marker proteins exist material and the carrier is sterilized are not part of this 40 comprise severe hyperlipidemia of various origin , homozy invention . gous familial hypercholesterolemia , heterozygous familial After step ( b ) , ( c ) , ( d ) or ( e ), the carrier may be stored hypercholesterolemia , defective Apo B - 100 , isolated Lipo until needed . protein ( a ) elevation , systemic lupus erythematosus (SLE ) , In another embodiment, the present invention relates to a Sjögren ' s syndrome, mixed connective tissue disease , solid carrier producible or produced by the method of the 45 dilated cardiomyopathy (DCM ) , diseases associated with invention . inhibitors to coagulation factors , idiopathic thrombocy In a further embodiment, the present invention relates to topenic purpura (ITP ) , thrombotic thrombocytopenic pur a solid carrier having biomolecules attached thereto , pura (TTP ) , autoimmune hemolytic anemia , hyperviscosity wherein said biomolecules are partially or completely cov - syndrome in hypergammaglobulinemia , myasthenia gravis , ered by / embedded in the composition according to the 50 Guillain - Barré syndrome, chronic inflammatory demyelinat invention . One embodiment of such a solid carrier, also ing polyneuropathy (CIDP ) , dysproteinemic polyneuropa termed “ post- coated carrier ” , comprising embedded biomol thies , bone marrow transplantation , endocrine orbitopathy, ecules is exemplified in the cross - section depicted in the diabetes mellitus type I ( IDDM ) , Goodpasture ' s syndrome, scheme of FIG . 1 . nephropathies due to immunoglobulin or immune complex In connection with the present invention the terms " par - 55 deposits , cryoglobulinemia , pemphigus , atopic dermatitis , tially covered ” or partially embedded ” preferably denote graft - versus- host (GvH ) diseases, host- versus - graft (HVG ) that at least 20 % of the biomolecules are covered or embed diseases , and various forms of vasculitis . ded in the composition according to the invention , prefer - Well- known marker proteins for specific diseases are e . g . ably at least 30 % , at least 40 % , at least 50 % , at least 60 % , pro - calcitonin , cytokines such as TNFa or IL6 , and c -reac at least 70 % , at least 80 % , more preferred at least 90 % , at 60 tive protein . least 95 % , at least 98 % or at least 99 % . The pathogens to which the biomolecules may bind Definitions and explanations given in connection with the include those causing infectious diseases, such as bacteria or composition according to and the methods of the invention fungi. Non -cellular pathogens causing infectious diseases and , inter alia , relating to biomolecules , carrier materials include viruses and prions. attachment and incubation procedures or sterilization muta - 65 Examples for such pathogens are HIV , HBV, HCV, Her tis mutandis apply to the solid carrier of the invention . Also , pesviruses such as e . g . the Herpes simplex , the Cytomegalo the particular superior features of the solid carrier of the and the Varicella zoster viruses, gram -positive bacteria e. g . US 9 , 797 , 895 B2 17 18 Streptococci, Staphylococci , gram -negative bacteria e . g . E . stored for an extended period (from one to five years ) coli , Pseudomonas aeruginosa . without any decrease in performance that may affect safety Also toxins may be indicative of a disease or a certain and efficacy when the products are used . Because full state of the body . A toxin is a poisonous substance , prefer period , ambient- aged samples usually do not exist for such ably produced by living cells or organisms. Toxins can be e . 5 products , it is generally necessary to conduct ' accelerated g . small molecules , peptides , or proteins that are capable of aging ' tests to provide experimental data in support of causing a disease on contact with or absorption by body performance and shelf - life claims for these products until tissues interacting with biological macromolecules such as full- period samples become available . enzymes or cellular receptors . Toxins vary greatly in their The primary reason for using accelerated -aging tech severity , ranging from usually minor and acute ( as in a bee 10 niques in the qualification testing of a medical device is to sting ) to almost immediately deadly ( as in botulinum toxin ) . bring the product to market at the earliest possible time. The In another preferred embodiment of the present invention , goal is to benefit both the patient - for example , through the solid post- coated carrier is sterilized as described above early availability of a life - enhancing device and the com for the use and the method of the invention , e . g . by beta or pany — by generating additional sales and market shares gamma irradiation . Suitable is e . g . beta - irradiation with a 15 without exposing either to any undue risk . dose of 25 kGy using an electron accelerator with 10 meV . Accelerated aging can be defined as a procedure that The solid carriers according to the present invention can seeks to determine the response of a device or material under be used in therapy and diagnosis . normal- usage conditions over a relatively long time, by A further aspect of the invention relates to the use of the subjecting the product for a much shorter time to stresses composition according to the invention for stabilizing solid 20 that are more severe or more frequently applied than normal carriers having biomolecules attached thereto for long - term environmental or operational stress . A significant enhance storage and / or sterilization . ment in test plan efficiency can be achieved using excessive A major challenge in the long -term storage and steriliza - environmental stress such as heat, oxygen , chemicals , or tion of biomolecules is to avoid irreversible changes to radiation . biomolecules having a desired biological activity . These 25 A simplified approach for accelerated aging is based on changes can result in molecular modifications of biomol- testing at a single accelerated temperature and then employ ecules that occur when the latter are subjected to stress, ing the rule stating that the rate of a chemical reaction will further resulting in a loss of the desired biological activity of increase by factor Qe for every 10° C . increase in tempera said biomolecules , in their reduced stability upon storage or ture . The typical relationship selected for commonly used in new immunological / antigenic properties which might put 30 medical polymers is Q10 = 2 ; that is , a doubling of the the recipients of such products at risk of allergic reactions reaction rate for each 10° C . increase in the temperature upon administration or application . This is especially impor above use or storage temperature . tant for proteins which are of complex structure and / or quite In order to achieve a sufficient shelf life for biomolecules labile , like e . g . many therapeutic proteins and monoclonal that are used for diagnosis or therapy , these biomolecules antibodies . 35 have to be frozen in order to prevent their denaturation Researchers have attempted to avoid irreversible changes (Cleland et al. , ( 2001 ) , Journal of Pharmaceutical Sciences that can occur to active compounds during sterilization by Vol. 90 , pp . 310 - 321) . Furthermore , the biomolecules of using ethylene oxide. However , ethylene oxide often reacts interest have to be incubated in stabilizing solutions con with proteins . In addition , because of the known tissue taining so -called lyoprotectants such as sugars ( e . g . sucrose , toxicity and the carcinogenic potential of the by -products of 40 trehalose , mannitol) or salts . However, due to the risk that ethylene oxide , the Food and Drug Administration has set biomolecules are denatured by freezing , a need exists for maximum residue limits for ethylene oxide and its major storage methods at 4° C . or at room temperature , especially reaction products ethylene glycol and ethylene chlorohydrin . for unstable biomolecules like e . g . IgM antibodies . One advantage of the present invention is that biomolecules Surprisingly it was found that by incubating biomol are protected by the composition according to the invention 45 ecules , e . g . antibodies, immobilized on a solid carrier in the so that sterilization can also be effected using ethylene stabilizing composition according to the present invention , oxide . these biomolecules can be stored up to 7 days at 45° C . Unlike ethylene oxide , radiation sterilization has the without significant loss of their biological activity . Accord advantages of high penetration ability , relatively low chemi- ing to the rule described earlier , this accelerated aging ' cal reactivity , and instantaneous effects without the need to 50 equals to a storage of 16 weeks at 5° C . Furthermore , the control temperature , pressure , vacuum or humidity . Radia - biomolecules can be sterilized without significant loss of tion sterilization is widely used in industry for a variety of their biological activity ( see appended examples) . products and both dosage levels and its biological effects are In another embodiment, the present invention relates to well known. It is generally agreed that electron - beam and medical and diagnostic products and devices comprising the gamma sterilization are equally effective in killing microbial 55 carrier of the invention . organisms. In a further alternative embodiment, the present invention While sufficient to effectively kill microorganisms, the provides the use of a solid carrier of the invention for the radiation generally alters the structure of biomolecules like preparation of medical or diagnostic products and / or proteins , DNA , RNA etc . as to render them biologically devices. Such devices being a combination of the biological inactive , if they are not protected or stabilized . Therefore , 60 component comprising the biomolecules stabilized accord there has been a significant need for a simple way to ing to the invention and a non - biological part are also termed effectively and safely sterilize biologically active com - biological- device combination product . pounds without deleteriously affecting their chemical, physi Solid carriers having biomolecules attached thereto that cal or physiological properties . This has been achieved in are embedded in / covered by the stabilizing composition accordance with the present invention . 65 according to the present invention can be used in a variety The introduction of new or modified products to the of diagnostic and medical/ therapeutic applications . Thera medical marketplace requires the assurance that they can be peutic applications or devices include medical implants , US 9 ,797 ,895 B2 19 20 catheters , stents , tubings, alone or in combination with other responds, at least in part , to a solid coated carrier of the components , matrices capable of eluting the attached invention or to which solid coated carriers of the invention biomolecules upon wetting with a liquid such as a body are attached . liquid , e . g . wound dressings , or medical devices used in This includes osteosynthesis devices , materials used for extracorporeal circulation . Diagnostic applications comprise 5 reconstructive surgery etc . as well as novel classes of stents . for example immunoassays like ELISA ( enzyme linked SuchSu novel classes of stents may have catalyzing features ( e . g . stimulatory, inhibitory or eliminating features) . In this immunosorbent assays ), protein chips, multi- analyte assays , regard , an exemplary stimulatory feature could be a carrier western blots , dot blots , immunohistochemistry , receptor bound antibody or fragment thereof that cross - links recep ligand assays and immuno - PCR . 10 tors on a cell surface and that can activate cells such as The therapy approaches may comprise in vivo as well as leukocytes . An exemplary inhibitory feature is a carrier ex vivo applications of the carrier of the invention in the bound antibody or fragment thereof which binds to receptors context of a medical device . Accordingly , a device compris on the cell surface and which can inactivate cells by either ing the solid post -coated carrier can be implanted into a inducing inactivating signals ( e . g . effecting leukocytes) or patient for an in vivo application . For an ex vivo applicationpucation 15 by preventing the binding of an activating molecule . AnAn a device comprising the solid coated carrier can be con example for an eliminating feature is a carrier- bound anti nected with the circulation of a body liquid to be treated . body or fragment thereofwhich can bind soluble factors and Blood derived from an artery or a vein of a patient may be thus reduce their concentration in the solution , e . g . deplete e . g . led through such device and subsequently piped back said factors ( e . g . Ig - Therasorb® or LDL - Therasorb® ) . into the patient ( connection with the blood stream ) . Alter - 20 Moreover, further alternative embodiments of the medical natively , samples of a body fluid may be incubated with the or diagnostic device are devices the interior walls of which carrier in vitro . In a subsequent step of the latter treatment are solid coated carriers of the invention or to which solid the body fluid can be reintroduced into the body of a patient. coated carriers of the present invention are attached . Such One embodiment of the medical device is suitable to devices include , but are not limited to ex vivo vaccination enable a contacting , filtering or cleaning of body fluids like 25 containers , apheresis devices, stem cell isolation devices , e . g . blood , lymph or liquor cerebrospinalis of a patient e . g . purging devices , syringes and devices for transient or per by connecting the device with the circulation of the body manent blood storage ( e . g . in blood banks , but also labora fluid of a patient and thereby treating a patient as described tory equipment in routine research laboratories ). An herein above. example for a diagnostic (medical ) device is an ELISA plate Moreover, the device may be suitable for the qualitative 30 wherein the surface of the plate (or at least of the reaction or quantitative detection of compounds as well as for wells ) corresponds, at least in part , to a solid coated carrier trapping certain cells or cell populations, e . g . stem cells , of the invention or has attached thereto solid coated carriers fetal cells or tumor cells , in a sample of a body fluid of a of the present invention . patient as also described in part herein below . Stem cells The present invention also relates to a sterilized container, comprise pluripotent, multipotent or totipotent stem cells . In 35 comprising a carrier, at least one biomolecule reversibly general, different cell lines , cell species or developmental attached to the carrier, the stabilizing composition according stages of cells can be distinguished by the presence of to the invention which partially or completely covers the different antigens on the cell surface . This enables for attached biomolecules ; and optionally a lid . The biomol selective trapping of the desired cells by choosing a biomol- ecules attachable to the carrier have been described above . ecule such as an antibody or fragment thereof attached to the 40 In a preferred embodiment, the at least one biomolecule carrier of the invention , wherein said biomolecules specifi - is attached to the carrier such that it can be quickly released cally binds to one of said antigens . In the case of trapping from the carrier , preferably immediately prior to use . fetal cells, application of the device of the invention avoids Depending on the kind of attachment, covalent or non potentially dangerous techniques such as amniocentesis in covalent, a quick release can e . g . be obtained by proteolytic that a blood sample obtained from the mother is used for 45 cleavage , changes in the pH value or temperature . trapping fetal cells without endangering the fetuses ' life . In a preferred embodiment, the carrier is solid , more Furthermore, the diagnosis of a cancerous disease and its preferably porous . In another preferred embodiment, the characterization is facilitated by the provision of enriched carrier is semi- solid and preferably comprises a hydrogel or tumor cell samples . a gelatinous protein mixture . In yet another preferred The term “ patient” as used throughout the present inven - 50 embodiment , the carrier is soluble and preferably comprises tion comprises ill or diseased as well as healthy subjects . A proteinaceous and / or carbohydrate structures that dissolve in patient in accordance with the present invention is any aqueous , optionally buffered solutions . person who receives medical attention , care , or treatment. The present invention further relates to a method for The person is most often but not always ill or injured and , producing a sterilized container for biomolecules, compris if so , in need of treatment by a physician or other medical 55 ing : ( a ) inserting a carrier into a container ; ( b ) reversibly professional. In other terms, the term " patient " is inter - attaching at least one biomolecule to said carrier ; ( c ) incu changeably used with “ subject” which may or may not be ill. bating the carrier with the at least one reversibly attached The subject can be an animal , preferably a mammal, most biomolecule in the liquid stabilizing composition according preferably a human . In accordance with the above , a patient to the invention , such that the at least one biomolecule is is also , for example , a healthy human who is , on an acute or 60 partially or completely covered by said stabilizing compo routine basis , diagnosed for a disease or health status. In sition ; ( d ) sealing the container; and ( e ) sterilizing the other terms, the invention may be used to find out whether container. a patient suffers from a certain disease (or a combination of In an alternative embodiment, the present invention diseases ) , is prone to develop such a disease or combination relates to a method for producing a sterilized container for thereof or not. 65 biomolecules, comprising : ( a ) reversibly attaching at least One embodiment of the device includes transient or one biomolecule to a carrier , ( b ) inserting the carrier with the permanent implants characterized by a surface which cor- at least one attached biomolecule into a container , ( c ) US 9 , 797 , 895 B2 21 22 incubating said carrier with the at least one reversibly body liquid of the patient with antibody covered solid attached biomolecule in the liquid stabilizing composition carriers of the invention under physiological conditions. according to the invention , such that the at least one biomol Physiological conditions include a pH value between 7 .0 ecule is partially or completely covered by said stabilizing and 7 . 7 and an osmolarity of 200 to 400 mosmo1/ 1 . composition , ( d ) sealing the container ; and ( e ) sterilizing the 5 Detection of whether the pathogen or marker protein container. indicative for the disease has been bound to the biomol In a further alternative embodiment, the present invention ecules can be effected by methods well known in the art and relates to a method for producing a sterilized container for described above . Exemplary methods comprise ELISA and biomolecules, comprising : (a ) reversibly attaching at least immunoprecipitation . one biomolecule to a carrier, ( b ) incubating the carrier with 10 In a preferred embodiment , the diagnostic method of the the at least one reversibly attached biomolecule in the liquid invention comprises the step of incubating the material of a stabilizing composition according to the invention , such that sample of the body liquid under cell culture conditions in the at least one biomolecule is partially or completely order to enrich a non - cellular pathogen such as a virus, cells covered by said stabilizing composition , ( c ) inserting the such as a bacterium , or a single cell eukaryotic pathogen carrier with the at least one reversibly attached biomolecule 15 prior to contacting the sample with the carrier of the inven into a container, ( d ) sealing the container; and ( e ) sterilizing tion ( step ( a ) ) . Cell culture conditions include a pH value the container. between 7 . 0 and 7 . 7 and an osmolarity of 200 to 400 The above methods may further comprise a step of drying mosmol/ l in a culture medium at 25 to 40° C . or removing the liquid part of the composition after incu . Moreover, the invention provides a diagnostic composi bation as described above . 20 tion comprising a solid coated carrier that is post- coated In a different embodiment, the present invention relates to according to the present invention . The diagnostic compo a container produced by the method of the present invention . sition of the invention will preferably be used for the Furthermore the invention relates to the use of a container diagnosis of a disease using methods described above in the of the invention for diagnostic or therapeutic applications as context of detection of marker proteins, preferably non described above . 25 cellular , pathogens , cells and toxins . In a further embodiment the invention provides a method Examples for diseases to be diagnosed with the diagnostic for diagnosing a disease comprising the steps of: composition of the invention have been described herein ( a ) contacting a sample obtained from a patient with a solid COMabove . carrier of the invention under suitable conditions to allow The invention also relates to a kit for the stabilization of specific binding of the biomolecules attached to the 30 immobilized biomolecules. The kit according to the inven carrier which specifically bind a marker protein indicative tion includes ( a ) at least one stabilizing composition accord for a disease , a , preferably non - cellular pathogen , a cell or ing to the present invention and (b ) instructions for contact a toxin to said pathogen or marker protein or cell or toxin ; ing immobilized biomolecules with an effective amount of and the at least one stabilizing composition to produce a stabi ( b ) detecting whether said marker protein indicative for the 35 lized biomolecule composition . disease , said preferably non - cellular pathogen , said cell or In a preferred embodiment, the stabilizing components said toxin has been bound to the biomolecules. include one or more solid materials (e .g . lyophilized or Samples include body liquids or fluids such as blood , powdered reagents or other compounds which are known in plasma, serum , saliva , urine , bronchoalveolar fluid , stomach the state of the art ) . and intestine secretion , cerebrospinal fluid , ascites , pleural 40 The various components of the kit may be packaged in effusion , exudate from wounds. one or more containers such as one or more vials . The vials Suitable conditions to allow specific binding of the may , in addition to the components , comprise preservatives biomolecules attached to the carrier which specifically bind or buffers for storage . a pathogen or marker protein indicative for a disease to said pathogen or marker protein include a pH value between 7 . 0 45 BRIEF DESCRIPTION OF THE DRAWINGS and 7 . 7 and an osmolarity of 200 to 400 mosmol/ l. Preferred biomolecules have been described above . Par - The figures show : ticularly preferred biomolecules comprise membrane bound FIG . 1 : A biomolecule , e . g . an antibody is attached to a and intracellular biomolecules such as receptors as well as solid carrier, preferably by use of spacer or linker molecules . soluble biomolecules or receptors or functional fragments 50 The antibody is embedded by the postcoating solution and thereof. Preferred functional fragments of membrane bound thereby protected of stress influences like irradiation . biomolecules contain the intracellular or the extracellular FIGS. 2 and 3 : The protective effect of amino acid portion of said biomolecules thus omitting their transmem - postcoatings increases with the number of different amino brane fraction . Preferably , the biomolecule or receptor is an acids used ( independent of the amino acid ) . The amino acids antibody or a fragment or derivative thereof retaining its 55 were selected as representatives of the following groups : binding activity and specificity as described above . Most amino acids with aromatic /positively charged / negatively preferably , the antibody is a monoclonal antibody . charged /polar non - charged /non polar aliphatic side -chain . Examples for diseases to be diagnosed with the diagnostic With postcoatings consisting of 5 different amino acids method of the invention comprise the diseases described after irradiation with 25 kGy approx . 65 % of the antigen herein above . The recited pathogen or marker protein may 60 binding ability compared to the not irradiated control is e .g . be detected by the use of antibodies like e. g . anti -p24 maintained ; after accelerated aging procedure (7 days at 45° (HIV ) ( see e . g . Schupbach et al. , J . Aquir . Immune Defic . C . ) approx . 85 % of the antigen binding ability compared to Syndr. ( 2005 ), Vol. 40 , pp . 250 - 256 ) or anti -IgB (HCMV ) the untreated control is maintained . ( see e . g . Just- Nubling G . et al. , Infection ( 2003 ) , 318 -323 ). FIGS. 4 and 5 : The protective effect of amino acid Suitable conditions for specific binding of a pathogen or 65 postcoatings cannot be further enhanced by increasing the marker protein indicative of the disease to the attached number of amino acids above 5 . After irradiation with 25 antibody may be achieved by contacting a sample of the kGy about 70 % antigen binding ability compared to US 9 , 797 ,895 B2 23 24 untreated control is maintained with all postcoatings con Sterilized samples (beta , 25 kGy ) maintain about 70 % of taining more than 5 amino acids; after accelerated aging ( 7 their antigen binding ability after 62 days at 45° C . Amino days at 45° C .) about 85 % is maintained . acid postcoatings containing only 2 amino acids maintain FIGS . 6 and 7 : The combination of amino acids shows a only about 40 % antigen binding ability during the storage protective effect of 65 % ( 4 amino acids) respectively 85 % ( 8 5 process, regardless of sterilization . Without postcoating only amino acids ), whereas the average effect of the single amino 20 - 30 % activity is preserved . acids is 22 % ( 4 amino acids ) respectively 33 % ( 8 amino FIGS . 20 and 21: The addition of 1 mM glycyrrhizic acid acids ) antigen binding ability compared to the unsterilized to the postcoating solutions enforces the protecting effect . control. The maximal effect of single amino acids is at 28 % Amino acid postcoatings containing at least 5 amino acids ( 4 amino acids) respectively 55 % (8 amino acids ) . 10 and glycyrrhizic acid provide protection during long time FIG . 8 : The addition of 1 mM Glycyrrhizic acid to amino storage . For the non sterilized samples, after 62 days at 45° acid postcoatings enhances the protective effect of all post C . about 90 % of the antigen binding ability is preserved . coatings to a maximum value of 70 - 80 % ( antigen binding Sterilized samples (beta , 25 kGy maintain about 80 % of abilityFI? compared0 . An amino to not acid irradiated postcoating control consisting) . of five 15 their antigen binding ability after 62 days at 45° C . Amino different amino acids ( 200 mM ) and 1 mM glycyrrhizic acid acid postcoatings containing 2 amino acids and glycyrrhizic shows a protective effect of 80 % antigen binding ability acid maintain about 70 % antigen binding ability during the compared to the untreated control after irradiation ( 25 KGy ) storage process , regardless of sterilization . Without post and 85 % after accelerated aging procedure , respectively . An coating only 20 - 30 % activity is preserved . postcoating consisting of albumin and mannitol shows only 20 FIG . 22 : Samples sterilized with gamma irradiation main 65 % remaining activity of after irradiation (25 kGy ) . After tain about 85 % activity when protected with an amino acid accelerated aging for 7 days at 45° C . there is almost no postcoating containing 18 amino acids; this effect is not activity remaining with albumin and mannitol, similar to the further enhanced with glycyrrhizic acid ; with 5 amino acids controls without any postcoating . the remaining activity is 75 % ; with 2 amino acids only 40 % FIG . 10 : A mixture of 18 amino acids was used to block 25 are maintained . The protection with 2 amino acids is unspecific binding to an ELISA plate without inhibiting improved by the addition of glycyrrhizic acid , here the specific antigen - antibody interactions . The blocking effi remaining activity is 65 % . Samples sterilized with ETO ciency is comparable to standard albumin blockings . FIG . 11 : An amino acid postcoating consisting of 18 maintain about 85 % activity when protected with an amino different amino acids ( 20 g / L ) and 0 . 25 mM glycyrrhizic 30 acidfor postcoating containing 18 amino acids ; this effect is not acid shows a protective effect of 83 % DNAse activity further enhanced with glycyrrhizic acid . Postcoatings con compared to the untreated control after irradiation ( 25 kGy) . taining 5 or 2 amino acids have only little protecting effect ; An postcoating consisting of albumin and mannitol shows the addition of glycyrrhizic acid enhances the protection 95 % remaining activity of after irradiation (25 KGy) . marginally . FIG . 12 : An amino acid postcoating consisting of 18 35 FIG . 23 : Amino acid postcoating of amino acids, dipep different amino acids (20 g /L in PBS ) shows a protective tides or mixtures thereof , optionally together with glycyr effect of 85 % antigen binding ability compared to the rhizic acid . untreated control after irradiation ( 25 KGy ) and 90 % after FIG . 24 accelerated aging procedure , respectively . With the addition An example is shown, where the vial itself is the carrier. of 1 mM glycyrrhizic acid to the amino acid postcoating the 40 First , the biomolecule was attached to the carrier by drying . protective effect is 92 % antigen binding ability compared to Subsequently , the stabilizer solution was added and also the untreated control after irradiation (25 kGy) and 95 % dried . The biomolecule (here interleukin 8 = IL8 ) loses most after accelerated aging procedure , respectively . of its biological function during subsequent sterilisation (25 FIG . 13 : Amino acid postcoatings consisting of 18 dif - kGy irradiation ) if no stabilizer is added . In contrast, a ferent amino acids ( 20 g / L in PBS ) show a protective effect 45 stabilizer solution with different amino acids protected the of 55 -60 % . Without postcoating , the amount of intact ds - biomolecule . Shown is the chemotactic activity of IL8 on DNA is reduced to 20 % , with albumin and mannitol 85 % are human neutrophil granulocytes . preserved . FIG . 25 FIG . 14 : anti -Hepatitis A test ( functional ELISA ) of an An example is shown , where the vial itself is the carrier . amino acid composition used for protecting anti Hepatitis 50 First, the biomolecule was attached to the carrier by drying . antibodies after lyophilisation and sterilization . Subsequently, the stabilizer solution was added and also FIG . 15 : The structural class of saponins has the potential dried . The biomolecule (here interleukin 8 = ILS ) loses most to enhance the protective effect of amino acid combinations of its biological function during accelerated storage (45° C .) FIG . 16 : Combinations of at least 3 amino acids and if no stabilizer is added . In contrast, a stabilizer solution with combinations of 2 amino acids with the addition of glycyr - 55 different amino acids protected the biomolecule . Shown is rhizic acid provide a maximal protection of an immobilized the chemotactic activity of IL8 on human neutrophil granu antibody when sterilized with 50 kGy beta irradiation . locytes. FIG . 17 : The amino acid composition provides protection FIG . 26 under different stress conditions . The best protection is An example is shown , where the vial itself is the carrier . provided for beta irradiation with different doses and arti - 60 First , the biomolecule was attached to the carrier by drying . ficial aging under elevated temperature . The protection for Subsequently , the stabilizer solution was added and also gamma irradiation or ethylene oxide sterilisation is less but dried . The biomolecule (here ds -DNA ) loses part of its still relevant. biological function during subsequent sterilisation ( 25 KGy FIGS . 18 and 19 : Amino acid postcoatings containing at irradiation ) if no stabilizer is added . In contrast , a stabilizer least 5 amino acids provide protection during long time 65 solution with different amino acids protected the biomol storage . For the non sterilized samples , after 62 days at 45° ecule . Shown is the detectable DNA amount in percent of C . about 80 % of the antigen binding ability is preserved . starting value . US 9 ,797 ,895 B2 25 26 FIG . 27 EXAMPLES An example is shown , where the stabilizer itself is the carrier . The biomolecule and the stabilizer solution were The examples illustrate the invention . added and dried together. The biomolecule (here interleukin Materials & Methods 8 = IL8 ) loses most of its biological function during subse - 5 All experiments were based on the same basic ELISA quent sterilisation ( 25 KGy irradiation ) if no stabilizer is assay design . added . In contrast, a stabilizer solution with different amino Adsorption of LO -MM - 3 to an ELISA Plate and Application acids protected the biomolecule . Shown is the chemotactic of Postcoatings activity of IL8 on human neutrophil granulocytes . A monoclonal antibody LO -MM -3 (anti - mouse -IgM , FIG . 28 Acris , SM1495P ) was adsorbed to the surface of a 96 well An example is shown , where the stabilizer itself is the ELISA plate (Greiner Bio -one , 655061) . LO -MM - 3 was carrier. The biomolecule and the stabilizer solution were diluted in PBS (without Ca2 + /Mg2 + , PAA , H15 -002 ) to a added and dried together. The biomolecule ( here an anti - concentration of 2 ug /mL . 100 uL of the antibody solution mouse IgG antibody ) loses most of its biological function 15 was pipetted to each well and incubated over night at 5° C . during subsequent storage and or sterilisation (25 kGy The plate was washed 2 times with washing buffer (25x irradiation ) if no stabilizer is added . In contrast , a stabilizer concentrate , Invitrogen , WB02 ) . solution with different amino acids protected the biomol 200 uL of each assayed stabilizing composition ( post ecule . Shown is the specific binding to the antigen . coating ) were pipetted per well. At least 4 wells were treated FIG . 29 20 identically with the same postcoating to calculate means and Influence of different desorption solutions on the biologi - standard deviations in the following analysis . The plate was cal activity of a biomolecule (here an anti -mouse IgG incubated with the postcoating solutions for 1 hour at antibody ) . With the exception of 0 . 5 M H2SO4 none of the ambient temperature . The postcoatings were discarded and other desorption solutions tested in this experiment had a the plate was dried at ambient temperature for at least 1 hour. significant influence on the biological activity of the biomol- 25 Amino acids used for postcoatings: L - alanine (Sigma ecule . Shown is the specific binding to the antigen . Aldrich , A7627 ), L -arginine (Sigma - Aldrich , A5131 ), L -as FIG . 30 paragine (Sigma -Aldrich , A0884 ), L -aspartate (Sigma -Al The desorption of a biomolecule ( here an anti- mouse IgG drich , A9256 ) , L -cysteine (Sigma - Aldrich , 1276 ) , antibody) is tested after the biomolecule was attached to an 30 L -glutamine ( AppliChem , A1420 ), L - glutamate (Sigma - Al open porous polyurethane foam ( supplier A , large pores ) . drich , G1251) , glycine (Merck , 1042010 ) , L - histidine While citrate buffer pH 4 .75 and 1 M NaCl only desorbed (Sigma - Aldrich , H8125 ) , L - isoleucine (Sigma - Aldrich , small amounts of the biomolecule , significantly more 12752 ) , L - leucine ( Sigma- Aldrich , L8000 ) , L - lysine biomolecule could be desorbed with 1M NaC1 + 0 .02 M ( Sigma - Aldrich , L5626 ) , L -methionine (Sigma - Aldrich , imidazole and phosphate buffered saline , respectively . o M9625 ) , L -phenylalanine (Sigma - Aldrich , P2126 ) , L - pro Shown is the specific binding to the antigen . line (Sigma - Aldrich , P0380 ), L - serine (Sigma - Aldrich , S4500 ), L -threonine (Sigma - Aldrich , T8625 ), L -tryptophan FIG . 31 (Sigma - Aldrich , T0254 ), L -tyrosine (Sigma - Aldrich , The desorption of a biomolecule (here an anti -mouse IgG T3754 ), L -valine (Sigma -Aldrich , V0500 ) antibody ) is tested after the biomolecule was attached to an Stress Exposure of the Coated Surface open porous polyurethane foam ( supplier B , smaller pores ). 40 Three identically treated plates were exposed to different Citrate buffer pH 4 .75 and phosphate buffered saline des environmental conditions. One plate was sterilized by irra orbed a little bit better than 1 M NaCl with or without 0. 02 diation (beta , 25 kGy) . The irradiation was conducted at M imidazole . Shown is the specific binding to the antigen . Beta -Gamma - Service , Bruchsal, Germany. One plate was FIG . 32 treated with an accelerated aging procedure (7 days at 45° The desorption of a biomolecule (here an anti -mouse IgG 45 C . ) . Based on a simplification of the Arrhenius equation it is antibody ) is tested after the biomolecule was attached to an assumed that by increasing the storage temperature by 10° open porous polyurethane foam ( supplier Smith & Nephews , C . , the speed of reaction , i. e . aging , is doubled (Hemmerich , small pores ) . The biomolecule (here an anti -mouse IgG 1998 : General Aging Theory and Simplified Protocol for antibody ) loses most of its biological function during sub - 5 Accelerated Aging ofMedical Devices ). This implies that an sequent storage and or sterilisation (25 kGy irradiation ) if no accelerated aging for 7 days at 45° C . equals a real time stabilizer is added . In contrast a stabilizer solution with aging of 16 weeks of cooled storage ( 5° C .) . different amino acids protected the biomolecule . The recov An identical plate without stress exposition served as a ery of the antibody is almost 100 % ( 5 ug /mL ). Shown is the control in each experiment. The protective effect was cal specific binding to the antigen . culated as the remaining antibody functionality after stress FIG . 33 conditions compared to the untreated control . The desorption of a biomolecule ( here an anti- mouse IgG antibody ) is tested after the biomolecule was attached to an stressed plate : 100 PVA - hydrogel . The biomolecule loses most of its biological functionality % ) = ? control plate function during subsequent storage and or sterilisation (25 OU kGy irradiation ) if no stabilizer is added . In contrast, a ELISA Detection of LO -MM -3 Functionality stabilizer solution with different amino acids protected the The dried postcoatings were removed from the wells by biomolecule . The recovery of the eluted antibody is very washing the plate 3 times . The ELISA plate was blocked by high . Shown is the specific binding to the antigen . 65 pipetting 300 uL blocking solution (10 g /L albumin in PBS ) FIG . 34 to each well and incubating for 1 hour at ambient tempera Chemical Structures of examples for cleavable linkers ture . The plate was washed 3 times . US 9 ,797 ,895 B2 27 28 The antigen to LO -MM -3 , CH11 (mouse IgM , MBL , Example 2 : The Combination of Amino Acids SY001 ) , was diluted to 125 ng/ mL and 200 uL of the antigen Reveals a Protective Effect not Attributed to One solution was pipetted to each well. The plate was incubated Particular Single Amino Acid for 1 hour at ambient temperature and washed 3 times . The bound antigen was detected by a detection antibody 5 Experiment: LO -MM - 9 (biotinylated anti -mouse - IgM , ABD serotec , The amino acids were dissolved either in 0 . 5 M NaOH MCA199B ) , which was diluted with PBS to 50 ng /mL . To (Merck , 106482 ) or 0 . 5 M HC1 (Merck , 100319 ) to obtain each well 200 uL were added and incubated for 1 hour at stock solutions with a maximal concentration . The amino ambient temperature . The plate was washed 3 times. acid stock solutions were mixed together to get a total amino Streptavidin (Horseradish peroxidase (HRP ) labeled , " acid concentration of 200 mM in the postcoating solution . Pierce , 21126 ) was diluted with PBS to 100 ng /mL . 200 uL The amino acids were used in equimolar ratio ( 4 amino were added to each well and incubated for 1 hour at ambient acids: 4x50 mM ; 8 amino acids: 8x25 mM ). For the single temperature . The plate was washed 3 times. amino acid solutions the amino acids were dissolved in aqua A ready -to - use ELISA substrate solution for HRP 5 dest. at the maximal soluble concentration , if possible 400 ( TMB - tetramethylbenzidine , Invitrogen , 00 - 2023 ) was m M . The pH of all postcoating solutions was set to approx . diluted 1 : 2 with aqua dest . and 200 uL were pipetted to each 7 . 0 . Concentrations of the single amino acids: Ala 400 mM , well . The plate was incubated for 20 minutes at ambient Glu 100 mM , Lys 400 mM , Thr 400 mM , Asn 200 mM , Asp temperature and protected from light. To stop the colour 100 mM , His 300 mM , Pro 400 mM , Ser 400 mM , Trp 60 reaction , to each well 50 uL of H2SO4 (diluted 1 : 5 with aqua 20 mM , Val 400 mM . dest . ,Merck , 1007311000 ) were added . The resulting yellow Adsorption of LO -MM - 3 to the plate and application of color was detected by measuring the absorption at a wave the postcoating ; sterilization and accelerated aging ; as well length of 450 nm (Fusion Photometer A153601 , Perki as the general ELISA procedure were conducted as nElmer ) . described in the Materials & Methods section . 25 Result : Example 1 : Postcoatings Consisting of at Least 5 As can be seen in FIGS. 6 and 7 , the protective effect of Amino Acids Show a Maximal Protection Against an amino acid mixture cannot be attributed to a protective Stress effect of one single amino acid of the mixture . The combination of amino acids shows a protective effect Experiment: 30 of 65 % ( 4 amino acids ) and 85 % ( 8 amino acids ) , whereas The amino acids were dissolved either in 0 . 5 M NaOH the average effect of the single amino acids of those mixtures (Merck , 106482 ) or 0 . 5 M HCl (Merck , 100319 ) to obtain is 22 % ( 4 amino acids ) and 33 % ( 8 amino acids ) antigen stock solutions with a maximal concentration . The amino binding as ability compared to the unsterilized control. The acid stock solutions were mixed together toto cotototolget a total aminomins maximal effect of single amino acids is at 28 % ( 4 amino acid concentration of 200 mM in the postcoating solution . 35 acids ) and 55 % ( 8 amino acids ) . The amino acids were used in equimolar ratio ( 2 amino Example 3 : The Addition of Glycyrrhizic Acid to acids : 2x100 mM ; 3 amino acids: 3x67 mM ; 4 amino acids : the Solution Further Increases the Protective Effect 4x50 mM , 5x40 mM , etc . ) of Amino Acid Postcoatings The pH of the amino acid mixtures was set to approx . 7 . 0 ; 40 and the mixtures were further diluted in PBS to get the final ent: concentration of 200 mM . The amino acids were dissolved either in 0 .5 M NaOH Adsorption of LO -MM - 3 to the plate and application of (Merck , 106482 ) or 0 .5 M HCl (Merck , 100319 ) to obtain the postcoating ; sterilization and accelerated aging ; as well stock solutions with a maximal concentration . The amino as the general ELISA procedure were conducted as 45 acid stock solutions were mixed together to get a total amino described in the Materials & Methods section . acid concentration of 200 mM in the postcoating solution . Results: The amino acids were used in equimolar ratio ( 2 amino As shown in FIGS. 2 and 3 , the protective effect of amino acids : 2x100 mM ; 3 amino acids: 3x67 mM ; 4 amino acids : acid postcoatings increases with the number of different 4x50 mM ; etc . ) . Glycyrrhizic acid ( ammonium salt , Fluka , amino acids used . The amino acids were selected as repre - 50 50531 ) was dissolved in 50 % ethanol (absolute , Sigma sentatives of the following groups: amino acids with aro - Aldrich , 32205 ) to obtain a 25 mM stock solution . Glycyr matic /positively charged /negatively charged /polar non rhizic acid was diluted 1 :25 to obtain a concentration of 1 charged / non polar aliphatic side - chain . It is not essential to mM in the postcoating solutions . use particular amino acids; they can be substituted with a The pH of the amino acid mixtures was set to approx . 7 . 0 ; different amino acid from the same group , or with similar 55 and the mixtures were further diluted in PBS to get the final characteristics respectively (Taylor , 1985 : The Classifica - amino acid concentration of 200 mM . tion of Amino Acid Conservation ) . Adsorption of LO -MM - 3 to the plate and application of With postcoatings consisting of 5 different amino acids the postcoating ; sterilization and accelerated aging ; as well after irradiation with 25 kGy approx . 65 % of the antigen as the general ELISA procedure were conducted as binding ability compared to the non - irradiated control is 60 described in the Materials & Methods section . maintained (FIG . 1 ) ; after accelerated aging procedure ( 7 Results : days at 45° C . ) approx . 85 % of the antigen binding ability The addition of glycyrrhizic acid to different amino acid compared to the untreated control is maintained ( FIG . 2 ) . postcoatings enhances the protective effect of all postcoat The protective effect of amino acid postcoatings cannot be ings to a maximum value of 75 - 80 % (antigen binding ability further enhanced by increasing the number of amino acids 65 compared to unirradiated control) as shown in FIG . 8 . above 5 . As shown in FIGS. 4 and 5 , postcoatings with 8 , 10 Glyzyrrhizic acid alone does not have a protective effect as or even 18 amino acids have virtually the same effect . a postcoating US 9 ,797 , 895 B2 29 30 Example 4 : Amino Acid Postcoatings Provide a incubated with the postcoating solutions for 1 hour at Protection Against Stress Conditions at Least ambient temperature . The postcoatings were discarded and Comparable to Postcoatings Containing the plate was dried at ambient temperature . Conventional Protecting Substances Like Albumin Blocking and Postcoating Solutions : and Mannitol 5 Blocking : 20 g / L human albumin (Biotest Pharma ) in PBS - ? Experiment: Postcoating : The amino acid postcoating was prepared as described in 20 g/ L human albumin + 10 g /L mannitol (Serag Wiesner , example 3 . Human albumin was used at a concentration of 219675) in PBS -/ 20 g / L and mannitol at 10 g / L , diluted in PBS . Blocking: Adsorption of LO -MM - 3 to the plate and application of 20 g/ L amino acids ( Ala , Asp , Arg , Gln , Glu , Gly , His , Ile , the postcoating; sterilization and accelerated aging ; as well Leu , Lys , Met , Phe , Pro , Ser, Thr, Trp , Tyr , Val) in as the general ELISA procedure were conducted as PBS - - described in the Materials & Methods section . Postcoating: Results : 15 20 g /L amino acids (Ala , Asp , Arg ,Gln ,Glu ,Gly , His , Ile , As depicted in FIG . 9 , amino acid postcoatings provide a Leu , Lys, Met , Phe , Pro , Ser , Thr, Trp , Tyr, Val) + 0 .25 protective effect comparable to or even better than postcoat mM glycyrrhizic acid ( ammonium salt, Fluke , 50531 ) ings based on albumin and mannitol, depending on the form in PBS - / of stress applied . Stress Exposure of the Coated Surface An amino acid postcoating consisting of five different 20 One plate was sterilized by irradiation (beta , 25 kGy) . The amino acids ( 200 mM ) and 1 mM glycyrrhizic acid shows a irradiation was conducted at Beta -Gamma - Service , Bruch protective effect of 80 % antigen binding ability compared to sal, Germany. the untreated control after irradiation (25 kGy) and 85 % after accelerated aging procedure , respectively. The post An identical plate without stress exposure served as a coating consisting of albumin and mannitol shows 65 % 25 control. The protective effect was calculated as the remain activity of the untreated control after irradiation ( 25 kGy) . ing DNAse functionality after stress conditions compared to After accelerated aging for 7 days at 45° C . there is almost the untreated control. no activity remaining with albumin and mannitol, similar to the controls without any postcoating . stressed plate . 100 30 functionality % ) = Example 5 : Amino Acid Mixtures Block Unspecific control plate Binding , e .g . to ELISA Plates Detection of DNAse Functionality Experiment: The plate was washed 3 times with PBS - / - . Ds- DNA LO -MM -3 was adsorbed to an ELISA plate as described 35 Standard (Sigma Aldrich , D1501 ) was diluted to 1 ug/ mL in in material & methods. The plate surface was blocked by PBS (with Ca² + /Mg2 + , Hyclone , SH3026401) . To each well incubating the wells with 300 uL blocking solution for 1 h 50 uL of the DNA - solution were added and incubated for 1 at ambient temperature . As blocking either 10 g / L human hour at 37° C . The fluorescent DNA - dye Picogreen (Mo albumin in PBS or 20 g / L of a mixture of 18 amino acids in lecular Probes , P7581) was diluted 1 : 1000 (PBS ) and 150 uL PBS were used . The following ELISA procedure was con - 40 of the dye were added to the DNA - solution in each well. ducted as described in the Materials & Methods section . The picogreen fluorescence was measured , the excitation Results : filter was set to 485 nm and the emission was detected at 530 Amino acid mixtures can also block unspecific binding to nm (Fusion Photometer A153601 , PerkinElmer ). The fluo surfaces , as shown in FIG . 10 . A mixture of 18 amino acids rescence signal correlates to the DNA concentration . DNAse blocks unspecific binding to an ELISA plate without inhib - 45 activity was determined as the reduction of DNA , i .e . iting specific antigen - antibody interactions . The blocking fluorescence signal. efficiency is comparable to standard albumin blockings . Results : As depicted in FIG . 11 , amino acid postcoatings provide Example 6 : Amino Acid Mixtures Provide a protective effect against stress ( irradiation ) also for Protection Against Stress for Enzymes Like DNAse 50 enzymes like DNAse. The protection is comparable to that of poscoatings based on albumin and mannitol Experiment: An amino acid postcoating consisting of 18 different Adsorption of DNAse to an ELISA Plate and Application of amino acids (20 g / L ) and 0 .25 mM glycyrrhizic acid shows Postcoatings a protective effect of 83 % DNAse activity compared to the The enzyme DNAse ( Sigma Aldrich , DN25 ) was 55 untreated control after irradiation ( 25 KGy ) . The postcoating adsorbed to the surface of a 96 well ELISA plate (Greiner consisting of albumin and mannitol shows 95 % activity of Bio - one , 655061) . DNAse was diluted in PBS (without the untreated control after irradiation ( 25 kGy ) . Ca2 +/ Mg2 + , PAA , H15 -002 ) to a concentration of 1 mg/ mL . 100 uL of the enzyme solution was pipetted to each well and Example 7 : Amino Acid Postcoatings Provide a incubated over night at 5° C . The plate was washed 2 times 60 Protection Against Stress Conditions for with PBS - / Therapeutic Antibodies Like IgG Infliximab Four wells were treated identically to calculate means and standard deviations in the following analysis . Unspecific Experiment: binding to the plate was blocked with 200 uL blocking The therapeutic antibody Infliximab (humanized IgG , solution per well and incubated for 1 hour at ambient 65 anti - Human - TNF - a , Centocor, DD7701504 ) was adsorbed temperature. The plate was washed 2 times with PBS -/ - . 200 to the ELISA plate surface . Infliximab was diluted with uL of each postcoating were pipetted per well. The plate was PBS - / - to a concentration of 1 ug/ mL and 100 uL of the US 9 , 797 , 895 B2 31 32 solution was pipetted to each well . The plate was incubated were added . The picogreen fluorescence was measured over night at 5° C . and washed 2x with washing buffer. immediately , the excitation filter was set to 485 nm and the Application of the postcoating ; sterilization and acceler - emission was detected at 530 nm (Fusion Photometer ated aging were conducted as described in the Materials & A153601, PerkinElmer ). The fluorescence signal correlates Methods section . 5 to the concentration of intact ds - DNA . ELISA Detection of Infliximab Functionality Results : The dried postcoatings were removed from the wells by As depicted in FIG . 13 . amino acid postcoatings with washing the plate 3 times . Thee ELISA plateplate was blockedblocked by provide a protective effect of 55 -60 % , independent of the pipetting 300 uL blocking solution ( 10 g / L albumin in PBS ) addition of glyzyrrhizic acid or mannitol. Without postcoat to each well and incubating for 1 hour at ambient tempera - 10 ing , the amount of intact ds -DNA is reduced to 20 % when ture . The plate was washed 3 times. irradiated with 25 kGy. Conventional stabilizing substances R & TheD , Catantigen 210 - toTA Infliximab), was diluted , TNF to - a1 ng( recombinant /mL and 200 human uL of , like85 % .albumin and mannitol provide a protection of about the antigen solution was pipetted to each well . The plate was incubated for 1 hour at ambient temperature and washed 3 15 times . Example 9 : Protective Effect of a Specific Amino The bound antigen was detected by a detection antibody Acid Composition (HRP labelled anti- human - TNF - a , R & D , Cat DTA00C ) . The ready - to -use -solution of the detection antibody was 20 mg human anti- Hepatitis A antibody (Beriglobin (hu diluted 1 : 2 with PBS . To each well 200 uL were added and 20 man IgG , AK ) , CSL Behring ) and 40 mg of a protective incubated for 1 hour at ambient temperature . The plate was composition were dissolved in water to a total volume of 525 washed 3 times . uL per sample and lyophilized . Afterwards , the samples 200 uL of a ready -to -use ELISA substrate solution for were dissolved in 1 mL water and tested for functionality HRP ( TMB = tetramethylbenzidine ) were pipetted to each using an HAV (Hepatitis A virus ) IgG ELISA . well . The plate was incubated for 20 minutes at ambient 25 Composition : temperature and protected from light. To stop the color 20 g Arginine reaction , to each well 50 uL of H , SO , (diluted 1 : 5 with aqua dest . ) were added . The resulting yellow colour was detected 20 g Histidine by measuring the absorption at a wavelength of 450 nm . 20 g Lysine Results : 30 3g Glutamic acid As depicted in FIG . 12 , amino acid postcoatings with or 2 g Tryptophane without glycyrrhizic acid provide a protective effect com parable to or even better than poscoatings based on albumin 20 g Glycine and mannitol, depending on the form of stress applied . 15 g Alanine An amino acid postcoating consisting of 18 different 35 0 . 2 g Tween 80 amino acids ( 20 g / L in PBS ) shows a protective effect of 1 g Glycyrrhizic acid ammonium salt 85 % antigen binding ability compared to the untreated The pH was adjusted to about 7 .2 using NaOH and /or control after irradiation ( 25 kGy ) and 90 % after accelerated Nol aging procedure , respectively . With the addition of 1 mM NaCl. Afterwards, the solutions were subjected to sterile glycyrrhizic acid to the amino acid postcoating the protec - 40 filtration . tive effect is 92 % antigen binding ability compared to the 400 uL of solution ( corresponding to 40 mg solid com untreated control after irradiation ( 25 kGy) and 95 % after poundspounas ) wereW mixed with 125 uL Beriglobin (corresponding accelerated aging procedure, respectively . The postcoating to 20 mg antibody ) consisting of albumin and mannitol shows 72 % activity of Lyophilisation : the untreated control after irradiation ( 25 kGy) . After accel- 45 Lyophilisation was carried out as follows: erated aging for 6 days at 45° C . there is only 15 % activity remaining with albumin and mannitol, similar to the controls initial freezing temperature - 40° C . ; without any postcoating . start of vacuum of 0 . 1 mBar after 3 h freezing time; temperature rise of about 1 . 5° C . /h for 23 h ; Example 8 : Amino Acid Postcoatings Provide a 50 6 h drving over night at 6° C . and 0 ,004 mBar . Protection Against Stress Conditions for Nucleic Sterilisation : Acids (dsDNA ) The lyophilized samples were irradiated with 25 kGy and Experiment : one with 50 kGy Beta -radiation . Ds- DNA (Sigma , D1501 ) was adsorbed to the ELISA 55 Results: plate surface . The DNA was dissolved in PBS . ( 1mg /mL ). The results of the HAV - ELISA are depicted in FIG . 14 . with 1 mM EDTA ( Fluka , 50531) to inhibit possible DNAse activity . The DNA was diluted with PBS 1 :64 to 15 ug/ mL After drying and sterilization white , non - odorant, inher and 100 uL of the solution was pipetted to each well . The ently stable cakes were obtained . 1 mL water was added to plate was incubated over night at 5° C . and washed 2x with 60 each sample and the dissolution behaviour was monitored . PBS - / Dissolution took place within less than 30 seconds and was Application of the postcoating and sterilization were free of aggregates. Even after 24 h neithermacroscopic nor conducted as described in the Materials & Methods section . microscopic aggregation was detectable . Detection of Intact Ds -DNA with Picogreen Conclusion : The dried postcoatings were removed from the wells by 65 Application of the composition resulted in only little loss washing the plate 3 times. To each well 100 UL Picogreen of the Hepatitis A antibody portion of Beriglobin as com dye (diluted 1 : 1000 in PBS - ' - , Molecular Probes , P7581) pared to the not irradiated control. US 9 ,797 , 895 B2 33 34 Example 10 : Test of Different Saponins as Experiment : Stabilizing Compounds. The Structural Class of Compositions Used : Saponins has a Stabilizing Effect on Antibodies Protective Composition A ( Compound Per Liter ) 20 g Arginine Materials & Methods 20 g Histidine All experiments were based on the same basic ELISA 20 g Lysine asassay design . ( see above ) 3 g Glutamine Adsorption of LO -MM - 3 to an ELISA plate and applica 2 g Tryptophan tion of postcoatings 20 g Glycine Stress exposure of the coated surface 10 15 g Alanine ELISA detection of LO -MM - 3 functionality 0 . 2 g Tween 80 Experiment: 1 g Glycyrrhizic acid ammonium salt Adsorption of LO -MM - 3 to the plate and application of The pH was adjusted to about 7 .2 using NaOH and /or the postcoating and sterilization ; as well as the general 15 HClfiltr . Afterwards, the solutions were subjected to sterile ELISA procedure were conducted as described in the Mate Adsorption of LO -MM - 3 to the plate and application of rials & Methods section . The irradiation dose ( electron the postcoating and sterilization , as well as the general beam ) was 50 kGy. ELISA procedure were conducted as described in the Mate Results : rials & The results of the experiment are depicted in FIG . 15 . The 20 Methods section . structural class of saponins has the potential to enhance the Results : protective effect of amino acid combinations . Preferred is The results of the experiment are depicted in FIG . 17 . The the use of the saponin glycyrrhizic acid . amino acid composition provides protection under different stress conditions . The best protection is provided for beta Example 11 : Stabilizing Compositions Comprising 25 irradiation with different doses and artificial aging under at Least 3 Different Amino Acids or at Least Two elevated temperature . The protection for gamma irradiation Different Amino Acids and a Saponin Such or ethylene oxide sterilisation is less but still relevant . Glycyrrhizic Acid Provide Very Good Protection Example 13 : Amino Acid Postcoatings Provide Materials & Methods 30 Protection During Long - Time Storage All experiments were based on the same basic ELISA assay design . (see above ) Experiment: Adsorption of LO -MM - 3 to an ELISA plate and applica The amino acids were dissolved either in 0 .5 M NaOH tion of postcoatings (Merck , 106482 ) or 0 . 5 M HC1 (Merck , 100319 ) to obtain Stress exposure of the coated surface 35 stock solutions with a maximal concentration . The amino ELISA detection of LO -MM - 3 functionality acid stock solutions were mixed together to get a total amino Experiment : acid concentration of 200 mM in the postcoating solution . The amino acids were dissolved either in 0 .5 M NaOH The amino acids were used in equimolar ratio . (Merck , 106482 ) or 0 . 5 M HCl (Merck , 100319 ) to obtain 18 amino acids : Ala , Arg , Asp , Gln , Glu , Gly , His , Ile , Leu , stock solutions with a maximal concentration . The amino 40 Lys, Met , Phe, Pro , Ser, Thr, Trp , Tyr , Val acid stock solutions were mixed together in different com - 5 amino acids ( 1 ) : Asp , Arg , Phe , Ser, Val binations to get total amino acid concentrations of 20 g /L in 5 amino acids ( 2 ): Ala , Glu , Lys, Thr, Trp the postcoating solutions. NNuu2 amino acids ( 1 ) : Asp , Val The pH of the amino acid mixtures was set to approx . 7 . 0 . 2 amino acids ( 2 ) : Ala , Glu Adsorption of LO -MM - 3 to the plate and application of 45 The pH of the amino acid mixtures was set to approx . 7 . 0 ; the postcoating and sterilization , as well as the general and the mixtures were further diluted in PBS to get the final ELISA procedure were conducted as described in the Mate concentration of 200 mM . rials & Methods section . The irradiation dose (electron Adsorption of LO -MM - 3 to the plate and application of beam ) was 50 kGy. the postcoating and sterilization ; as well as the general Results : 50 ELISA procedure were conducted as described in the Mate The results of the experiment are depicted in FIG . 16 . rials & Methods section . Long - time storage was simulated Combinations of at least 3 amino acids and combinations of by an accelerated aging procedure . The plates were stored at 2 amino acids with the addition of glycyrrhizic acid provide 45º C . and antibody activity was determined after 0 , 10 , 25 , a maximal protection (more than 75 % ) of an immobilized 41 and 62 days of storage . This equals real time aging at 5° antibody when sterilized with 50 kGy beta irradiation . 55 C . of 0 , 6 , 12 , 24 and 36 months. Results : Example 12 : A Preferred Amino Acid Composition Amino acid postcoatings containing at least 5 amino acids Provides Protection Under Different Stress provide protection during long time storage . For the non Conditions sterilized samples, after 62 days at 45° C . about 80 % of the 60 antigen binding ability is persevered . Sterilized samples Materials & Methods (beta , 25 kGy ) maintain about 70 % of their antigen binding All experiments were based on the same basic ELISA ability after 62 days at 45° C . Amino acid postcoatings assay design . ( see above ) containing only 2 amino acids maintain only about 40 % Adsorption of LO -MM -3 to an ELISA Plate and Application antigen binding ability during the storage process, regardless of Postcoatings 65 of sterilization . The addition of 1 mM glycyrrhizic acid to Stress Exposure of the Coated Surface the postcoating solutions enforces the protecting effect: For ELISA Detection of LO -MM - 3 Functionality the non - sterilized samples containing at least 5 amino acids US 9 ,797 ,895 B2 35 36 and glycyrrhizic acid , after 62 days at 45° C . about 90 % of 7 amino acids: Arg , His , Lys, Glu , Trp , Gly , Ala the antigen binding ability is preserved . Sterilized samples 2 dipeptides: Gly - Tyr, Gly -Gln (beta , 25 kGy ) maintain about 80 % of their antigen binding The pH of the amino acid mixtures was set to approx . 7 . 0 . ability after 62 days at 45° C . Amino acid postcoatings Adsorption of LO -MM - 3 to the plate and application of containing 2 amino acids and glycyrrhizic acid maintain 5 the postcoating and sterilization , as well as the general about 70 % antigen binding ability during the storage pro ELISA procedure were conducted as described in the Mate cess . rials & Methods section . The irradiation dose ( electron beam ) was 50 kGy. Example 14 : Amino Acid Postcoatings Provide Results : Protection During Different Sterilization Processes 10 Samples sterilized with 50 kGy beta irradiation maintain about 60 % activity when protected with an amino acid Experiment : postcoating containing only 7 amino acids; this effect is The amino acids were dissolved either in 0 . 5 M NaOH further enhanced with the addition of dipeptides such as (Merck , 106482 ) or 0 .5 M HCl (Merck , 100319 ) to obtain Gly - Tyr or dipeptide combinations. Glycyrrhizic Acid does stock solutions with a maximal concentration . The amino 15 not enhance the protective effect of the amino acid dipeptide acid stock solutions were mixed together to get a total amino combination further . acid concentration of 200 mM in the postcoating solution . The amino acids were used in equimolar ratio . Example 16 : Interleukin - 8 in Glass - Vials was 18 amino acids : Ala , Arg , Asp , Gln , Glu ,Gly , His , Ile , Leu , Sterilized , the Vial Itself is the Carrier Lys, Met, Phe, Pro , Ser, Thr, Trp , Tyr, Val 20 5 amino acids: Asp , Arg , Phe, Ser , Val Experiment: 2 amino acids : Asp , Val Interleukin - 8 (IL - 8 , R & D , 208 - IL ) was diluted in PBS The pH of the amino acid mixtures was set to approx . 7 .0 ; (without Ca2+ /Mg2 + , PAA , H15 -002 ) to 10 ug/ mL . 5 uL of and the mixtures were further diluted in PBS to get the final the solution (50 ng IL - 8 ) were added to glass vials and concentration of 200 mM . rotated for 4 hours until dried . 25 uL of a stabilizing solution Adsorption of LO -MM -3 to the plate and application of (20 g /L amino acid mixture and 1 mM glyzyrrhizic acid the postcoating and sterilization , as well as the general (ammonium salt , Fluke , 50531 ) ) were added and rotated / ELISA procedure were conducted as described in the Mate - dried overnight. rials & Methods section . One plate was irradiated ( gamma, The vials were sterilized with 25 kGy (beta irradiation ) . 25 kGy ) . The irradiation was conducted at Beta - Gamma- 30 Unsterilized controls were stored under cool conditions . Service , Bruchsal, Germany . Another plate was sterilized by Assay : EO ( ETO BO1 cycle ) ; the sterilization was conducted at Neutrophile granulocytes were isolated from 10 % ACDA Rose GmbH , Trier, Germany. whole blood . 20 mL ACDA blood ( 10 % ) were sedimented Results : with 2 mL HES (Grifols 662650 ) . The supernatant was Samples sterilized with gamma irradiation maintain about 35 pipetted to 7 mL Percoll (L6143 ) and centrifuged 20 min at 85 % activity when protected with an amino acid postcoating 2000xg . The isolated granulocytes were resuspended in 1 % containing 18 amino acids; this effect is not further enhanced autologous serum and set to a cell count of 0 .5x10° / mL . with glycyrrhizic acid ; with 5 amino acids the remaining As positive controls 5 uL IL - 8 - solution ( 50 ng ) were activity is 75 % ; with 2 amino acids only 40 % are main - dissolved in 25 uL of the stabilizing solution (A and B ) . To tained . The protection with 2 amino acids is improved by the 40 each sterile vial 1 mL PBS (with Ca2 + /Mg2 +, Hyclone, addition of glyccyrhizic acid ; here the remaining activity is SH3026401 ) (with 1 % autologous serum ) were added to 65 % . Samples sterilized with ETO maintain about 85 % dissolve the dried film . activity when protected with an amino acid postcoating To detect the chemotactic activity of the samples, the containing 18 amino acids ; this effect is not further enhanced complete IL - 8 solutions from the sterile and non - sterile vials with glycyrrhizic acid . Postcoatings containing 5 or 2 amino 45 and the controls were pipetted into 12 -well -plates . Migration acids have only little protecting effect ; the addition of filters ( 3 um , Corning , 3462 ) were inserted and 500 uL of the glycyrrhizic acid enhances the protection marginally . granulocyte suspension was pipetted into the filters . The plates were incubated for 30 min at 37° C . The number of Example 15 : Postcoatings Consisting of Amino migrated cells was detected by counting the cells in each Acids and Dipeptides Provide Protection Against 50 well via FACS and counting beads ( Invitrogen , C36950 ) . High Irradiation Doses Results : see FIG . 26 Materials & Methods The biomolecule (here interleukin 8 = IL8 ) loses most of All experiments were based on the same basic ELISA its biological function during subsequent sterilisation (25 assay design . (see above ) 55 kGy irradiation ) if no stabilizer is added . In contrast, a with Adsorption of LO - MM - 3 to an ELISA plate and applica - different amino acids protected the biomolecule . Shown is tion of postcoatings the chemotactic activity of IL8 on human neutrophil granu Stress exposure of the coated surface locytes. ELISA detection of LO -MM - 3 functionality Experiment: 60 Example 17 : Interleukin - 8 in Glass - Vials was The amino acids were dissolved either in 0 . 5 M NaOH Sterilized , the Stabilizer Itself is the Carrier (Merck , 106482 ) or 0 . 5 M HCl (Merck , 100319 ) to obtain stock solutions with a maximum concentration . The amino Experiment: acid stock solutions were mixed together to get a total amino Interleukin - 8 (IL -8 ) was diluted in PBS (without Ca²+ 1 acid concentration of 20 g / L in the postcoating solution . The 65 Mg2+ , PAA , H15 - 002 ) to 10 ug/ mL . 5 uL of the solution ( 50 dipeptides alone were used with a concentration of 10 g / L ng IL - 8 ) and 25 uL of a stabilizing solution ( 20 g / L amino and in combination with amino acids with 2 g / L . acid mixture and 1 mM glyzyrrhizic acid (ammonium salt, US 9 ,797 ,895 B2 37 38 Fluke, 50531 ) ) were mixed and pipetted into glass vials . The diluted 1 : 2 in H20 and 200 uL were added to each well . The vials were rotated / dried over night . plate was incubated 15 min at ambient temperature and was The vials were sterilized by irradiation with 25 kGy . protected from light. To stop the color reaction 50 uL diluted Unsterilized controls were stored under cool conditions. H2SO4 (diluted 1 : 5 with aqua dest ., Merck , 1007311000 ) Assay : 5 were added . The absorption of the plate was detected at 450 Neutrophile granulocytes were isolated from 10 % ACDA nm ( Fusion Photometer A153601 , PerkinElmer) . whole blood . 20 mL ACDA blood ( 10 % ) were sedimented Results: with 2 mL HES (Grifols 662650 ) . The supernatant was see FIG . 30 pipetted to 7 mL Percoll (L6143 ) and centrifuged 20 min at 2000xg . The isolated granulocytes were resuspended in 1 % 10 The biomolecule (here an anti -mouse IgG antibody ) loses autologous serum and set to a cell count of 0 .5x10° /mL . most of its biological function during subsequent storage As positive controls 5 uL IL - 8 -solution (50 ng ) were and or sterilisation ( 25 kGy irradiation ) if no stabilizer is dissolved in 25 uL of a stabilizing solution ( A and B ) . To added . In contrast , a stabilizer solution with different amino each sterile vial 1 mL PBS (with Ca2 + /Mg2 + , Hyclone , acids protected the biomolecule . Shown is the specific SH3026401 ) ( with 1 % autologous serum ) were added to 15 binding to the antigen . dissolve the dried film . To detect the chemotactic activity of the samples , the Example 19 : Anti -Mouse -IgG was Sterilized , the complete IL - 8 - solutions from the sterile and non - sterile vials Carrier is an Polyurethane Foam and the controls were pipetted into 12 -well - plates .Migration filters ( 3 um ) were inserted and 500 uL of the granulocyte 20 Experiment: suspension was pipetted into the filters . The plates were from a fine porous polyurethan (PU ) foamam incubated 30 min at 37° C . The number of migrated cells (Smith & Nephew , 66012608) samples with a defined diam was detected by counting the cells in each well ( via FACS eter ( 1 cm ) were punched . Anti -Mouse - IgG (biotinylated , and counting beads) . Jackson ImmunoResearch , 115 - 065 - 003 ) was attached to Results : 25 the samples : the antibody was diluted to 5 ug /mL either in see FIG . 29 PBS ( without Ca2 + /Mg2 + , PAA , H15 -002 ) or in a stabilizing The biomolecule (here interleukin 8 = IL8 ) loses most of solution ( 20 g / L amino acid mixture and 1 mM glyzyrrhizic its biological function during subsequent sterilisation (25 acid (ammonium salt , Fluke , 50531 ) ) and the PU samples kGy irradiation ) if no stabilizer is added . In contrast, a were covered with antibody solutions . The samples were stabilizer solution with different amino acids protected the 30 incubated 1 h at 37° C . biomolecule . Shown is the chemotactic activity of ILS on The antibody solution was removed and the PU samples human neutrophil granulocytes . were air dried for 2 h . The samples were sterilized via beta irradiation ( 25 kGy) and unsterile controls were stored under Example 18 : Anti- Mouse -IgG in Glass - Vials was 35 cool conditconditions . Sterilized , the Stabilizer Itself is the Carrier Assay : Experiment: An ELISA plate (Greiner Bio -one , 655061 ) was coated Anti -Mouse - IgG (biotinylated , Jackson ImmunoRe with the antigen (mouse IgG , Innovativ Research , Ir -Ms search . 115 - 065 - 003 ) was diluted in PBS (without Ca2 + 1 GB) : the antigen was diluted to 1 ug/ mL , 100 uL were Mg2 + . PAA . H15 - 002 ) to 4 ug /mL . 25 uL ( 100 ng ) of the 40 pipetted to each well and incubated over night at 4° C . The antibody solution and 25 uL of a 2x concentrated stabilizing plate was washed 2x with washing buffer ( 25x concentrate , solution (20 g / L amino acid mixture and 1 mM glyzyrrhizic Invitrogen , W802) . The plate was blocked with Albumin acid (ammonium salt, Fluke , 50531) ) were mixed and ( 5 % ) and washed again 3x . pipetted into glass vials . The vials were rotated /dried over The PU samples were covered with PBS and incubated 1 night. 45 h at ambient temperature . The sample solutions were col The vials were sterilized by irradiation with 25 kGy. lected and diluted 1 :20 and further serial diluted 1 : 4 with Unsterilized controls were stored under cool conditions . PBS. To calculate the antibody concentration of the samples Assay : a serial dilution of fresh antibody was prepared . An ELISA plate (Greiner Bio -one , 655061) was coated The samples and standard were pipetted to the ELISA with the antigen (mouse IgG , Innovativ Research , Ir -Ms - 50 plate (2x200 uL each ) an incubated 1 h at ambient tempera Gf) : the antigen was diluted to 1 ug /mL , 100 uL were ture . The plate was washed 3x . To each well 200 uL pipetted to each well and incubated over night at 4° C . The Streptavidin solution (Horseradish peroxidase (HRP ) plate was washed twice with washing buffer (25x concen labeled , Pierce , 21126 , diluted to 0 . 1 ug /mL in PBS ) were trate , Invitrogen , W802 ) . The plate was blocked with Albu added and incubated 1 h at ambient temperature . The plate min ( 5 % ) and washed again 3 times . 55 was washed 3x . HRP cromogenic substrate TMB To all sample vials 200 °L PBS were added to dissolve the ( TMB = tetramethylbenzidine , Invitrogen , 00 -2023 ) was dried film (theoretically 5 ug /mL ) . The samples were diluted diluted 1: 2 in H20 and 200 uL were added to each well. The to 10 ng /mL with PBS . To calculate the antibody concen - plate was incubated 15 min at ambient temperature and was tration a serial dilution of fresh antibody was prepared . protected from light. To stop the color reaction 50 uL diluted The samples and standard were pipetted to the ELISA 60 H2SO4 (diluted 1: 5 with aqua dest ., Merck , 1007311000 ) plate (2x200 uL each ) an incubated 1 h at ambient tempera were added . The absorption of the plate was detected at 450 ture . The plate was washed 3x . To each well 200 ul nm ( Fusion Photometer A153601 , PerkinElmer ) . Streptavidin solution (Horseradish peroxidase (HRP ) Results : labeled , Pierce, 21126 , diluted to 0 . 1 ug/ mL in PBS) were The biomolecule (here an anti -mouse IgG antibody ) loses added and incubated 1 h at ambient temperature . The plate 65 most of its biological function during subsequent storage was washed 3x . HRP chromogenic substrate TMB and or sterilisation ( 25 kGy irradiation ) if no stabilizer is ( TMB = tetramethylbenzidine , Invitrogen , 00 - 2023 ) was added . In contrast a stabilizer solution protected the biomol US 9 ,797 ,895 B2 39 40 ecule . The recovery of the antibody is almost 100 % (5 wherein the composition of (i ) does not contain dipep ug /mL ) . Shown is the specific binding to the antigen . tides or tripeptides. 2 . The method of claim 1 , wherein stabilizing biomol Example 20 : Anti -Mouse - IgG was Sterilized, the ecules includes stabilizing the structure and /or activity of Carrier is an PVA Hydrogel 5 biomolecules , enhancing the shelf - life of biomolecules and / or protecting biomolecules against stress -mediated damage . Experiment: 3 . The method of claim 1 , wherein the composition A 7 % ( m / v ) solution of polyvinylalcohol (PVA , Sigma, comprising at least 3 different amino acids comprises at least 341584 - 25G ) in water ( heated to 85° C . ) was prepared . The 4 or at least 5 different amino acids. solution was cooled down to ambient temperature . Anti 10 4 . The method of claim 3 , wherein the composition mouse IgG (biotinylated , Jackson ImmunoResearch , 115 comprising at least 5 different amino acids comprises at least 065 -003 ) was diluted to 200 ug /mL in PBS . one amino acid of each group of, The hydrogel mixture was composed as follows: ( a ) an amino acid with non polar , aliphatic R groups ; 6 .75 mL PVA solution ( 7 % ) ( b ) an amino acid with polar , uncharged R groups ; 4 .5 uL anti mouse IgG ( 200 ug /mL ) 15 ( c ) an amino acid with positively charged R groups ; 2 .25 either PBS or stabilizing solution ( ( 20 g / L amino ( d ) an amino acid with negatively charged R groups ; and acid mixture and 1 mM glyzyrrhizic acid (ammonium ( e ) an amino acid with aromatic R groups. salt , Fluke , 50531 ) ) in PBS ) 5 . The method of claim 3 , wherein the composition PVA hydrogels were poured into small petri dishes ( diam - comprising at least 5 different amino acids comprises amino eter 35 mm , 2 mL solution ) . The hydrogel films were air 20 acids selected from dried for 48 h . The samples were sterilized via beta irradia ( a ) alanine, glutamate , lysine, threonine and tryptophan ; tion ( 25 kGy ) and unsterile controls were stored under cool ( b ) aspartate , arginine , phenylalanine , serine and valine; conditions . ( c ) proline , serine , asparagine , aspartate , threonine , and Assay : phenylalanine ; An ELISA plate (Greiner Bio -one , 655061) was coated 25 ( d ) tyrosine , isoleucine , leucine, threonine, and valine ; or with the antigen (mouse IgG , Innovativ Research , Ir- Ms ( e ) arginine , glycine, histidine , alanine , glutamate , lysine , Gf) : the antigen was diluted to 1 ug/ mL , 100 uL were and tryptophan pipetted to each well and incubated over night at 4° C . The 6 . The method of claim 1, wherein the composition plate was washed 2x with washing buffer (25x concentrate , comprising at least three different amino acids, or the Invitrogen , W802) . The plate was blocked with Albumin 30 composition comprising at least two different amino acids ( 5 % ) and washed again 3x . and a saponin , comprises less than 1 % by dry weight The PVA hydrogels were placed in 6 well plates and cysteine . covered with 2 mL PBS (without Ca2 + /Mg2 + , PAA , H15 7 . The method of claim 1 , wherein the composition 002 ) . After 30 min , 1 h and 2 h the PBS was collected and comprising at least three different amino acids, or the replaced with fresh PBS . 35 composition comprising at least two different amino acids Serial dilutions of the samples and the standard were and a saponin , further comprises less than 1 % Tween . pipetted into the ELISA plate ( 2x200 uL each ) and incubated 8 . The method of claim 1 , wherein the saponin is glycyr for 1 h at ambient temperature . The plate was washed 3x . To rhizic acid or a derivative thereof. each well 200 uL Streptavidin solution (Horseradish peroxi- 9 . A method of producing a solid carrier having biomol dase (HRP ) labeled , Pierce, 21126 , diluted to 0 . 1 ug/ mL in 40 ecules attached thereto , comprising the steps of PBS) were added and incubated 1 h at ambient temperature . (a ) reversibly attaching the biomolecules to the solid The plate was washed 3x . Chromogenic substrate TMB carrier ; and ( TMB = tetramethylbenzidine , Invitrogen , 00 - 2023 ) was (b ) incubating the carrier of step (a ) in a composition diluted 1 : 2 in H20 and 200 uL were added to each well . The selected from : plate was incubated 15 min at ambient temperature and was 45 ( i ) a composition comprising at least three different protected from light. To stop the color reaction 50 uL diluted amino acids, or H2SO4 ( diluted 1 : 5 with aqua dest . , Merck , 1007311000 ) ( ii ) a composition comprising at least two different were added . The absorption of the plate was detected at 450 amino acids and a saponin , nnnm ( Fusion Photometer A153601 , PerkinElmer ). wherein the composition of ( i ) does not contain dipep Results : 50 tides or tripeptides . The biomolecule (here an anti -mouse IgG antibody ) loses 10 . The method according to claim 1 or 9 , further com most of its biological function during subsequent storage prising sterilizing the solid carrier after step (b ). and or sterilisation (25 kGy irradiation ) if no stabilizer is 11 . The method according to claim 10 wherein the ster added . In contrast a stabilizer solution protected the biomol- ilization of the carrier is effected by ethylene oxide , beta ecule . The recovery of the eluted antibody is very high . 55 radiation , gamma radiation , X -ray , heat inactivation , auto Shown is the specific binding to the antigen . claving or plasma sterilization . The invention claimed is : 12 . The method of claim 1 , wherein the biomolecules are 1 . A method for producing stabilized biomolecules , com - reversibly attached to the solid carrier via a cleavable linker, prising and wherein the biomolecule can be released from the solid ( a ) reversibly attaching the biomolecules to a solid carrier; 60 carrier by using a means for cleaving the linker . and 13 . The method of claim 12 , wherein the cleavable linker ( b ) embedding the biomolecules in a composition selected is selected from the group consisting of ethylene glycol from : bis (succinimidylsuccinate ), bis [ 2 - (succinimidooxycarbony ( i) a composition comprising at least three different loxy )ethyl ] sulfone , dithiobis (succinimidylpropionate ), dis amino acids , or 65 uccinimidyl tartarate , succinimidyl 2 - ([ 4 , 4 '- azipentanamido ] ( ii ) a composition comprising at least two different ethyl) - 1 , 3 '- dithiproprionate , and sulfosuccinimidyl - 2 - ( m amino acids and a saponin , azido - o - nitrobenzamido ) - ethyl- 1 , 3 '- proprionate . US 9 , 797 , 895 B2 41 42 14 . The method according to claim 1 or 9 further com ( b ) detecting whether said marker protein indicative for prising the disease , said non - cellular pathogen , said cell or said ( c ) subjecting the solid carrier to drying . toxin has been bound to the biomolecules . 15 . A solid carrier produced by the method of claim 9 . 20 . A method for producing stabilized biomolecules , 16 . The solid carrier of claim 15 , wherein the biomol- 5 comprising ecules are proteins , peptides, nucleic acids, carbohydrates, ( a ) adsorbing biomolecules directly to the surface of a lipids, fatty acids, polyalcohols and combinations or modi solid carrier, and fications thereof, wherein the proteins preferably are anti ( b ) covering the biomolecules in a composition selected bodies, enzymes , receptors , cytokines, hormones , mem from : brane proteins, growth factors , albumins, globulins , 10 ( i ) a composition comprising at least three different transport proteins or blood coagulation factors . amino acids, or 17 . The solid carrier of claim 15 , wherein the biomol ( ii ) a composition comprising at least two different ecules specifically bind to a marker protein indicative for a amino acids and a saponin , disease, a non - cellular pathogen , a cell or a toxin . wherein the composition of ( i) does not contain dipep 18 . A method of preparing a medical device comprising 15 tides or tripeptides, the solid carrier of claim 15 , wherein the medical device is so that the biomolecules are partially or completely cov selected from the group consisting of an implant, a tubing , ered by the composition comprising at least three a catheter , a stent, a tubing, a wound dressing and a medical different amino acids, or the composition comprising at device used in extracorporeal circulation . least two different amino acids and a saponin , wherein 19 . A method for diagnosing a disease comprising the 20 the covered biomolecules can be released from the steps of: carrier by adding a liquid to solubilize /dissolve the ( a ) contacting a sample obtained from a patient with a covered biomolecules . solid carrier according to claim 16 under suitable 21 . The method of claim 20 , further comprising : conditions to allow specific binding of the biomol sterilizing the covered biomolecules ; and ecules attached to the carrier to said marker protein releasing the sterilized covered biomolecules from the indicative for a disease , said non -cellular pathogen , carrier. said cell or said toxin ; and * * * *