|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 Figure 7 solid carrier in spacer / linker Y antibody O postcoating atent Oct. 24 , 2017 Sheet 2 of 30 US 9 ,797 ,895 B2 . W - - - - - - - - - - - * * * * * * * * * * * * * - - - - - - - - - . * * * * * * * * * * WinWVwLXDomu *. 011 The , Lys .* Glu . Ala .* * VVS - . Val 1 4, -0.+, 5 4 ,Ser .? * * Ang . Asp .15- ,7 " 77.-;', prior to this even i mentera .1- .* . M AMMA the then that . J? SK7 . vi *,. Glu Aa .1* . .. re * * * * * * * * * * * * .. .. WY * * * * * * * * * * * * * * * * * * * * * * . -*,. Val ,Ser . * Are . Asp .1 *. -:;., .1 * *. : * .: * : .21' * * 101 * : . : Glu , Ala . :. * * * * * buneogsodpoyQURUY '. .*-11:00 ?.??:????? OMSZYILMpajeipewa)uopjezilasJayle&WW-0710Aggleuapouny jobunsisuooslupeogsod . 746 sproeouwegoj . 1.'-*4 Ser . Gy . esp *., *. 1. Glu Aa . - - - - - - - - . 1.47777- . ' . .,* e Os .- .TTTTT *. TITI I FT *.-, ' ' ':,.; . buge001sod " *. Without . 1. 88 89 .1- )control unsterilized of W -.TT *. Xuqy buipuig vaguy * _ . _ _ _ _ _ _ . _ - _ - _ - _ U V . U . 14, atent Oct. 24 , 2017 Sheet 3 of 30 US 9 ,797 , 895 B2 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . - . - . -. - . -. - . - . - . - . - . - . - - . - - - . * , * . * . * . - . - . 4 . - . -. - . - . - . - . - . -. - . - . -. F . hewan terakuratanakiri vipi * * iivii k set * * * * * * * * * * * * * * * * * * * * * ** * * * * * ** * * . TOO *.- Thr , Lys * , Glu , Ala * rrrrrrrrrr rrrrrrr * WW9 vest. HAYA * XX ai , ch . Arg , Asp * Thr , Lys Gu Ala - '. Vzien .' 4.AA .' . WYYYYYYYYY Val .Ger V .: : Arg , Asp * . *. ." Jul . .*, , Glu , Ala 0.9jesyoensjenbaosyteskep . - - AminoAcidPostcoating Ouidepaille18932jayeC-WW01J0Aduonouns joOussuossbuneousod 3AA enot . gotouwespoe * . GY , úsp . .' * . * - . GU Ala - * . 2AA . .,:-' * . Val . usp .' . .:- .! . 3:53 : {} } * . .' . BOUM . * - - * . * . * . * * * * * * * * * * * 8888889880** * * * * ** ** * * * * * )control untreated of % ( Ability Binding Antigen numeria. : . Warnir atent Oct. 24 , 2017 Sheet 4 of 30 US 9 ,797 , 895 B2 1. VYYY w . 1977/7WY -1. e .1 4 . :.5"4 'Ik .- . Gin,GluGlyHe Mhe.Po,SerThr . deleu,Lysvel WV81 . 44+4,WY du . wwii. "'. .-*4 .' . ,YKYYYYis14+ .'" . 2 . Y . -VY .34504- * * Lys:Var,Mhe Ser,TrgVal . ti . WOL . ",73.154144 . .144YYYYYY444444 . - AlaGu.Lys,TheArgGlyAsnAspHáskisLeuAB Lys,MoSer -* * end 172018 * 11. *.2244202424444490444- * ., * ? */.34' Figure4 postcoatingsconsistingof AminoAcidPostcoating - -.* Be,leuPheThe *Y - ":44 .: . 7 . : . 2383 . * * . * 100 . *:. o * . * . * * .9 : * .: GAA ,*.701"27 * * . * . Asp,Argthe Ser;Val * . * * ** ** * * * . ., ??. + + + + + + + + + + + + + + ( lofuo poziuasun jo % ) Aíy ????g ????? - - - - - - - + . nzi i mer . U . S . Patent Oct . 24 , 2017 Sheet 5 of 30 US 9 , 797 ,895 B2 4SYWWW * * . ' * * * Mhe.PoSer,Ther Trp,TheVa 18AA Ser,TrpValleLeuLysNet. 10AA . LysNet,MheGinGluGyhs.leLeuPrehrPoSer * L * . * emmin * ! ! - -Asp,ArgPheAlaGuLysThir.GluGlyAsnHisLeu 5 . 8AA Figure5 postcoatingsconsistingof AminoAcidPostcoating FunctionalityofLQ-MM3aftersacceleratedaging (7daysat45°Cequals16weeks5°C acidsamino5morethan Tro. SAA SerVal )control untreated of % ( Kugy öupun uomoy atent Oct. 24 , 2017 Sheet 6 of 30 US 9 ,797 , 895 B2 * . * * * . * . 4 , 1 . , - , . , . - . - . - . - . - . - . - . - . - . - . - . - . - . - . - . - ' ' ' ' ' ' m 444Y "* . - "-.* YYYYYY4X445,5*18 :. .* *: . * :i* 7.,"14* - - - 5447 * :* " 1", :* *4. 9 " . : FuncionalityofLOMM3aftersterilization(irradiatedwith25KGy): Postcoatingconsistingofdaminoacid 474.4 gano sppyeouweafbuisalorpoieduos