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ferrocenecompounds.xxxix

metal-organic compoundsm328#2003International Union of CrystallographyDOI:10.1107/S0108270103011703Acta Cryst.(2003).C 59,m328±m330Ferrocene compounds.XXXIX.11-FerrocenylisochromaneMario Cetina,a Senka akovicÂ,b Vladimir Rapic Âb *and Amalija GolobicÏc aFaculty of Textile Technology,University of Zagreb,Pierottijeva 6,HR-10000Zagreb,Croatia,b Laboratory of Organic Chemistry,University of Zagreb,Faculty of Food Technology and Biotechnology,Pierottijeva 6,HR-10000Zagreb,Croatia,and cLaboratory of Inorganic Chemistry,Faculty of Chemistry and Chemical Technology,University of Ljubljana,PO Box 537,SI-1001Ljubljana,Slovenia Correspondence e-mail:*************Received 14April 2003Accepted 27May 2003Online 22July 2003In the title compound,[Fe(C 5H 5)(C 14H 13O)],the plane of the heterocyclic ring is almost perpendicular to the plane of the substituted cyclopentadienyl ring,and the heterocyclic ring adopts a half-chair conformation.The conformation of the nearly parallel cyclopentadienyl (Cp)rings [the dihedral angle between their planes is 2.7(1) ]is almost halfway between eclipsed and staggered,and the rings are mutually twisted by about 19.4(2) (mean value).The mean lengths of the CÐC bonds in the substituted and unsubstituted cyclopentadienylring are 1.420(2)and 1.406(3)AÊ,respectively,and the FeÐC distances range from 2.029(2)to 2.051(2)AÊ.The phenyl and unsubstituted cyclopentadienyl rings are involved in CÐH ÁÁÁ%interactions,with intermolecular H ÁÁÁcentroiddistances of 2.85and 3.14AÊfor CÐH ÁÁÁ%(Ph),and 2.88A Êfor CÐH ÁÁÁ%(Cp).In two of these interactions,the CÐH bond points towards one of the ring bonds rather than towards the ring centroid.In the crystal structure,the CÐH ÁÁÁ%interactions connect the molecules into a three-dimensional framework.CommentOptically active ferrocene derivatives are widely employed as chiral ligands in asymmetric reactions,and there is continuing interest in the development of ef®cient procedures for the preparation of these derivatives in enantiopure forms (Gonsalves &Chen,1995;Bolm et al.,1998;Pioda &Togni,1998;Perea et al.,1999).Ferrocene derivatives exhibiting centro-and planar chirality are very convenient substrates forbiotransformations (KoÈllner et al.,1998;Richards &Locke,1998;Schwink &Knochel,1998;Patti &Nicolosi,1999;akovicÂet al.,2003).In the course of our research on enzyme-catalyzed resolution of centrochiral ferrocene compounds,racemic 2-( -hydroxyferrocenyl)benzenethanol and 1-ferro-cenylisochromane,(I),were prepared by reduction of methyl2-(ferrocenoyl)benzeneacetate ( akovicÂ,2000).The molecular structure of (I)is the ®rst reported structure to contain an isochromanyl group attached to the ferrocenyl moiety (Fig.1).Moreover,the Cambridge Structural Database (Allen,2002)lists only three structures containing an iso-chromanyl group at all (Yamato et al.,1984;Unterhalt et al.,1994;Eikawa et al.,1999).The heterocyclic six-membered ringActa Crystallographica Section CCrystal Structure CommunicationsISSN0108-2701Figure 2Part of the crystal structure of (I),showing the formation of (010)sheets built from C14ÐH14A ÁÁÁCg 1i and C18ÐH18ÁÁÁCg 2ii interactions (Cg 1and Cg 2are the centroids of rings C12/C13/C16±C19and C6±C10,respectively).CÐH ÁÁÁ%interactions are indicated by dashed lines.[Symmetry codes:(i)x ,12Ày ,z À12;(ii)1+x ,y ,1+z.]Figure 1A view of (I),with the atom-numbering scheme.Displacement ellipsoids for non-H atoms are drawn at the 20%probability level.1Part XXXVIII:Cetina et al.(2003).adopts a distorted half-chair conformation,in which atoms O1and C15are 0.426(1)and À0.348(2)AÊfrom the plane of the other ring atoms (C11±C14);the C11ÐC12ÐC13ÐC14torsion angle is 2.4(2) .The bond lengths in the heterocyclic and fused phenyl rings (Table 1)mostly agree with the equivalent bond lengths in the structures of 1,1H -oxybis(iso-chromane)(Eikawa et al.,1999)and (S )-1-(phenyl)ethyl-ammonium (S )-isochromane-1-carboxylate (Unterhalt et al.,1994).The exception is the C12ÐC13bond,which is shorter($0.04AÊ)in the latter structure.Heterocyclic ring atoms O1,C11and C15and cyclopentadienyl (Cp)ring atom C1lie in the same plane,the C15ÐO1ÐC11ÐC1torsion angle being 178.95(14) .The dihedral angle between the mean plane of these four atoms and the C1±C5Cp ring is 47.8(1) .Furthermore,the plane of the heterocyclic ring is almost perpendicular to the plane of the C1±C5ring and is parallel to the plane of the fused phenyl ring.The corresponding dihedral angles are 87.3(1)and 4.0(1) .The exocyclic C2ÐC1ÐC11bond angle is larger than the C5ÐC1ÐC11angle (Table 1).The Cp rings are planar and almost parallel to each other [the dihedral angle between their planes is 2.7(1) ],and the FeÐC distances are in the range2.034(2)±2.051(2)AÊfor the substituted (C1±C5)and 2.029(2)±2.049(2)AÊfor the unsubstituted (C6±C10)ring,the average values being 2.042(2)and 2.038(2)AÊ,respectively.The CÐC bonds are slightly longer in the substituted ring than in the unsubstituted ring [1.410(3)±1.429(2)versus 1.397(3)±1.414(3)AÊ],and the bond angles in both rings range from 107.52(15)to 108.33(18) .The geometry of the ferrocenyl moiety agrees well with the structures of ferrocene (Seiler &Dunitz,1979)and of theferrocene derivatives we have reported previously (Cetina et al.,2002,2003).The main conformational difference was observed in the orientation of the Cp rings.In (I),the rings are twisted from an eclipsed conformation by 19.4(2) (mean value).The values of the corresponding CÐCg 3ÐCg 2ÐC pseudo-torsion angles (Cg 3and Cg 2are the centroids of the C1±C5and C6±C10rings,respectively),de®ned by joining two eclipsing Cp C atoms through the ring centroids,range from 19.0(2)to 19.7(2) .The conformation is almost exactly halfway between eclipsed and staggered,as demonstrated by the C1ÐCg 3ÐCg 2ÐC9torsion angle of 163.4(1) .This angle would be 180 for a staggered conformation and 144 for a fully eclipsed conformation.The centroids of the Cp rings are almost equidistant from the Fe atom;the FeÐCg 3and FeÐCg 2distances are 1.647(1)and 1.650(1)AÊ,respectively,while the Cg 3ÐFeÐCg 2angle is 178.2(1) .There are a number of CÐH ÁÁÁ%interactions (Table 2and Fig.2).Atom H14A of the heterocyclic ring is positioned almost perpendicularly above the phenyl-ring centroid (Cg 1)of the adjacent molecule.The six relevant H ÁÁÁC distances fallin the narrow range 3.06±3.28AÊ,and the H ÁÁÁCg i distance is signi®cantly shorter than any of the H ÁÁÁC distances[symmetry code:(i)x ,12Ày ,z À12;Table 2].The CÐH ÁÁÁ%interaction between phenyl atom H18and the unsubstituted Cp ring exhibits a completely different geometry.The H18ÁÁÁC7ii distance is shorter than the H ÁÁÁCg ii distance [symmetry code:(ii)1+x ,y ,1+z ].The second shortest H ÁÁÁC contact is that to atom C6,and the CÐH bond points towards the C6ÐC7bond of the Cp ring rather than towards the ring centroid (Cg 2).Similarly,the longest interaction,C5ÐH5ÁÁÁCg 1iii [symmetry code:(iii)1Àx ,Ày ,2Àz ],points towards the C12ÐC13bond.Both the H5ÁÁÁC12iii and the H5ÁÁÁC13iii contacts are shorter than the H ÁÁÁCg iii distance.The molecules linked by these CÐH ÁÁÁ%interactions build a three-dimensional framework (Fig.3).ExperimentalNaBH 4(253mg,6.7mmol)was added gradually to a solution of methyl 2-(ferrocenoyl)benzeneacetate (326mg,0.9mmol)in a mixture of EtOH and Et 2O (1:1v /v ;5ml).The mixture was re¯uxed for 2h and worked up in the usual manner.Separation by preparative thin-layer chromatography on silica gel (Merck,Kieselgel 60HF 254)yielded 2-( -hydroxyferrocenyl)benzeneethanol (237mg;yield 78%)and orange crystals of 1-ferrocenylisochromane (57mg;yield 20%;m.p.365±366K).Single crystals of the title compound were obtained by slow evaporation from a cyclohexane solution at room tempera-ture.IR (CH 2Cl 2,cm À1):)3081(w )and 3020(w )(CÐH,ferrocene),2942(m )(CÐH,aliphatic),1278(m )(CÐOÐC);1H NMR (DMSO,p.p.m.): 7.18(d ,1H,H16),7.12(d ,1H,H17),7.14(d ,1H,H18),7.16(d ,1H,H19),4.23(s ,5H,unsubstituted ferrocene ring),4.13±4.20(m ,4H,substituted ferrocene ring),3.97(m ,1H,H15A ),3.77(m ,1H,H15B ),2.78(m ,2H,H14),5.58(s ,1H,H11);13C NMR (DMSO,p.p.m.): 137.29(C12),132.91(C13),128.5(C17),126.25(C18),125.98(C16),125.36(C19),90.21(C1),73.63(C11),68.62(unsub-stituted ferrocene ring),68.56±66.31(substituted ferrocene ring),61.55(C15),27.99(C14).Acta Cryst.(2003).C 59,m328±m330Mario Cetina et al.[Fe(C 5H 5)(C 14H 13O)]m329metal-organic compoundsFigure 3Part of the crystal structure of (I),showing the cyclic motif generated by the C5ÐH5ÁÁÁCg 1iii interaction (Cg 1is the centroid of ring C12/C13/C16±C19),which links the (010)sheets into a three-dimensional framework.CÐH ÁÁÁ%interactions are indicated by dashed lines,and the unit-cell box has been omitted for clarity.[Symmetry code:(iii)1Àx ,Ày ,2Àz .]metal-organic compoundsm330Mario Cetina et al.[Fe(C 5H 5)(C 14H 13O)]Acta Cryst.(2003).C 59,m328±m330Crystal data[Fe(C 5H 5)(C 14H 13O)]M r =318.18Monoclinic,P 21a ca =11.5053(2)A Êb =18.5095(3)A Êc =7.1941(1)AÊ =106.933(1)V =1465.62(4)AÊ3Z =4D x =1.442Mg m À3Mo K radiationCell parameters from 3411re¯ections =2.6±27.5 "=1.02mm À1T =293(2)K Prism,orange0.80Â0.40Â0.15mm Data collectionNonius KappaCCD area-detector diffractometer 9and 3scansAbsorption correction:multi-scan (DENZO±SMN ;Otwinowski &Minor,1997)T min =0.630,T max =0.85716885measured re¯ections3334independent re¯ections 2662re¯ections with I >2'(I )R int =0.064 max =27.4 h =À14314k =À23323l =À939Re®nementRe®nement on F 2R [F 2>2'(F 2)]=0.030wR (F 2)=0.076S =1.033334re¯ections 190parametersH-atom parameters constrainedw =1/['2(F 2o )+(0.0341P )2+0.3545P ]where P =(F 2o +2F 2c )/3(Á/')max =0.001Á&max =0.25e A ÊÀ3Á&min =À0.23e AÊÀ3All H atoms were included in calculated positions as riding atoms,with SHELXL 97(Sheldrick,1997)defaults viz.CÐH =0.93AÊfor aromatic H atoms,0.98AÊfor methine H atoms,and 0.97A Êfor methylene H atoms.For all H atoms,the isotropic displacement parameters were set at 1.2times the equivalent anisotropic displacement parameters of the attached non-H atoms.Data collection:COLLECT (Nonius,2000);cell re®nement:DENZO±SMN (Otwinowski &Minor,1997);data reduction:DENZO±SMN ;program(s)used to solve structure:SHELXS 97(Sheldrick,1997);program(s)used to re®ne structure:SHELXL 97(Sheldrick,1997);molecular graphics:PLATON (Spek,2003);soft-ware used to prepare material for publication:SHELXL 97.The re¯ection data were collected at the Faculty of Chem-istry and Chemical Technology,University of Ljubljana,Slovenia.We acknowledge with thanks the ®nancial contri-bution of the Ministry of Education,Science and Sport of the Republic of Slovenia (grant Nos.X-2000and PS-511-103),which made the purchase of the apparatus possible.Supplementary data for this paper are available from the IUCr electronic archives (Reference:GD1251).Services for accessing these data are described at the back of the journal.ReferencesAllen,F.H.(2002).Acta Cryst.B 58,380±388.Bolm,C.,MunÄiz-Ferna Ândez,K.,Seger,A.,Raabe,G.&Gunther,K.(1998).Chem.63,7860±7867.Cetina,M.,Hergold-BrundicÂ,A.,Nagl,A.,Jukic Â,M.&Rapic Â,V .(2003).Struct.Chem.14,289±293.Cetina,M.,MrvosÏ-Sermek,D.,Jukic Â,M.&Rapic Â,V .(2002).Acta Cryst.E 58,m676±m678.akovicÂ,S.(2000).PhD thesis,University of Zagreb,Croatia. akovicÂ,S.,Lapic Â,J.&Rapic Â,V .(2003).Biocatal.Biotransform.In the press.Eikawa,M.,Sakaguchi,S.&Ishii,Y.(1999).Chem.64,4676±4679.Gonsalves,K.E.&Chen,X.(1995).Ferrocenes ,edited by A.Togni &T.Hayashi,ch.10,pp.497±527.Weinheim:VCH.KoÈllner,C.,Pugin,B.&Togni,A.(1998).J.Am.Chem.Soc.120,10274±10275.Nonius (2000).COLLECT.Nonius BV ,Delft,The Netherlands.Otwinowski,Z.&Minor,W.(1997).Methods in Enzymology ,Vol.276,Macromolecular Crystallography ,Part A,edited by C.W.Carter Jr &R.M.Sweet,pp.307±326.New York:Academic Press.Patti,A.&Nicolosi,G.(1999).Tetrahedron :Asymmetry ,10,2651±2654.Perea,J.J.A.,Lotz,M.&Knochel,P .(1999).Tetrahedron :Asymmetry ,10,375±384.Pioda,G.&Togni,A.(1998).Tetrahedron :Asymmetry ,9,3903±3910.Richards,C.J.&Locke,A.(1998).Tetrahedron :Asymmetry ,9,2377±2407.Schwink,L.&Knochel,P .(1998).Chem.Eur.J.4,950±968.Seiler,P .&Dunitz,J.D.(1979).Acta Cryst.B 35,2020±2032.Sheldrick,G.M.(1997).SHELXS 97and SHELXL 97.University ofGoÈttingen,Germany.Spek,A.L.(2003).J.Appl.Cryst.36,7±13.Unterhalt,B.,Krebs,B.,Lage,M.&Nocon,B.(1994).Arch.Pharm.327,799±804.Yamato,M.,Hashigaki,K.,Kokubu,N.&Nakato,Y.(1984).J.Chem.Soc.Perkin Trans.1,pp.1301±1304.Table 2Hydrogen-bonding geometry (AÊ, ).Cg 1and Cg 2are the centroids of rings C12/C13/C16±C19and C6±C10,respectively.D ÐH ÁÁÁA D ÐH H ÁÁÁA D ÁÁÁA D ÐH ÁÁÁA C14ÐH14A ÁÁÁCg 1i 0.97 2.85 3.627(2)138C18ÐH18ÁÁÁCg 2ii 0.93 2.88 3.660(2)142C18ÐH18ÁÁÁC7ii 0.93 2.86 3.760(3)163C18ÐH18ÁÁÁC6ii 0.93 3.03 3.874(3)151C5ÐH5ÁÁÁCg 1iii 0.93 3.14 3.984(2)152C5ÐH5ÁÁÁC12iii 0.93 2.98 3.840(2)154C5ÐH5ÁÁÁC13iii0.933.023.949(2)177Symmetry codes:(i)x Y 12Ày Y z À12;(ii)1 x Y y Y 1 z ;(iii)1Àx Y Ày Y 2Àz .Table 1Selected geometric parameters (AÊ, ).O1ÐC111.4236(19)O1ÐC15 1.432(2)C1ÐC11 1.503(2)C11ÐC12 1.523(2)C12ÐC16 1.388(2)C12ÐC13 1.397(2)C13ÐC19 1.392(3)C13ÐC14 1.504(3)C14ÐC15 1.501(3)C16ÐC17 1.384(3)C17ÐC18 1.380(3)C18ÐC19 1.379(3)C11ÐO1ÐC15110.39(13)C2ÐC1ÐC11127.61(14)C5ÐC1ÐC11124.78(14)O1ÐC11ÐC1109.37(13)O1ÐC11ÐC12111.64(13)C1ÐC11ÐC12112.14(13)C13ÐC12ÐC11119.77(14)C12ÐC13ÐC14120.38(16)C15ÐC14ÐC13111.24(15)O1ÐC15ÐC14110.04(15)。

Antibody structure, instability, and formulation

MINIREVIEWAntibody Structure,Instability,and FormulationWEI WANG,SATISH SINGH,DAVID L.ZENG,KEVIN KING,SANDEEP NEMAPﬁzer,Inc.,Global Biologics,700Chesterﬁeld Parkway West,Chesterﬁeld,Missouri63017Received14March2006;revised17May2006;accepted4June2006Published online in Wiley InterScience().DOI10.1002/jps.20727 ABSTRACT:The number of therapeutic monoclonal antibody in development hasincreased tremendously over the last several years and this trend continues.At presentthere are more than23approved antibodies on the US market and an estimated200ormore are in development.Although antibodies share certain structural similarities,development of commercially viable antibody pharmaceuticals has not been straightfor-ward because of their unique and somewhat unpredictable solution behavior.This articlereviews the structure and function of antibodies and the mechanisms of physical andchemical instabilities.Various aspects of formulation development have been examinedto identify the critical attributes for the stabilization of antibodies.ß2006Wiley-Liss,Inc.and the American Pharmacists Association J Pharm Sci96:1–26,2007Keywords:biotechnology;stabilization;protein formulation;protein aggregation;freeze drying/lyophilizationINTRODUCTIONProtein therapies are entering a new era with the inﬂux of a signiﬁcant number of antibody pharmaceuticals.Generally,protein drugs are effective at low concentrations with less side effects relative to small molecule drugs,even though,in rare cases,protein-induced antibody formation could be serious.1Therefore,this category of therapeutics is gaining tremendous momentum and widespread recognition both in small and large drugﬁrms.Among protein drug therapies,antibodies play a major role in control-ling many types of diseases such as cancer, infectious diseases,allergy,autoimmune dis-eases,and inﬂammation.Since the approval of theﬁrst monoclonal antibody(MAb)product -OKT-3in1986,more than23MAb drug products have entered the market(Tab.1).The estimated number of antibodies and antibody derivatives constitute20%of biopharmaceutical products currently in development(about200).2The global therapeutic antibody market was predicted to reach$16.7billion in2008.3There are several reasons for the increasing popularity of antibodies for commercial develop-ment.First,their action is speciﬁc,generally leading to fewer side effects.Second,antibodies may be conjugated to another therapeutic entity for efﬁcient delivery of this entity to a target site, thus reducing potential side effects.For instance, Mylotarg is an approved chemotherapy agent composed of calicheamicin conjugated to huma-nized IgG4,which binds speciﬁcally to CD33for the treatment of CD33-positive acute myeloid leukemia.Another example is the conjugation of immunotoxic barnase with the light chain of the anti-human ferritin monoclonal antibody F11as potential targeting agents for cancer immuno-therapy.4Third,antibodies may be conjugated to radioisotopes for speciﬁc diagnostic purposes. Examples include CEA-Scan for detection of color-ectal cancer and ProstaScint for detection of prostate stly,technology advancement has made complete human MAb available,which are lessimmunogenic.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY20071 Correspondence to:Wei Wang(Telephone:(636)-247-2111;Fax:(636)-247-5030;E-mail:wei.2.wang@pﬁ)Journal of Pharmaceutical Sciences,Vol.96,1–26(2007)Pharmacists AssociationT a b l e 1.C o m m e r c i a l M o n o c l o n a l A n t i b o d y P r o d u c t s#B r a n d n a m e M o l e c u l eM A bY e a r C o m p a n y R o u t e I n d i c a t i o n M A b C o n c B u f f e r E x c i p i e n t s S u r f a c t a n t p H1A v a s t i n B e v a c i z u m a bH u m a n i z e d I g G 1,149k D a2004G e n e t e c h a n d B i o O n c o l o g y I V i n f u s i o nM e t a s t a t i c c a r c i n o m a o f c o l o n o r r e c t u m ,b i n d s V E G F 100m g a n d 400m g /v i a l (25m g /m L )s o l u t i o n 5.8m g /m L m o n o b a s i c N a P h o s H 2O ;1.2m g /m L d i b a s i c N a P h o s a n h y d r o u s (4m L ,16m L ﬁl l i n v i a l )60m g /m L a -T r e h a l o s e d i h y d r a t e (4m L ,16m L ﬁl l i n v i a l )0.4m g /m L P S 20(4m L ,16m L ﬁl l i n v i a l )6.22B e x x a rT o s i t u m o m a b a n d I -131T o s i t u m a b M u r i n e I g G 2l2003C o r i x a a n d G S KI V I n f u s i o nC D 20p o s i t i v e f o l l i c u l a r n o n H o d g k i n s l y m p h o m aK i t :14m g /m L M A b s o l u t i o n i n 35m g a n d 225m g v i a l s ;1.1m g /m L I 131-M A b s o l u t i o n10m M p h o s p h a t e (M A b v i a l )145m M N a C l ,10%w /v M a l t o s e ;I 131-M A b :5–6%P o v i d o n e ,1–2,9–15m g /m L M a l t o s e ,0.9m g /m L N a C l ,0.9–1.3m g /m L A s c o r b i c a c i d 7.23C a m p a t h A l e m t u z u m a bH u m a n i z e d ,I g G 1k ,150k D a2001I l e x O n c o l o g y ;M i l l e n i u m a n d B e r l e xI V i n f u s i o nB -c e l l c h r o n i c l y m p h o c y t i c l e u k e m i a ,CD 52-a n t i g e n 30m g /3m L s o l u t i o n3.5m g /3m L d i b a s i c N a P h o s ,0.6m g /3m L m o n o b a s i c K P h o s 24m g /3m L N a C l ,0.6m g /3m L K C l ,0.056m g /3m L N a 2E D T A 0.3m g /3m L P S 806.8–7.44C E A -S c a n (l y o )A c r i t u o m a b ;T c -99M u r i n e F a b ,50k D a1996I m m u n o m e d i c s I V i n j e c t i o n o r i n f u s i o nI m a g i n g a g e n t f o r c o l o r e c t a l c a n c e r1.25m g /v i a l L y o p h i l i z e d M A b .R e c o n s t i t u t e w 1m L S a l i n e w T c 99m 0.29m g /v i a l S t a n n o u s c h l o r i d e ,p o t a s s i u m s o d i u m t a r t r a t e t e t r a h y d r a t e ,N a A c e t a t e .3H 2O ,N a C l ,g l a c i a l a c e t i c a c i d ,H C l S u c r o s e5.75E r b i t u x C e t u x i m a bC h i m e r i c h u m a n /m o u s e I g G 1k ,152kD a 2004I m C l o n e a n d B M S I V i n f u s i o n T r e a t m e n t o fE GF R -e x p r e s s i n g c o l o r e c t a l c a r c i n o m a 100m g M A b i n 50m L ;2m g /m L s o l u t i o n1.88m g /m L D i b a s i c N a P h o s Á7H 2O ;0.42m g /m L M o n o b a s i c N a P h o s ÁH 2O8.48m g /m L N a C l 7.0–7.46H e r c e p t i n (l y o )T r a s t u z u m a bH u m a n i z e d I g G 1k1998G e n e t e c h I V i n f u s i o n M e t a s t a t i c b r e a s t c a n c e r w h o s e t u m o r o v e r e x p r e s s H E R 2p r o t e i n 440m g /v i a l ,21m g /m L a f t e r r e c o n s t i t u t i o n 9.9m g /20m L L -H i s t i d i n e H C l ,6.4m g /20m L L -H i s t i d i n e400m g /20m L a -T r e h a l o s e D i h y d r a t e 1.8m g /20m L P S 2067H u m i r a A d a l i m u m a bH u m a n I g G 1k ,148k D a2002C A T a n d A b b o t t S CR A p a t i e n t s n o t r e s p o n d i n g t o D M A R D s .B l o c k s T N F -a l p h a40m g /0.8m L s o l u t i o n (50m g /m L )0.69m g /0.8m L M o n o b a s i c N a P h o s Á2H 2O ;1.22m g /0.8m L D i b a s i c N a P h o s Á2H 2O ;0.24m g /0.8m L N a C i t r a t e ,1.04m g /0.8m L C i t r i c a c i d ÁH 2O 4.93m g /0.8m L N a C l ;9.6m g /0.8m L M a n n n i t o l 0.8m g /0.8m L P S 805.28L u c e n t i s R a n i b i z u m a bH u m a n i z e d I g G 1k f r a g m e n t2006G e n e n t e c h I n t r a v i t r e a l i n j e c t i o n A g e -r e l a t e d m a c u l a r d e g e n e r a t i o n (w e t )10m g /m L s o l u t i o n10m M H i s t i d i n e H C l10%a -T r e h a l o s e -D i h y d r a t e 0.01%P S 205.52WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY 2007DOI 10.1002/jps9M y l o t a r g (l y o )G e m t u z u m a b o z o g a m i c i nH u m a n i z e d I g G 4k c o n j u g a t e d w i t h c a l i c h e a m i c i n2000C e l l t e c h a n d W y e t h I V i n f u s i o nH u m a n i z e d A b l i n k e d t o c a l i c h e a m i c i n f o r t r e a t m e n t o f C D 33p o s i t i v e a c u t e m y e l o i d l e u k e m i a 5m g p r o t e i n -e q u i v a l e n t l y o p h i l i z e d p o w d e r /20-m L v i a l M o n o b a s i c a n d d i b a s i c N a P h o s p h a t e D e x t r a n 40,S u c r o s e ,N a C l 10O n c o S c i n tS a t u m o m a b p e n d e t i d eM u r i n e I g G 1k c o n j u g a t e d t o G Y K -D T P A1992C y t o g e n I V i n j e c t i o nI m a g i n g a g e n t f o r c o l o r e c t a l a n d o v a r i a n c a n c e r0.5m g c o n j u g a t e /m L s o l u t i o n (2m L p e r v i a l )P h o s p h a t e b u f f e r s a l i n e 6.011O r t h o c l o n e O K TM u r o m o m a b -C D 3M u r i n e ,I g G 2a ,170k D a1986O r t h o B i o t e c h I V i n j e c t i o nR e v e r s a l o f a c u t e k i d n e y t r a n s p l a n t r e j e c t i o n (a n t i C D 3-a n t i g e n )1m g /m L s o l u t i o n2.25m g /5m L m o n o b a s i c N a P h o s ,9.0m g /5m L d i b a s i c N a P h o s 43m g /5m L N a C l 1m g /m L P S 807Æ0.512P r o s t a S c i n tI n d i u m -111c a p r o m a b p e n d e t i d e M u r i n e I g G 1k -c o n j u g a t e d t o G Y K -D T P A1996C y t o g e n I V i n j e c t i o nI m a g i n g a g e n t f o r p r o s t a t e c a n c e r0.5m g c o n j u g a t e /m L s o l u t i o n (1m L p e r v i a l )P h o s p h a t e b u f f e r s a l i n e 5–713R a p t i v a (l y o )E f a l i z u m a bH u m a n i z e d I g G 1k2003X o m a a n d G e n e n t e c h S C C h r o n i c m o d e r a t e t o s e v e r e p l a q u e p s o r i a s i s ,b i n d s t o C D 11a s u b u n i t o f L F A -1150m g M A b /v i a l ;125m g /1.25m L (100m g /m L )a f t e r r e c o n s t i t u t i o n w i t h 1.3m L S W F I 6.8m g /v i a l L -H i s t i d i n e H C l ÁH 2O ;4.3m g /v i a l L -H i s t i d i n e123.2m g /v i a l S u c r o s e 3m g /v i a l P S 206.214R e m i c a d e (l y o )I n ﬂi x i m a bC h i m e r i c h u m a n /m u r i n e M A b a g a i n s t T N F a l p h a (a p p .30%m u r i n e ,70%c o r r e s p o n d s t o h u m a n I g G 1h e a v y c h a i n a n d h u m a n k a p p a l i g h t c h a i n c o n s t a n t r e g i o n s )1998C e n t o c o r I V i n f u s i o nR A a n d C r o h n ’s d i s e a s e (a n t i T N F a l p h a )100m g /20-m L V i a l ,10m g /m L o n r e c o n s t i t u t i o n2.2m g /10m L M o n o b a s i c N a P h o s H 2O ,6.1m g /10m L D i b a s i c N a P h o s Á2H 2O 500m g /10m L S u c r o s e 0.5m g /10m L P S 807.215R e o P r o A b c i x i m a bF a b .C h i m e r i c h u m a n -m u r i n e ,48k D a 1994C e n t o c o r /L i l l y I V i n j e c t i o n a n d i n f u s i o n R e d u c t i o n o f a c u t e b l o o d c l o t r e l a t e d c o m p l i c a t i o n s 2m g /m L s o l u t i o n 0.01M N a P h o s p h a t e 0.15M N a C l 0.001%(0.01m g /m L )P S 807.216R i t u x a n R i t u x i m a bC h i m e r i c m o u s e /h u m a n I g G 1k w i t h m u r i n e l i g h t a n d h e a v y c h a i n v a r i a b l e r e g i o n (F a b d o m a i n ),145kD a1997I D E C a n d G e n e n t e c h I V i n f u s i o nN o n H o d g k i n ’s l y m p h o m a .(a n t i C D 20-a n t i g e n )10m g /m L s o l u t i o n7.35m g /m L N a C i t r a t e Á2H 2O9m g /m L N a C l 0.7m g /m L P S 806.5(C o n t i n u e d )ANTIBODY FORMULATION3DOI 10.1002/jpsJOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY 200717S i m u l e c t (l y o )B a s i l i x i m a bC h i m a r i c I g G 1k ,144kD a1998N o v a r t i s I V i n j e c t i o n a n d i n f u s i o nP r e v e n t i o n o f a c u t e k i d n e y t r a n s p l a n t r e j e c t i o n ,I L -2r e c e p t o r a n t a g o n i s t10m g a n d 20m g /v i a l ,4m g /m L o n r e c o n s t i t u t i o n 3.61m g ,7.21m g M o n o b a s i c K P h o s ;0.50m g ,0.99m g N a 2H P O 40.8m g ,1.61m g N a C l ;10m g ,20m g S u c r o s e ;40m g ,80m g M a n n i t o l ;20m g 40m g G l y c i n e 18S y n a g i s (l y o )P a l i v i z u m a bH u m a n i z e d I g G 1k ,C D R o f m u r i n e M A b 1129,148k D a 1998M e d I m m u n e I M i n j e c t i o nP r e v e n t r e p l i c a t i o n o f t h e R e s p i r a t o r y s y n c y t i a l v i r u s (R S V )50m g a n d 100m g /v i a l ,100m g /m L o n r e c o n s t i t u t i o n47m M H i s t i d i n e ,3.0m M G l y c i n e 5.6%M a n n i t o l19T y s a b r i N a t a l i z u m a bH u m a i n z e d I g G 4k2004B i o g e n I D E C I V I n f u s i o nM S r e l a p s e 300m g /15m L s o l u t i o n 17.0m g M o n o b a s i c N a P h o s ÁH 2O ,7.24m g d i B a s i c N a P h o s Á7H 2O f o r 15m L 123m g /15m L N a C l3.0m g /15m L P S 806.120V e r l u m a N o f e t u m o m a b M u r i n e F a b 1996B o e h r i n g e r I n g e l h e i m a n d D u P o n t M e r c k I V i n j e c t i o n I m a g i n g a g e n t f o r l u n g c a n c e r10m g /m L s o l u t i o nP h o s p h a t e b u f f e r s a l i n e?21X o l a i r (l y o )O m a l i z u m a bH u m a n i z e d I g G 1k ,149k D aG e n e n t e c h w N o v a r t i s a n d T a n o xS CA s t h m a ,i n h i b i t s b i n d i n g o f I g E t o I g E r e c e p t o r F C e R I202.5m g /v i a l ,D e l i v e r 150m g /1.2m L o n r e c o n s t i t u t i o n w i t h 1.4m L S W F I 2.8m g L H i s t i d i n e H C l ÁH 2O ;1.8m g L H i s t i d i n e145.5m g S u c r o s e 0.5m g P S 2022Z e n a p a x D a c l i z u m a bH u m a n i z e d I g G 1,144k D a1997R o c h e I V i n f u s i o nP r o p h y l a x i s o f a c u t e o r g a n r e j e c t i o n i n p a t i e n t s r e c e i v i n g r e n a l t r a n s p l a n t s .I n h i b i t s I L -2b i n d i n g t o t h e T a c s u b u n i t o f I L -2r e c e p t o r c o m p l e x 25m g /5m L M A b S o l u t i o n3.6m g /m L M o n o b a s i c N a P h o s ÁH 2O ;11m g /m L D i b a s i c N a P h o s Á7H 2O4.6m g /m L N a C l 0.2m g /m L P S 806.923Z e v a l i nI b r i t u m o m a b -T i u x e t a nM u r i n e I g G 1k -t h i o u r e a c o v a l e n t l i n k a g e t o T i u x e t a nI D E C I V i n f u s i o nC D 20a n t i g e n .(K i t w i t h Y t t e r i u m -90i n d u c e s c e l l u l a r d a m a g e b y b e t a e m i s s i o n )3.2m g /2m L s o l u t i o n 09%N a C l 7.1T a b l e 1.(C o n t i n u e d )#B r a n d n a m e M o l e c u l eM A bY e a r C o m p a n y R o u t e I n d i c a t i o n M A b C o n c B u f f e r E x c i p i e n t s S u r f a c t a n t p H4WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY 2007DOI 10.1002/jpsDevelopment of commercially viable antibody pharmaceuticals has,however,not been straight-forward.This is because the behavior of antibodies seems to vary,even though they have similar structures.In attempting to address some of the challenges in developing antibody therapeutics, Harris et al.5reviewed the commercial-scale formulation and characterization of therapeutic recombinant antibodies.In a different review, antibody production and puriﬁcation have been discussed.2Nevertheless,the overall instability and stabilization of antibody drug candidates have not been carefully examined in the litera-ture.This article,not meant to be exhaustive, intends to review the structure and functions of antibodies,discuss their instabilities,and sum-marize the methods for stabilizing/formulating antibodies.ANTIBODY STRUCTUREAntibodies(immunoglobulins)are roughly Y-shaped molecules or combination of such molecules(Fig.1). Their structures are divided into two regions—the variable(V)region(top of the Y)deﬁning antigen-binding properties and the constant(C)region (stem of the Y),interacting with effector cells and molecules.Immunoglobulins can be divided into ﬁve different classesÀIgA,IgD,IgE,IgM,and IgG based on their C regions,respectively desig-nated as a,d,e,m,and g(ﬁve main heavy-chain classes).6Most IgGs are monomers,but IgA and IgM are respectively,dimmers and pentamers linked by J chains.IgGs are the most abundant,widely used for therapeutic purposes,and their structures will be discussed as antibody examples in detail.Primary StructureThe structure of IgGs have been thoroughly reviewed.6The features of the primary structure of antibodies include heavy and light chains, glycosylation,disulﬁde bond,and heterogeneity. Heavy and Light ChainsIgGs contain two identical heavy(H,50kDa)and two identical light(L,25kDa)chains(Fig.1). Therefore,the total molecular weight is approxi-mately150kDa.There are several disulﬁde bonds linking the two heavy chains,linking the heavy and light chains,and residing inside the chains (also see next section).IgGs are further divided into several subclasses—IgG1,IgG2,IgG3,and IgG4(in order of relative abundance in human plasma),with different heavy chains,named g1, g2,g3,and g4,respectively.The structural differences among these subtypes are the number and location of interchain disulﬁde bonds and the length of the hinge region.The light chains consist of two types—lambda(l)and kappa(k). In mice,the average of k to l ratio is20:1,whereas it is2:1in humans.6The variable(V)regions of both chains cover approximately theﬁrst 110amino acids,forming the antigen-binding (Fab)regions,whereas the remaining sequences are constant(C)regions,forming Fc(fragment crystallizable)regions for effector recognition and binding.6The N-terminal sequences of both the heavy and light chains vary greatly between different antibodies.It was suggested that the conserved sequences in human IgG1antibodies Figure1.Linear(upper panel)and steric(lower panel)structures of immunoglobulins(IgG).ANTIBODY FORMULATION5DOI10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007are approximately95%and the remaining5% is variable and creates their antigen-binding speciﬁcity.5The V regions are further divided into three hypervariable sequences(HV1,HV2,and HV3)on both H and L chains.In the light chains,these are roughly from residues28to35,from49to59,and from92to103,respectively.6Other regions are the framework regions(FR1,FR2,FR3,and FR4).The HV regions are also called the complementarity determining regions(CDR1,CDR2,and CDR3). While the framework regions form the b-sheets, the HV sequences form three loops at the outer edge of the b barrel(also see Section2.2).Disulﬁde BondsMost IgGs have four interchain disulﬁde bonds—two connecting the two H chains at the hinge region and the other two connecting the two L chains to the H chains.6Exceptions do exist.Two disulﬁde bonds were found in IgG1and IgG4 linking the two heavy chain in the hinge region but four in IgG2.7In IgG1MAb,HC is linked to the LC between theﬁfth Cys(C217)of HC and C213on the LC.In IgG2and IgG4MAbs,it is the third Cys of HC(C123)linking to the LC.7A disulﬁde bond between HC C128and LC C214 was found for mouse catalytic monoclonal anti-bodies(IgG2a).8IgGs have four intrachain disulﬁde bonds, residing in each domain of the H and L chains, stabilizing these domains.The intrachain disul-ﬁde bonds in V H and V L are required in functional antigen binding.9Native IgG MAbs should not have any free sulfhydryl groups.7However, detailed examination of the free sulfhydryl groups in recombinant MAbs(one IgG1,two IgG2,and one IgG4)suggests presence of a small portion of free sulfhydryl group(approximately0.02mol per mole of IgG2or IgG4MAb and0.03for IgG1.7In rare cases,a free cysteine is found.A nondisulﬁde-bonded Cys at residue105was found on the heavy chain of a mouse monoclonal antibody,OKT3 (IgG2a).10OligosaccharidesThere is one oligosaccharide chain in IgGs.6This N-linked biantennary sugar chain resides mostly on the conserved Asn297,which is buried between the C H2domains.5,11For example,the oligosaccharide resides on Asn-297of the C H2 domain of chimeric IgG1and IgG3molecules12but on Asn299in a monoclonal antibody,OKT3 (IgG2a).10The oligosaccharide,often microheter-ogeneous,is typically fucosylated in antibodies produced in CHO or myeloma cell lines5and may differ in other cell lines.2,11There are many factors that dictate the nature of the glycan microheterogenity on IgGs.These include cell line,the bioreactor conditions and the nature of the downstream processing.An additional oligo-saccharide can be found in rare cases.A human IgG produced by a human-human-mouse hetero-hybridoma contains an additional oligosaccharide on Asn75in the variable region of its heavy chain.13In addition,O-linked carbohydrates could also exist in this antibody.Proper glycosylation is critical for correct functioning of antibodies.11It was demonstrated that removal of the oligosaccharide in IgGs(IgG1 and IgG3)made them ineffective in binding to C1q, in binding to the human Fc g RI and activating C; and generally more sensitive to most proteases than their corresponding wild-type IgGs(one exception).12This is because the binding site on IgG for C1q,theﬁrst component of the complement cascade,is localized in the C H2domains.11 Furthermore,the glycosylation can affect the antibody conformation.12Oligosaccharides in other regions can also play a critical role.Removal of an oligosaccharide in a Fv region of the CBGA1antibody resulted in a decreased antigen-binding activity in several ELISA systems.13In addition,this oligosaccharide might play critical role in reducing the antigenicity of the protein.14The sugar composition of the oligosaccharide is also critical in antibody functions.It has been shown that a low fucose(Fuc)content in the complex-type oligosaccharide in a humanized chimeric IgG1is responsible for a50-fold higher antibody-dependent cellular cytotoxicity(ADCC) compared with a high Fuc counterpart.15 HeterogeneityPuriﬁed antibodies are heterogeneous in struc-ture.This is true for all monoclonal antibodies (MAbs)due to differences in glycosylation pat-terns,instability during production,and terminal processing.5For example,ﬁve charged isoforms were found in recombinant humanized monoclo-nal antibody HER2as found by capillary iso-electric focusing(cIEF)and sodium dodecyl sulfate–capillary gel electrophoresis(SDS–CGE).16Six separate bands were focused under6WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007DOI10.1002/jpsIEF for two mouse monoclonal antibodies IgG2a (k)and IgG1(k).17A mature monoclonal antibody, OKT3(IgG2a),contain cyclized N-terminus (pyroglutamic acid,À17D)in both H and L chains, processed C-terminus(no Lys,À128D)of the H chains,and a small amount of deamidated form.10 Similar observation was also reported for a huma-nized IgG1(k).18In rare cases,gene cross-over may lead to formation of abnormal heavy chains.For example,a puriﬁed monoclonal anti-IgE antibody contains a small amount of a variant H chain, which had16fewer amino acid residues than the normal H chain(position is between Arg108of the L chain and Ala124of the H chain).19 Secondary and Higher-Order StructureThe basic secondary and higher-order structural features of IgGs have been reviewed.6Only a small portion of the three-dimensional structures of IgGs has been solved.20The antibody’s secon-day structure is formed as the polypeptide chains form anti-parallel b-sheets.The major type of secondary structure in IgGs is these b-sheets and its content is roughly70%as measured by FTIR.21The light chain consists of two and the heavy chain contains four domains,each about 110amino acid long.6,20All these domains have similar folded structures—b barrel,also called immunoglobulin fold,which is stabilized by a disulﬁde bond and hydrophobic interaction(pri-mary).These individual domains($12kDa in size)interact with one another(V H and V L;C H1 and C L;and between two C H3domains except the carbohydrate-containing C H2domain)and fold into three equal-sized spherical shape linked by a ﬂexible hinge region.These three spheres form a Y shape(mostly)and/or a T shape.22The less globular shape of IgGs is maintained both by disulﬁde bonds and by strong noncovalent interactions between the two heavy chains and between each of the heavy-chain/light-chain pairs.23Through noncovalent interactions,a less stable domain becomes more stable,and thus,the whole molecule can be stabilized.24A detailed study indicates that the interaction between two CH3domains are dominated by six contact residues,ﬁve of these residues(T366,L368, F405,Y407,and K409)forming a patch at the center of the interface.25These noncovalent interactions are spatially oriented such that variable domain exchange(switching V H and V L; inside-out IgG;ioIgG)induces noncovalent multimerization.26The six hypervariable regions in CDR(L1,L2, L3,H1,H2,and H3)form loops of a few predictable main-chain conformations(or canonical forms), except H3loop,which has too many variations in conformation to be predicted accurately.27,28 There is a slight difference in the loop composition and shape between the two types of light chains.20 However,no functional difference was found in antibodies having l or k chain.6Basic Functions of AntibodiesThe basic functions of antibodies have been reviewed.6There are two functional areas in IgGs—the V and C regions.The V regions of the two heavy and light chains offer two identical antigen-binding sites.The binding of the two sites (bivalent)can be independent of each other and does not seem to depend on the C region.29The exact antigen-binding sites are the CDR regions with participation of the frame work regions.30 Binding of antigens seems through the induced-ﬁt mechanism.31,32The induced-ﬁt mechanism allows multispeciﬁcity and polyreactivity.It has been suggested that about5–10residues usually contribute signiﬁcantly to the binding energy.32 The C regions of antibodies have three main effector functions(1)being recognized by receptors on immune effector cells,initiating antibody-dependent cell cytotoxicities(ADCC),(2)binding to complement,helping to recruit activated pha-gocytes,and(3)being transported to a variety of places,such as tears and milk.6In addition,C domains also modulate in vivo stability.23,29,33The function of Fc is affected by the structure of Fab. Variable domain exchange(switching V H and V L; inside-out IgG;ioIgG)affected Fc-associated func-tions such as serum half-life and binding to protein G and Fc g RI.26The hinge region providesﬂexibility in bivalent antigen binding and activation of Fc effector functions.26Two chimeric IgG3antibodies lacking a genetic hinge but with Cys residues in CH2 regions was found to be deﬁcient in their inter-molecular assembly,and both IgG3D HþCys and IgG3D Hþ2Cys lost greatly their ability to bind Fc g RI and failed to bind C1q and activate the complement cascade.34Alternative Forms of AntibodiesIn addition to species-speciﬁc antibodies,other antibody forms are generated to meet various needs.In the early development of antibody therapies,antibodies were made from murineANTIBODY FORMULATION7DOI10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007sources.However,these antibodies easily elicit formation of human anti-mouse antibody (HAMA).Therefore,humanized chimeric antibo-dies were generated.Chimeric monoclonal anti-bodies(60–70%human)are made of mouse variable regions and human constant regions.2 Such antibodies can still induce formation of human anti-chimeric antibody(HACA).Highly humanized antibodies,CDR-grafted antibodies, are made by replacing only the human CDR with mouse CDR regions(90–95%human).2These antibodies are almost the same in immunogeni-city potential as completely human antibodies, which may illicit formation of human anti-human antibody(HAHA).Other alternative forms of antibodies have also been generated and these different forms have been reviewed.35Treatment with papain would cleave the N-terminal side of the disulﬁde bonds and generate two identical Fab fragments and one Fc fragment.Fab0s are50kDa(V HþC H1)/ (V LþC L)heterodimers linked by a single disul-ﬁde bond.Treatment with pepsin cleaves the C-terminal side of the disulﬁde bonds and pro-duces a F(ab)02fragment.The remaining H chains were cut into several small fragments.6Cleavage by papain occurs at the C-terminal side of His-H22836or His-H227.37Reduction of F(ab0)2will produce two Fab0.23Fv fragments are noncovalent heterodimers of V H and V L.Stabilization of the fragment by a hydrophilicﬂexible peptide linker generates single-chain Fv(scFvs).2Fragments without constant domains can also be made into domain antibodies (dAbs).These scFvs are25–30kDa variable domain (V HþV L)dimers joined by polypeptide linkers of at least12residues.Shorter linkers(5–10residues)do not allow pairing of the variable domains but allow association with another scFv form a bivalent dimer (diabody)(about60kDa,or trimer:triabody about 90kDa).38Two diabodies can be further linked together to generate bispeciﬁc tandem diabody (tandab).39Disulﬁde-free scFv molecules are rela-tively stable and useful for intracellular applica-tions of antibodies—‘‘intrabodies.’’38The smallest of the antibody fragments is the minimal recognition unit(MRU)that can be derived from the peptide sequences of a single CDR.2ANTIBODY INSTABILITYAntibodies,like other proteins,are prone to a variety of physical and chemical degradation path-ways,although antibodies,on the average,seem to be more stable than other proteins.Antibody instabilities can be observed in liquid,frozen,and lyophilized states.The glycosylation state of an antibody can signiﬁcantly affect its degradation rate.40In many cases,multiple degradation path-ways can occur at the same time and the degrada-tion mechanism may change depending on the stress conditions.41These degradation pathways are divided into two major categories—physical and chemical instabilities.This section will explore the possible degradation pathways of antibodies and their inﬂuencing factors.Physical InstabilityAntibodies can show physical instability via two major pathways—denaturation and aggregation. DenaturationAntibodies can denature under a variety of conditions.These conditions include temperature change,shear,and various processing steps. Compared with other proteins,antibodies seem to be more resistant to thermal stress.They may not melt completely until temperature is raised above708C,21,42,43while most other mesophilic proteins seem to melt below708C.44Shear may cause antibody denaturation.For example,the antigen-binding activity of a recombinant scFv antibody fragment was reduced with aﬁrst-order rate constant of0.83/h in a buffer solution at a shear of approximately20,000/s.45Lyophilization can denature a protein to var-ious extents.An anti-idiotypic antibody(MMA 383)in a formulation containing mannitol,sac-charose,NaCl,and phosphate was found to loose its in vivo immunogenic properties(only10–20% of normal response rate)upon lyophilization.46 Since the protein showed no evidence of degrada-tion after lyophilization,no change in secondary structure by CD(29%b-sheet,14%a-helix,and 57%‘‘other’’),the loss of activity was attributed to the conformational change.Indeed,tryptophan ﬂuorescence properties were different between the lyophilized and unlyophilized antibodies.46 AggregationAntibody aggregation is a more common manifes-tation of physical instability.The concentration-dependent antibody aggregation was considered the greatest challenge to developing protein formulations at higher concentrations.47This is8WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007DOI10.1002/jps。

大口黑鲈的营养需要研究进展____

动物科学现代农业科技2011年第21期大口黑鲈(Micropterus salmoides ),俗称加州鲈,原产于美国加利福尼亚州,隶属鲈形目(Perciformes ),太阳鱼科(Ceutrarchidae )。

20世纪80年代初引入我国,由于其生长快、病害少、耐低温、肉多刺少、味道鲜美及营养丰富等优点,已成为我国养殖的主要淡水鱼品种之一。

大口黑鲈属典型淡水肉食性鱼,迄今尚未成功开发出营养平衡的全价专用饲料,尤其全程使用饲料一直是业界的一大难题,表现在中后期经常出现生长慢、厌食、肝脏疾病等问题[1]。

虽然大口黑鲈的养殖在国内外均有一定的规模,而且饲料成本占养殖成本的比例较高,但有关大口黑鲈营养需要的研究仍十分缺乏[2]。

在国外,大部分大口黑鲈的养殖,均采用比较容易获得的其他肉食性鱼类如鲑鱼和鳟鱼的饲料,而非采用针对大口黑鲈自身营养需要配制的专用饲料[3]。

在国内,养殖户投喂的饲料多以冰鲜下杂鱼和其他动物性饲料为主,这对海洋资源无疑是一种浪费,同时对养殖环境的污染也十分明显,容易引起各种疾病的暴发[4]。

按大口黑鲈2010年的产量测算,我国潜在的鲈鱼专用饲料需求可达20万t/年[1]。

对配合饲料的需要日益增加,亟待进一步全面开展其营养需要的研究。

因此,该文综述了国外内大口黑鲈营养需要的研究进展,并参考其他鱼类的营养需要,比较全面地总结了大口黑鲈对饲料中各营养素的需要量,以期为大口黑鲈专用饲料的研发和配制提供参考。

1大黑鲈对各种营养成分的需要量1.1蛋白质和氨基酸由于没有专门为大口黑鲈开发的商用饲料,目前在国外均采用其他肉性鱼类的饲料(蛋白质含量>40%,鱼粉含量50%~70%)[5-8]。

最早关于大口黑鲈饲料蛋白质营养需要的研究见于1981年[5]。

研究发现,0~1龄的大口黑鲈对饲料中蛋白质的需要量为39.9%~40.8%(基于饲料干物质)。

以饲料中水分含量为10%来计算的话,蛋白质含量为36%~37%(饲料湿重)即可满足1龄及之前的大口黑鲈鱼的生长。

比伐卢定简介

比伐卢定旳作用机制
比伐卢定不与血浆蛋白，红细胞结合，不与P450作用，在血浆中游离存在，静脉注射后5min能够达峰。以浓度方式延长血清中旳活化部分凝血酶时间（aPPT)、凝血时间（TT)、凝血酶原时间（PT)。血清中游离旳及血栓中结合旳凝血酶均能被克制，不受血小板释放物质旳影响。进入人体内被蛋白酶清除，经肾脏排出，在肾功能正常状体下半衰期为25min。
比伐卢定-----进口品未进国内，深圳信立泰产品将于今年年底上市，单纯抗IIa因子克制剂，主推PCI手术
比伐卢定旳作用机制
活性部位
活性部位 N
C
结合部位
结合部位
凝血酶
活性部位
N
C
结合部位
直接旳、特异旳、可逆性克制凝血酶(Ⅱa)
Maraganore JM ,etc . Biochemistry ,1990 ,29 (30) :7095.
PCI
PPI
比伐卢定旳利用
急性心肌梗死（AMI） HIT:血小板降低症
VBT：管内放射手术
ห้องสมุดไป่ตู้
比伐卢定旳作用机制
因凝血酶可水解本品多肽顺序中 Arg3 和 Pro4 之间旳肽键 ,使本品失活，所以本品对凝血酶旳克制作用是可逆而短暂旳。
Phe Pro Arg Pro Gly Gly Gly Gly Ans Gly
Glu Ile Glu Glu Phe Asp Gly Asn Glu Try Leu Maraganore JM ,etc . Biochemistry ,1990 ,29 (30) :7095.
比伐卢定旳理化性质
中文名：比伐卢定英文名称：bivalirudin, AngiomaxTM（Medicines 企业）抗凝成份:一种20肽旳水蛭素衍生片段,分子量： 2180,剂型：注射剂

Water-Splitting Electrocatalysis in Acid Conditions Using RuthenateIridate Pyrochlores-s

Figure
S1:
Refined
powder
XRD
patternsfor来自(a)(Na0.33Ce0.67)2(Ir)2O7
(b)
(Na0.33Ce0.67)2(Ir0.5Ru0.5)2O7 and (c) (Na0.33Ce0.67)2(Ru)2O7. In (a) * and # indicate small amounts of IrO2 and CeO2 impurity, respectively.
3
TGA/DSC (using a Mettler-Toledo TGA/DSC1 instrument) were recorded under a flow of air with a 10 °C min-1 heating rate. All show 5 % surface water/hydroxide mass loss to 600 ° C. The Ircontaining materials additionally show mass loss at >900 °C which corresponds to reduction to Ir metal; this generally increases with increasing Ir content in the series of materials (athough the mass loss is not always complete at 1000 °C.
4
Polarisation curves were obtained by logarithmically spaced galvanostatic steps with electrochemical impedance spectroscopy at each step. The EIS curves were fitted using a resistor, inductor, and Cole-element in series as the equivalent circuit using ZView. Exhaust gas was fed to a Spectra mass spectrometer and continuously monitored throughout the entire test protocol for oxygen evolution and carbon corrosion products from competing side reactions. Electrochemical work at Diamond Light Source used a Hg/Hg2SO4 (MMS) reference electrode in 0.5M H2SO4 which was calibrated against a hydrogen reference electrode off site at 0.68V vs RHE such that 1.00 V vs MMS signal was equivalent to 1.68 V vs RHE. Table S2 shows the mass activity and onset for oxygen evolution data collected during voltammetry. Also included in the table are the fit parameters found for data collected at 10 mA cm2 during polarisation. The parameters indicate DC resistance, R0, charge transfer resistance, Rct, capacitive time constant, τ, and phase, α.

不同酶消化法提取猪原代肝细胞的效果比较

532024.4·试验研究0 引言猪圆环病毒（PCV ）是Circoviridae 科Circovirus 属的一种无囊膜的单链环状DNA 病毒。

在已知的4个血清型中，PCV2为猪易感的致病性病毒[1]。

PCV2感染会诱导宿主免疫抑制引起猪圆环病毒病（PCVD ），包括断奶仔猪多系统衰竭综合征、新生仔猪先天性脑震颤、皮炎与肾病综合征、猪呼吸道病综合征、母猪繁殖障碍等，给全世界养猪业带来较大的经济损失，是世界各国的兽医与养猪业者公认的造成重大影响的猪传染病[2]。

PCV2的感染在猪生长发育的不同阶段有不同的组织嗜性。

但无论是胎儿阶段还是出生后，肝细胞都是PCV2感染和复制的靶细胞。

因此，PCV2也被视为一种能够诱导猪肝炎的病毒[3]。

且PCV2诱导的肝细胞凋亡在PCV2引发的相关病变和疾病的发病机制中具有关键性作用[4]。

因此，方便、快捷地获取大量有活性的猪肝细胞对于研究PCVD 的致病机制具有重大意义。

目前获取肝细胞常用的方法主要包括机械分离细胞法、非酶分离细胞法、离体酶消化法和酶灌流法等[5]。

因此，本试验采用简便、经济、无需特殊设备、仅需部分肝组织的离体酶消化法，比较不同酶消化分离猪原代肝细胞的效果，为一般实验室提取分离大量有活性的猪肝细胞提供参考。

1 材料与方法1.1 材料1.1.1 主要试剂新鲜猪肝组织，Hank's 平衡盐溶液（HBSS ），磷酸盐缓冲液（无菌PBS ），4%多聚甲醛（PFA ），收稿日期：2024-01-27基金项目：国家自然科学基金项目：复杂器官与组织在脾脏内的功能性再生（32230056）作者简介：周徐倩（1999-），女，汉族，浙江温州人，硕士在读，研究方向：组织工程与再生医学。

*通信作者简介：董磊（1978-），男，汉族，安徽阜阳人，博士，教授，研究方向：组织工程与再生医学、生物材料。

周徐倩，董磊．不同酶消化法提取猪原代肝细胞的效果比较[J]．现代畜牧科技，2024，107（4）：53-55．　doi ：10.19369/ki.2095-9737.2024.04.014．　ZHOU Xuqian ，DONG Lei ．Comparison of the Eﬀect of Diﬀerent Enzyme Digestion Methods on Extraction of Porcine Primary Hepatocytes[J]．Modern Animal Husbandry Science & Technology ，2024，107（4）：53-55．不同酶消化法提取猪原代肝细胞的效果比较周徐倩，董磊*（南京大学，江苏南京 210023）摘要：猪肝细胞是猪圆环病毒的靶细胞，简单快速地提取猪原代肝细胞对于研究猪圆环病毒病的致病机制具有重要意义。

泛昔洛韦

密度：1.4g/cm3 熔点：102-104°C 沸点： 5 5 0 . 2 ºC 闪点： 2 8 6 . 6 ºC 折射率：1.628 外观：灰白色粉末溶解性：易溶于丙酮或甲醇，难溶于乙醇或异丙醇
摩尔折射率：82.66 摩尔体积（cm3/mol）：239.8 等张比容（90.2K）：639.3 表面张力（dyne/cm）：50.4 极化率（10-24cm3）：32.77
进入人体内后迅速转变成喷昔洛韦，喷昔洛韦可被病毒编码的胸苷激酶磷酸化成OCV单磷酸，再经宿主的磷酸化成为喷昔洛韦三磷酸盐，三磷酸盐在病毒感染的细胞内迅速形成，缓慢代谢，致半衰期延长，参与HBV DNAp的三磷酸鸟苷(Pgtp)竞争，并进入DNA，作用于DNA合成的起始和延伸步骤，抑制DNA的合成，对水痘-带状疱疹病毒、单纯疱疹病毒1型和2型和HBV均有较强的抑制作用。
泛昔洛韦
药品
目录
01 化合物简介
03 药典信息
02 药品简介 04 安全信息
泛昔洛韦，是一种有机化合物，化学式为C14H19N5O4，是第二代开环核苷类抗病毒药，主要用于疱疹病毒感染，尤其是带状疱疹。
化合物简介
基本信息理化性质
分子数据化学数据
化学式：C14H19N5O4 分子量：321.332 CAS号：-87-4
疏水参数计算参考值（XlogP）：无氢键供体数量：1 氢键受体数量：8 可旋转化学键数量：9 互变异构体数量：3 拓扑分子极性表面积：122 重原子数量：23 表面电荷：0 复杂度：404 同位素原子数量：0 确定原子立构中心数量：0
药品简介
作用机理
药代动力学
不良反应
泛昔洛韦口服迅速吸收，生物利用度77%，在体内很快转达变为喷昔洛韦，t1/2约为2h，约60-65%经肾排出。在水痘-带状疱疹病毒感染的细胞内有一个较长的半衰期（9-10小时），单纯疱疹病毒1型和2型感染的细胞内半衰期分别为10小时和20小时。

Comparedrugreleaseprofiles

Compare drug release profiles of water poor soluble drugs from a novel chitosan and polycarbophil interpolyelectrolytes complexation (PCC) and hydroxylpropyl - methylcellulose (HPMC) based matrix tabletsZhilei Lu*, Weiyang Chen, Eugene Olivier, Josias H., HammanDepartment of Pharmaceutical Sciences, Tshwane University of Technology, Private Bag X680, P retoria, 0001, South Africa资料个人收集整理，勿做商业用途*Corresponding author:Zhilei Lu (Dr.)Department of Pharmaceutical Sciences,Tshwane University of Technology,Private Bag X680,Pretoria, 0001,South Africa (e-mail: luzj@tut.ac.za)AbstractThe aim of this study was to compare the drug release behaviours of water poor soluble drugs from an interpolyelectrolyte complex (IPEC) of chitosan with polycarbophil (PCC) and hydroxylpropylmethylcellulose (HPMC) based matrix tablets. A novel interpoly - electrolyte complex (IPEC) of chitosan with polycarbophil (PCC) was synthesized and characterized. Water poor soluble drugsHydrochlorothiazide and Ketoprofen were used in this study as model drugs.Polymers (including PCC, HPMC K100M and HPMC K100LV) based matrix tablets drug controlled release system were prepared using direct compression method.The results illustrate PCC based-matrix tablets offer a swelling controlled release system for water poor soluble drug and drug release mechanism from this matrix drug delivery system can be improved by addition of microcrystalline cellulose (Avicel).Analysis of the in vitro release kinetic parametersof the matrix tablets, PCC based matrix tablets exhibited similar or higher drug release exponent (n) and mean dissolution time (MDT) values compared to the HPMC based matrix tablets. It demonstrated that PCC polymer can be successfully used as a matrixcontrolled release system for the water poor soluble model drugs such as hydroxylpropylmethylcellulose (HPMC). 资料个人收集整理，勿做商业用途1 IntroductionOver the last three decades years, as the expense and complications involved in marketing new drug entities have increased, with concomitant recognition of the therapeutic advantages of controlled drug delivery, greater attention has been focused on the development of novel and controlled release drug delivery systemsto provide a long-term therapeutic of drugs at the site of action following a single dose (Mandal, 2000; Jantzen and Robinson, 2002). Many formulation techniques have been used tobuild t”he barrier into the peroral dosage form to provide slow release of the maintenance dose. These techniques include the use of coatings, embedding of the drug in a wax, polymeric or plastic matrix, microencapsulation, chemical bindingto ion-exchange resins and incorporation into an osmotic pump (Collett and Moreton, 2002:293). Among different technologies used in controlled drug delivery, polymeric matrix systems are the most majority because of the simplicity of formulation, ease of manufacturing, low cost and applicability to drugs with wide range of solubility (Colombo, et al., 2000; Jamzad and Fassihi, 2006). 资料个人收集整理，勿做商业用途Drugs release profiles from polymeric matrix system can influence by different factors, but the type, amount, and physicochemical properties of the polymers used play a primary role (Jamzad and Fassihi, 2006). Hydroxylpropyl-methylcellulose (HPMC) is the most important hydrophilic carrier material used for oral drug sustained delivery systems (Colombo, 1993; Siepmann and Peppas, 2001). BecauseHPMC is water soluble polymer, it is generally recognized that drug release fromHPMC matrices follows two mechanisms, drug diffusion through the swelling gellayer and release by matrix erosion of the swollen layer (Ford et al., 1987; Raoet al., 1990; Colombo, 1993; Tahara et al., 1995; Reynolds, Gehrke et al., 1998; Siepmann et al., 1999; Siepmann and Peppas, 2001). However, diffusion, swelling and erosion are most important rate-controlling mechanisms of commercial available controlled release products (Langer and Peppas, 1983), the major advantages of swelling/erosionHPMC based matrix drug delivery system are: (i) minimum the drug burst release; (ii) the different physicochemical drugs release rate approach a constant; (iii) the possibility to predict the effect of the device design parameters (e.g. shape, size and composition of HPMC-based matrix tablets) on the resulting drug release rate, thus facilitating the development of new pharmaceutical products (Colombo, 1993;Siepmann and Peppas, 2001资).料个人收集整理，勿做商业用途Interpolyelectrolyte complexes (IPEC) are formed as precipitates by two oppositely charged polyelectrolytes in an aqueous solution, have been reported as a new class of polymer carriers, which play an important role in creating new oral drug delivery systems (Peppas and Khare, 1993; Berger et al., 2004). A variety chemical structure and stoichiometry of both components in interpolyelectrolyte complexes depends onthe pH values of the media, ionic strength, concentration, mixing ratio, and temperature (Peppas, 1986; Dumitriu and Chornet, 1998; Berger et al., 2004;Moustafine et al., 2005a). Chitosan is a positively charged (amino groups) deacetylated derivative of the natural polysaccharide, chitin (Paul and Sharma, 2000).Chitosan has already been successfully used to form complexes with natural anionic polymers such as carboxymethylcellulose, alginic acid, dextran sulfate,carboxymethyl dextran, heparin, carrageenan,pectin methacrylic acid copolymers ? (Eudragit polymers) and xanthan (Dumitriu and Chornet, 1998, Berger et al., 2004,Sankalia et al., 2007, Margulis and Moustafine, 2006)资. 料个人收集整理，勿做商业用途In this study, a novel polymer - IPEC between chitosan and polycarbophil (PCC) was synthesized, characterized and used as direct compressedexcipients in the matrix tablet. Although it have been well known that various IPEC have been used as a polymer carriers in drug controlled release system (Peppas and Khare, 1993, Garcia and Ghaly, 1996, Lorenzo-Lamoza et al., 1998, Soppirnath and Aminabhavi, 2002,Chen et al., 2004, Nam et al., 2004, Moustafine et al., 2005b), IPEC chitosan and polycarbophil was used as a polymer carriers have been investigated by Lu et al., (2006, 2007a, 2007 b, 2008a; 2008b资料个人收集整理，勿做商业用途The aim of this study was to comparein vitro water poor soluble drugs release profile of HPMC based matrices system to PCC based matrices system at same formulation.Water poor soluble model drugs Hydrochlorothiazide and ketoprofen were used in thisstudy. Two types HPMC (K100M and K100LV) and PCC polymers were used indirect compressedpolymers based matrix drug release system. The results of the hydration and erosion studies showed PCC based matrix systems have superior swelling properties. Drug release exponent (n) of each formulation PCC based matrices tablets are higher than HPMC based matrices tablets at pH 7.4 buffer solutions. It demonstrated that PCC has high potential to use in polymer based matrix drug con trolled released delivery for water poor soluble drugs资料个人收集整理，勿做商业用途2. Materials and methods2.1 MaterialsChitosan (Warren Chem Specialities, South Africa, Deacetylation Degree =91.25%),Polycarbophil (Noveon, Cleveland, USA), Hydroxylpropylmethylcellulose (MethocelK100M, K100LV Premium, Colorcon Limited, Kent, England), Ketoprofen (Changzhou Siyao Pharma. China), Hydrochlorothiazide (Huzhou Konch Pharmaceutical Co., Ltd. China), Microcrystalline cellulose (Avicel, pH101, FMC corporation NV, Brussels, Belgium), Sodium carboxymethyl starch (Explotab, Edward Mendell Co., Inc New York, USA). All other chemicals were of analytical grade and used as receive资料个人收集整理，勿做商业用途2.2 Preparation of interpolyelectrolytes complexation between chitosan and P olycarb op hil (PCC)资料个人收集整理，勿做商业用途Chitosan (30 g) was dissolved in 1000 ml of a 2% v/v acetic acid solution andpolycarbophil (30 g) was dissolved in 1000 ml of a 2% v/v acetic acid solution. Thechitosan solution was slowly added to the polycarbophil solution underhomogenisation (5200 rpm, ZKA , Germany) over a period of 20 minutes. Themixture was then mechanically stirred for a period of 1 hour at a speed of 1200 rpm(Heidolph RZR2021, Germany). The gel formed was separated by centrifuging for 5 min at 3000 rpm and then washed several times with a 2% v/v acetic acidsolution toremove any unreacted polymeric material. The gel was freeze dried for a period of 48 hours (Jouan LP3, France) and the lyophilised powder was screened through a 300prn sieve资料个人收集整理，勿做商业用途2.3Differential scanning calorimetry (DSC)DSC thernograns of the PCC were recorded with a Shinadzu DSC50 (Kyoto, Japan) instrument. The thermal behaviour was studied by sealing 2 mg samples of the material in aluminium crimp cells and heating it at a heating rate of 10o C per min under the flow of nitrogen at a flow rate of 20 ml/min. The calorimeter was calibrated with 2 mg of indium (Kyoto, Japan, melting point 156.4o C) at a heating rate of 10o C per min.资料个人收集整理，勿做商业用途2.4Fourier transforn infrared (FT-IR)Fourier transforn infrared (FT-IR) spectral data of the PCC polyner was obtained on a FTS-175C spectrophotoneter (BIO-RAD, USA) using the KBr disk nethod. 资料个人收集整理，勿做商业用途2.5Preparation of the natrix tabletsIn order to conpare the release profiles of water poor soluble drugs fron polyner based natrix tablets, nonolithic natrix type tablets containing hydrochlorothiazide or ketoprofen were prepared by conpressing a nixture of the ingredients with varying concentrations of the PCC, HPMC K100M and HPMC K100LV as indicated in Table 1. The ingredients of the different fornulationswere nanually pre-nixed by stirring in a 1000 nl glass beaker for 30 ninutes with a spatula. After the addition of 0.05 g of nagnesiun stearate (0.5% w/w), the powder nass was nixed for 10 nin. The powdernixture was conpressed using a rotating tablet press (Cadnach, India) fitted with round, shallow pun ches to p roduce matrix type tablets with a 6 mm diameter资料个人收集整理，勿做商业用途26 Weight, hard ness, thick ness and friability of tablets资料个人收集整理，勿做商业用途Weight variation was tested by weighing 20 randomly selected tablets individually, the n calculati ng the average weight and comparing the in dividual tablet weights to the average. The specification for weight variation is 10% from the average weight if the average weight < 0.08 g (USP 2006资料个人收集整理，勿做商业用途The hardnessof ten randomly selected matrix type tablets of each formulation was determined using a hardness tester (TBH 220 ERWE K A, Germany). The force (N) n eeded to break each tablet was recorded料个人收集整理，勿做商业用途The thick ness of each of 10 ran domly selected matrix type tablets were measured witha vernier calliper (accuracy = 0.02 mm). The thickness of the tablet should be within 5% variation of the average value资料个人收集整理，勿做商业用途A friability test was con ducted on the tablets using an Erweka Friabilator (TA3R,Germany). Twenty matrices were randomly selected from each formulation and any loose dust was removed with the aid of a soft brush. The selected tablets were weighed accurately and p laced in the drum of the friabilator. The drum was rotated at 25 rpm for 4 minu tes after which the matrices were removed. Any loose dust was removed from the matrices before they were weighed again. The friability maximal limit is 1% (USP 2006) was calculated using the following equation资料个人收集整理，勿做商业用途F (%) = W before (g)「W曲(g)X 100%(1)W after (g)Where F is the friability, W before is the initial weight of the matrices and W after is the weight of the matrices after test ing资料个人收集整理，勿做商业用途2.7 Swelli ng and erosi on studiesSwelling and erosion studies were carried out for all formulations matrix tablets. The matrices were weighed in dividually before they were pl aced in 900 ml p hos phate buffer (pH 7.4) at 37.0 0.寸C.± The medium was stirred with a paddle at a rotation speed of 50 rpm in a USP dissolution flask. At each time point, three tablets of each formulatio n were removed from the dissoluti on flask and gen tly wiped with a tissue toremove surface water, weighed and then placed into a plastic bowel. The matrix tablets were dried at 60°C until constant weight was achieved. The mean weights were determ ined for the three tablets at each time in terval. The data obta ined from this exp erime nt was used to calculate the swelli ng in dex and p erce ntage mass loss.料个人收集整理，勿做商业用途2.7.1Swelli ng indexThe swelli ng in dex (or degree of swelli ng) was calculated accord ing to the followi ngequati on资料个人收集整理，勿做商业用途s,=WJ—00W dWhere SI is the swelling index, W s and W d are the swollen and dry matrix weights, resp ectively, at immersio n timet in the buffer soluti on.资料个人收集整理，勿做商业用途2.7.2P erce ntage of matrix erosi onThe p erce ntage of matrix erosi on is calculated in relatio n to the in itial dry weight of the matrices, accord ing to the followi ng equation 资料个人收集整理，勿做商业用途Erow 册件"00%Where: dry weight (t) is the weight at time t.28 Assay of hydrochlorothiazide and ket oprofen in matrix tablets.料个人收集整理，勿做商业用途The drug content of the matrix type tablets was determ ined by crush ing 10 ran domly selected tablets from each formulatio n in a mortar and p estle. App roximately 80 mg po wder from the hydrochlorothiazide or ket oprofen containing matrices were weighed accurately and individually transferred into a 200 ml volumetric flasks, which were then made up to volume with p hos phate buffer soluti on (pH 7.4). This mixture was stirred for 30 minutes to allow compiete release of the drug. After filtration through a 0.45 阿filter membrane, the solution was assayed using ultraviolet (UV) spectrophotometry (Helios a Thermo , England) at a wavelength of 271 nm for hydrochlorothiazide and 261 nm for ketoprofen. The assay for drug content wasperformed in triplicate for each formulation. The percentage drug content of the tablets was calculated by mea ns of the followi ng equation:料个人收集整理，勿做商业用途DC (% w/w^W dru^x100%WmtWhere DC is the drug content, W drug is the weight of the drug and W mt is the weight of the matrix tablet .资料个人收集整理，勿做商业用途2.9 Release an alysisThe USP (2006) dissoluti on app aratus 2 (i.e. p addle) was used to determ ine the in vitro drug release from the different polymers based matrix tablets. The dissolution medium (900 ml) consisted of phosphate buffer solution (pH 7.4) at 37 0.5 o C and a ± rotation speed of 50 rpm was used. Three hydrochlorothiazide or ketoprofen matrix tablets of each formulatio n were in troduced into each of three dissoluti on vessels (i.e.?in triplicate) in a six station dissolution apparatus (TDT-08L, Electrolab , India).Samp les (5 ml) were withdraw n at sp ecially in tervals, and 5 ml of p reheated dissolution medium was replaced immediately. Sink conditions were maintained throughout the study. The samp les were filtered through a 0.45 阿membra ne, hydrochlorothiazide or ketoprofen content in the solution was determined using ultraviolet (UV) spectrop hotometry at a wavele ngth of 271 or 261 nm, res pectively.An alyses were p erformed in tripli cate资料个人收集整理，勿做商业用途2.9.1 Kin eticCon trolled release drug delivery systems may be classified accord ing to their mecha ni sms of drug release, which in cludes diffusi on-con trolled, dissoluti on con trolled, swelli ng con trolled and chemically con trolled systems (La nger et al., 1983). Drug release from sim pie swellable and erosi on systems may be described by the well-known power law expression and is defined by the following equation(Ritger and Pepp as, 1987; P illay and Fassihi, 1999资料个人收集整理，勿做商业用途Where M t is the amount of drug released at time t, M is the overall amount of drug released, K is the release con sta nt; n is the release or diffusi onal exponent; M/M is the cumulative drug concen trati on released at time t (or fractio nal drug release)料个人收集整理，勿做商业用途The release exponent (n) is used for the in terpretati on of the release mecha nism from poly meric matrix con trolled drug release systems (Peppas 1985). For the case of < 0.45 corrosFickdantdiffusi on release (Case I<an89homalous (non-Fickia n) transport, n = 0.89 toa zero-order (Case II) release kin etics, and n > 0.89to a super Case II transport (Ritger and Pepp as, 1987资料个人收集整理，勿做商业用途 The dissoluti on data were modelled by using the Po wer law equati on (Eq 7) withgraphs analysis software (Origin Scientific Graphing and Analysis software, Version 7, Origi nLab Corpo rati on, USA) using the Gaussia n-Newt on (Leve nberg-Hartely) app roach 资料个人收集整理，勿做商业用途2.9.2 Mea n dissolutio n time (MDT)MDT is a statistical moment that describes the cumulative dissolution process and provides a quantitative estimation of the drug release rate. It is defined by the following equation (Reppas and Nicolaides, 2000; Sousa^t al., 2002):资料个人收集整理，勿做商业用途nMDTt i M t/M^ i =±Where MDT is the mean dissolution time, M t is the amount of the drug released at time t; t i is the time (min) at the midpoint between i and i-1 and M 乂 is the overall amount of the drug released.料个人收集整理，勿做商业用途cyli ndrical, i n sp ecially, n diffusi on al), 0.45 < n2.9.3 Differe nt factor f i and Similarityfactor f 2 The different factor f i is a measure of the relative error between two dissoluti on curves and the similarity factor f 2 is a measure of similarity in the p erce ntage dissoluti on betwee n two dissoluti on curves (Moore and Fla nn er, 1996). Assu ming that the p erce ntage dissolved values for two p rofiles cannot be higher tha n 100, the differe nt factor f 1 can have values from 0 (whe n no differe nee the two curves exists) to 100 (when maximum differenee exists). With the same assumption holding, the similarity factor f 2 can have values from 100 (when no differenee between the two curves exists) to 0 (when maximum differenee exists) (Pillay and Fassihi, 1999;Moore and Fla nn er, 1996; Re ppas and Nicolaides, 2000). In this study, these factors were used to confirm the relative of release p rofiles of water poor soluble model drugs from poly mers based matrix tablets of the same formulati ons. They are defi ned bythe followi ng equati ons:资料个人收集整理，勿做商业用途 f^100xn Z |Rt —Tt t 吕 n z R f2"0^「100hXG (Rt -T , I V ny 丿］Where n is the number of sample withdrawal points, R t is the percentage of the refere nee dissolved at time t, T is the p erce ntage of the test dissolved at time 资料个人收集整理，勿做商业用途 3 Results and discussion 3.1 Prep arati on and characterisati on ofPCC The ion ic bond of the interpo "electrolyte comp lex (IP EC) betwee n chitosa n andpo lycarb op hil was con firmed by means p reviously p ublished differe ntial sea nning calorimetry (DSC) (Lu et al., 2007b) and Fourier tran sform in frared (FT-IR). Fig.1shows the FT-IR sp ectra of chitosa n, po lycarb op hil and the PCC poly mer.资料个人收集整理，勿做商业用途-1A peak that appears at 1561 cm on the IR spectrum of the PCC, which might be assigned to the carboxylate groups that formed ionic bonds with the protonated amino groups of chitosan as previously illustrated for the interaction between Eudragit E andEudragit L (Moustafine at al., 2005). This ionic bond seems to be the primary bin di ng force for the formatio n of a comp lex betwee n chitosa n and po lycarb op hil.资料个人收集整理，勿做商业用途Chitosan is a cationic polymer of natural origin with excellent gel and film forming properties. Polycarbophil can also be considered as polyanions with negatively charged carboxylate groups. Mixing chitosan and polycarbophil in acidic solution (2% acetic acid solution was used in the study), ionic bonds should form between the protonated free amino groups of chitosan and carboxylate groups of polycarbophil.According to the results obtained from DSC and FT-IR, the possible process of formatio n of interpo "electrolyte comp lexes may be described as illustrated in Fig.2资料个人收集整理，勿做商业用途3.2Physical characteristics and drug content of poly mers based matrix tablets^ 料个人收集整理，勿做商业用途As summarised in Table 2, the physical characteristics of matrix tablets showed the good thickness uniformity, as ranged from 3.40 0.04 to 4.12± 0.0±4mm, a variationof matrix tablets weight from 73.3 2.4 mg to±87.9 4.0±mg, furthermore the weight variation of all formulation tablets is very low (< 10% from the average weight) (USP 2006). Hardness of the matrix tablets shows a range from 68 ±14 to 94 ±12N.The tablets also pasted the friability test (<1%), confirm that all formulations tablets are within USP (2006) limits. Drugs content of all formulations ranged from 4.60 0.65 to 5.01 0.1±1%.资料个人收集整理，勿做商业用途3.3Swelli ng and erosi on prop erties of the poly mers based matrices tablets资料个人收集整理，勿做商业用途Investigation of matrix hydration and erosion by gravimetrical analysis is a valuable exercise to better understand the mechanism of release and the relative importance of participating parameters (Jamzad and Fassihi, 2006). Fig.3 and Fig.4 illustrate the water uptake profiles and Fig.5， Fig.6 illustrate percentage of matrix erosion of all formulation tablets, respectively. Swelling properties of the all formulation matrix tablets based on the content of PCC, HPMC K100M and HPMC K100LV in the matrices tablets. Water uptake and percentage of matrix erosion values of these matrix tablets show superior swelling characteristics either HPMC K100LV based matrix tablets, or PCC based matrix tablets. 资料个人收集整理，勿做商业用途IPEC betwee n chitosa n and po lycarb op hil is a three -dime nsional n etworks water insoluble poly (acrylic acid) polymer with free hydroxy groups. Hydroxy groups ofPCC contribute hydrophilic capacity significantly and polymer erosion characteristics depend on the reaction ratio of chitosan and polycarbophil while polymer synthesis.While the PCC based matrixes were put into the buffer solution, the electrostatic repulsion between fixed charges (hydroxy groups) uncoiled the polymers chains.The counterion diffusion inside the PCC gel creates an additional osmotic pressure difference across the gel, consequently lead to higher water uptake (Peppas and Khare, 1993; Lu, et al., 2007b). During the matrix erosion, the ionic bonds between chitosan and polycarbophil were not broken by the matrix swelling. PCC based matrix tablets (F1 and F7 formulation) have superior swelling behaviors compare to the HPMC based matrices. Swelling index values of F1 and F7 formulation matrix tablets are 1599.62±216.68 % and 1579.82 ±118.05 % at 12 hours, respectively.Furthermore, addition of microcrystalline cellulose (Avicel) can increase matrices erosion significantly. Compare the erosion behaviors of F1, F7 and F2, F8formulation (containing 20% Avicel), F1 and F7 matrix tablets erode 5.74 1.62 % and 6.59 1±.18 % on 12 hours only, cont rary F2 and F8 matrix tablets erode 55.59 1.43 and 100 % respectively. Microcrystalline cellulose (Avicel) is widely used in pharmaceutical, primarily as a binder/diluent, also has some disintegrant properties on oral tablet and capsule formulations where it is used in both wet granulation and direct-compression process (Wheatley, 2000). In this study, matrix erosion behaviours were act by microcrystalline cellulose facilitating the transport of liquid into the pore of matrix tablets. It demonstrates that PCC polymer have capacity to form swelli ng only or swelli ng-erosi on matrix drug delivery system.资料个人收集整理，勿做商业用途It also was confirmed that PCC based matrix tablets have much better swelling behaviors than HPMC based matrix tablets by comparing swelling curves in Fig.3 andFig.4. Swelling index values of F1 and F7 formulation matrix tablets are 1599.62216.68 % and 1579.82 11±8.05 % at 12 hours, contrary F3 and F9 matrix tablets are545.96 ±4.32% and 547.72 2±6.27%. HPMC K100LV based matrix tablets have excellence erosion curves in this study, F5, F6, F11 and F12 formulation matrixtablets eroded 100% on 12 hours, but F2 and F8 (PCC based tablets) formulation matrix tablets can eroded 55.59 ±1.43 and 100 % with microcrystalline cellulosefacilitating. 资料个人收集整理，勿做商业用途3.4Drug releaseIn vitro drug release was performed in pH 7.4 phosphate buffer solution for 12 hours.Results of percentage drug release versus time for hydrochlorothiazide and ketoprofen in different formulations matrices tablets are presented in Fig.7 and Fig. 8, while theMDT and drug release kinetics values were present in Table 3资.料个人收集整理，勿做商业用In this study, water poor soluble model drugs hydrochlorothiazide and ketoprofen release from polymers based matrix tablets was controlled by the polymer matrices swelling or swelling combination with erosion. Percentage of drug release, matrix swelling and erosion of F7 were summarised in Fig 9. The percentageketoprofen release curve follows the percentagematrix tablets swelling curve, it demonstrates that PCC based matrix drug delivery system is the swelling dependent drug release system for water poor soluble model drugs. Same as F7 matrix tablets, F1 matrix tablets is also a swelling only drug delivery system, in these matrix systems drugs release behaviour primarily depend on the matrix swelling characteristics. Because as the superior swelling capacity of PCC based matrix tablets, liquid environments inside of the matrix provide that the model drugs release are zero order drug release.As described in Table 3, release exponentsn)( of F1 and F7 are 0.83 0.03 a±nd 0.99 ± 0.02 during the experimental time, respectively. 资料个人收集整理，勿做商业用途Addition of microcrystalline cellulose (Avicel) influence the model drugs release profiles from PCC based matrix tablets significantly. Cumulative drug release of F2 and F8 formulation tablets is 93.7 4.13 % a±nd 99.6 4.2±5% at 12 hours, relativelyF1 and F7 formulation tablets is 73.8 1.13 % an±d 47.2 4.5±3 % only. This can be explained by drugs release mechanism were swelling and erosion instead of swelling only, consequently accelerate the drugs release. The adjustable capacity of PCC based matrix drug delivery system by addition of microcrystalline cellulose (Avicel) dem on strates the poten tial useful of PCC poly mer in drug con trolled release field 资料个人收集整理，勿做商业用途Compare to the PCC based matrix tablets, the drugs release profiles of HPMC based matrix tablets were adjusted difficultly. The relatives f1 and f2 values of difference polymers including PCC, HPMC 100M, HPMC 100LV based matrix tablets containing hydrochlorothiazide under same formulation were show in Table 4. As describedf1 and f2 values in Table 4, F3 and F4, F5 and F6 formulation tablets have similar drug release behaviours, but F1 and F2 formulation tablets illustrate different drug release behaviours. This phenomenacan be explained by the superior water uptake capacity of PCC polymer, more water containing can easier broken the physical tensility between the polymer particles. 资料个人收集整理，勿做商业用途However, HPMC 100LV polymer has excellence erosion characteristics, in this study model drugs release from HPMC 100LV based matrix tablets illustrate matrix erosion dependent properties. In generally, drug release from swelling and erosion matrix system shows zero order release pare the drug release exponentsn(), release constant (k1), and mean dissolution time (MDT) of F2 to F5, F6, they have not significantly different as described in Tablet 3, furthermore the relatives f1 and f2 values between F2 and F5, F6 in Table 4 show they are similar release profiles. It imply PCC based matrix tablets can become a swelling and erosion drug delivery system by the addition of microcrystalline cellulose (Avicel), this drug delivery system illustrate similar drug release p rofiles as HPMC 100LV based matrix tablets资料个人收集整理，勿做商业用途Although it is very complex process that the model drugs release from swelling and。

盐酸伐昔洛韦的合成路线

Valacyclovir盐酸伐昔洛韦的合成路线制药工程一班刘金贵3010207301Valacyclovir一中文名:维德思,盐酸伐昔洛韦,盐酸万乃洛韦二化学名称为：L-缬氨酸-2-（6-氧代-2-氨基-1，6-二氢- 9H -嘌呤-9-基）甲氧基乙基酯盐酸盐三化学结构式(如下图) 分子式C13-H20-N6-O4 分子量324.342四药代动力学本品口服进入人体后迅速分解为L-缬氨酸和阿昔洛韦，前者在体内参与正常生理生化代谢，后者在被疱疹病毒感染的细胞中，血中阿昔洛韦达峰时间为0.88～1.75小时。

口服生物利用度为67±13%，是阿昔洛韦的3～5倍。

药物进入体内后广泛分布，可分布至多种组织中，其中胃、小肠、肾、肝、淋巴结和皮肤组织中浓度最高，脑组织中的浓度最低。

药物在体内全部转化为阿昔洛韦，代谢物主要从尿中排除，其中阿昔洛韦占46%～59%，8-羟基-9-鸟嘌呤占25%～30%，9-羟基甲氧基鸟嘌呤占11%～12%。

阿昔洛韦原形为单相消除，血消除半衰期（t1/2β）为2.86±0.39小时。

五药理毒理药理作用:伐昔洛韦是一特异性疱疹病毒抑制剂，为阿昔洛韦(嘌呤核苷类似物)的L-缬氨酸酯。

伐昔洛韦在体内可能是通过伐昔洛韦水解酶迅速几乎完全转化为阿昔洛韦和缬氨酸。

阿昔洛韦在体外具有抑制单纯疱疹病毒(HSV)Ⅰ型和Ⅱ型、水痘带状疱疹病毒(VZV)、巨细胞病毒(CMV)、Epstein-Barr病毒(EBV)和人类疱疹病毒6(HHV-6)的作用。

病毒的胸苷激酶使其磷酸化，成为单磷酸化合物，再由细胞激酶磷酸化变成二磷酸和三磷酸化合物。

三磷酸化合物是抗病毒的活性物质，可抑制病毒的DNA聚合酶，终止其DNA合成，显示抗病毒效力。

由于本品是ACV 的氨基酸酯，没有游离羟基提供给磷酸化，因而在未转化为ACV之前，并无抗病毒活性，这一点使其不象其它前体药物如地昔洛韦(desciclovir)等另外增加对细胞的毒性。

7-羟基-5-甲氧基香豆素

7-羟基-5-甲氧基香豆素
7-羟基-5-甲氧基香豆素（7-Hydroxy-5-methoxy-coumarin）是一种天然的化合物，也称为法芙丽妥（Favipiravir），是近年来被广泛研究的一种治疗新冠病毒的药物。

它是一种香豆素衍生物，化学结构中含有一个7-羟基和一个5-甲氧基，具有多种生物活性。

此外，它还被广泛应用于农业、食品和医药等领域。

生物活性
1. 抗病毒作用：法芙丽妥是近年来研究较多的新冠病毒治疗药物之一，早期研究表
明它能够有效抑制新冠病毒复制，缩短患者病程。

2. 抗氧化作用：法芙丽妥具有明显的抗氧化作用，能够清除自由基，保护细胞免受
氧气自由基侵害。

3. 抗炎作用：法芙丽妥还具有抗炎作用，能够抑制炎症反应，减轻炎症损伤。

4. 抗肿瘤作用：法芙丽妥与其他天然产物结合使用，能够抑制细胞增殖并诱导细胞
凋亡，具有显著的抗肿瘤作用。

应用领域
1. 医药领域：法芙丽妥是一种常用的药物中间体，广泛用于合成各类生物活性化合物，如抗病毒药物、抗肿瘤药物等。

2. 农业领域：法芙丽妥可以作为植物生长调节剂，促进植物生长，提高产量。

3. 食品领域：法芙丽妥也被应用于食品添加剂中，具有防腐、抗氧化等作用。

总之，7-羟基-5-甲氧基香豆素是一种具有广泛应用前景的生物活性分子，具有多种
生物学和药理学作用，是目前治疗新冠病毒的研究热点之一。

随着相关研究的深入，法芙
丽妥将有望成为新型抗病毒药物的重要代表。

常用蛋白酶切割位点

HRV 3C Protease
Leu-Glu-Val-Leu-Phe-Gln▼Gly-Pro
L-E-V-L-F-Q▼G-P
Novagen
Ni-NTA (6His recomb. enzyme)
SUMO Protease
recognize the tertiary structure of the ubiquitin-like (UBL) protein, SUMO
Amersham-Biosciences, Novagen,
SIGMA, Roche
Benzamidine-Agarose
Factor Xa
Ile-Glu/Asp-Gly-Arg▼?
I-E/D-G-R▼
Amersham-Biosciences,
New England Biolabs,
Roche
Benzamidine-Agarose
Invitrogen – Life Technologies
Ni-NTA (6His recomb. TEV)
PreScission
Leu-Glu-Val-Leu-Phe-Gln▼Gly-Pro
L-E-V-L-F-Q▼G-P
Amersham-Biosciences
GSTrap for GST fusion enzyme
KEX2对arg的专一性高，要求最重要。
Arg前为lys效率最高，不切-Arg-lys，Pro影响KEX2切割
羧肽酶
羧肽酶B可以切割C端的Lys或Arg；羧肽酶A可以切割C端除了Lys、Arg、Pro的氨基酸，但如果倒数第二个氨基酸为Pro两种羧肽酶均不能作用
1.胰蛋白酶属肽链内切酶，能把多肽链中Lys和Arg残基中的羧基侧切断。

细胞蛇的研究进展

2007年,英国牛津大学的刘骥陇等在研究果蝇U 小体和P 小体(U 小体和P 小体是真核生物细胞质中的无膜细胞器)的功能关系时,用4种针对Cup (P 小体中的一种蛋白质)的抗体,对雌性果蝇的卵巢组织进行免疫组织化学染色,染色结果除了预期标记上的P 小体外,还标记出了长条形的丝状结构[1]。

这种结构的形状和数量与纤毛很相似,导致当时以为在果蝇中找到了有纤毛的新细胞类型。

但后来的一系列实验表明,该结构与纤毛没有关系,于是将其命名为“细胞蛇”。

最初是抗Cup 抗体不纯产生假象,意外发现的细胞蛇,而采用亲和层析纯化后的抗Cup 抗体无法再DOI:10.16605/ki.1007-7847.2020.10.0258细胞蛇的研究进展收稿日期:2020-10-22;修回日期:2020-11-19;网络首发日期:2021-07-27基金项目:宁夏自然科学基金项目(2020AAC03179);国家自然科学基金资助项目(31560329)作者简介:李欣玲(1999—),女,广西贵港人,学生;*通信作者:俞晓丽(1984—),女,宁夏银川人,博士,副教授,主要从事干细胞与生殖生物学研究,E-mail:********************。

李欣玲,张樱馨,李进兰,潘文鑫,王彦凤,杨丽蓉,王通,俞晓丽*(宁夏医科大学生育力保持教育部重点实验室临床医学院基础医学院,中国宁夏银川750000)摘要:细胞蛇是近年来细胞生物学研究的热门方向之一,由于其在细胞的增殖、代谢和发育上具有一定的生物学功能,因此,对一些疾病如癌症等的临床诊断或治疗具有一定的指导意义。

细胞蛇是由三磷酸胞苷合成酶(cytidine triphosphate synthetase,CTPS)聚合而成的无膜细胞器,其形成过程及功能在不同类型的细胞中不尽相同。

例如:细胞蛇能促进癌细胞增殖,并使患者病情恶化;过表达的细胞蛇可抑制神经干细胞增殖,影响大脑皮层发育;在卵泡细胞中,细胞蛇相当于CTPS 的存储库,在卵子发生过程起到促进细胞增殖和代谢的作用。

leventinib化学式

leventinib化学式
Leventinib是一种口服多靶点酪氨酸激酶抑制剂，它的化学名称是Lenvatinib Mesylate，其化学式为C21H19ClN4O4，分子量为426.85克/摩尔。

Leventinib是一种小分子化合物，通常用于治疗甲状腺癌和肾细胞癌。

它通过抑制多种受体酪氨酸激酶，包括血管内皮生长因子受体（VEGFR）、成纤维细胞生长因子受体（FGFR）和表皮生长因子受体（EGFR），从而抑制肿瘤的血管生成和生长。

Leventinib的化学结构使其具有潜在的抗肿瘤活性，并且正在进行广泛的临床研究以探索其在其他类型的癌症治疗中的潜力。

总的来说，Leventinib的化学式为C21H19ClN4O4，它是一种具有潜在抗肿瘤活性的口服多靶点酪氨酸激酶抑制剂。

布鲁氏菌抗体琼脂扩散(琼扩)试剂的说明书

布鲁氏菌抗体琼脂扩散(琼扩)试剂的说明书1. 引言1.1 概述布鲁氏菌抗体琼脂扩散（琼扩）试剂是一种用于检测布鲁氏菌感染的重要工具。

布鲁氏菌感染是一种人畜共患疾病，严重影响了全球动物农牧业和人类健康。

早期诊断和及时治疗对于控制和预防布鲁氏菌感染至关重要。

本说明书将详细介绍该试剂的产品特点、适用范围、操作步骤等内容，帮助用户正确使用琼扩试剂进行布鲁氏菌感染的快速检测。

1.2 文章结构本文分为五个主要部分：引言、布鲁氏菌抗体琼脂扩散(琼扩)试剂的说明书、试剂性能评估、贮存和稳定性考察以及结论。

引言将介绍文章的背景和目的，而后面各部分则会进一步讨论试剂的相关信息，并对其在实际应用中的表现进行评估。

1.3 目的本说明书旨在提供清晰明确的指导，使用户能够准确理解和正确操作布鲁氏菌抗体琼脂扩散(琼扩)试剂。

通过详细介绍试剂的产品特点、使用范围和操作步骤，帮助用户正确完成布鲁氏菌感染的快速检测。

此外，本文还将通过试剂性能评估、贮存和稳定性考察等部分，提供对该试剂在实际应用中的有效性和可靠性进行确证和评价。

最后，在结论部分将总结主要研究发现，并展望未来研究方向与应用前景。

以上为“1. 引言”部分的详细内容。

注：请注意，由于回答格式限制，上述内容可能无法完全复制到普通文本格式中。

如果您需要更详尽的信息，请直接参考或编辑原始JSON目录形式。

2. 布鲁氏菌抗体琼脂扩散(琼扩)试剂的说明书2.1 产品介绍布鲁氏菌抗体琼脂扩散(琼扩)试剂是一种用于检测人体中是否存在布鲁氏菌感染的试剂。

该试剂基于琼脂扩散原理，利用特定的抗原和抗体反应产生可见的沉淀线，用于初步筛查和辅助诊断布鲁氏菌感染。

2.2 使用范围本试剂仅供医疗实验室或专业技术人员使用，用于布鲁氏菌感染的初步筛查及辅助诊断。

不适用于自我诊断或家庭使用。

2.3 操作步骤以下是使用布鲁氏菌抗体琼脂扩散(琼扩)试剂进行检测的操作步骤：1. 准备工作：将试剂从冰箱中取出并适当回温至室温（约20°C-25°C）。

荧光抗体稀释液(Evans Blue法)

北京雷根生物技术有限公司
荧光抗体稀释液(Kolmers 法)
简介：
荧光抗体稀释液用于荧光染色时一抗(primary antibody)、二抗(secondary antibody)的稀释和配制。

当荧光抗体的染色如果背景较高，可以用Leagene 荧光抗体稀释液(Kolmers 法)稀释至临界浓度，使特异性染色呈阳性而非特异性染色呈阴性。

Leagene 荧光抗体稀释液(Kolmers 法)由伊文思蓝、盐等组成，为蓝色液体，减少非特异性荧光，宜做常规应用。

组成：
自备材料：
1、载玻片
2、盖玻片
3、荧光显微镜
操作步骤(仅供参考)：
1、按实验具体要求操作。

2、稀释抗体后应4℃避光保持。

注意事项：
1、荧光物质均易发生淬灭，染色后的样品宜避光保存。

2、在使用荧光抗体稀释液的情况下，虽然可减缓淬灭，仍需尽量避光。

有效期： 12个月有效。

编号名称 IH0347 IH0347 Storage 荧光抗体稀释液(Evans Blue 法) 50ml 100ml 4℃ 避光使用说明书 1份。

Elabscience水溶性长臂生物素标记试剂盒使用说明书

（本试剂盒仅供体外研究使用，不用于临床诊断！）产品货号：E-LK-B008产品规格：1次标记/3次标记/10次标记Elabscience®水溶性长臂生物素标记试剂盒使用说明书Water-soluble Long-arm Biotin Labeling Kit使用前请仔细阅读说明书。

如果有任何问题，请通过以下方式联系我们：销售部电话************，************技术部电话************具体保质期请见试剂盒外包装标签。

请在保质期内使用试剂盒。

联系时请提供产品批号(见试剂盒标签)，以便我们更高效地为您服务。

产品介绍Elabscience®水溶性长臂生物素标记试剂盒提供了生物素标记所需全部试剂，用于简单有效地对蛋白质、抗体和其他含有伯氨基(NH2-)分子进行生物素标记。

长臂降低多个生物素化分子与同一亲和素分子复合物结合时的空间位阻，生物素结合的空间位阻能够有效优化标记和检测效果。

试剂盒内的生物素已经活化，可直接使用，每次可标记0.1-2 mg 抗体。

试剂盒中包含用于抗体标记脱盐的Filtration tube，不用透析，操作简便，熟练操作90 min可完成整个标记过程。

基本信息水溶性长臂生物素(Sulfo-NHS-LC-LC-Biotin)结构式：分子量：670分子式：C26H40O10N5S2Na间隔臂长度：30.5 Å产品特点✓试剂全面: 本试剂盒提供了生物素标记所需全部试剂。

✓快速: 整个过程仅需90 min。

✓方便: 通过Filtration tube 即可脱盐，无需透析或者凝胶过滤。

✓使用灵活: 既可用于微量标记又可大量标记，每次可标记0.1-2 mg抗体。

✓理想的标记效果: 已经优化确定了最适的标记比例，降低标记不足或由于过度标记而失活的可能性。

✓细胞标记: 本试剂盒提供的水溶性生物素可标记细胞表面蛋白，而不穿透细胞膜损伤细胞。

试剂盒组成及保存未拆封的试剂盒可在2-8℃保存一年；溶解后的Sulfo-NHS-LC-LC-Biotin 能在2-8℃保存一周。

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=====================================================================Acq. Operator : Li Shan(LCMS-02) Seq. Line : 53Acq. Instrument : HY-LCMS-02 Location : P2-E-02Injection Date : 6/2/2015 6:11:45 PM Inj : 1 Inj Volume : 3.000 µl Acq. Method : D:\AGLIENT 1260\DATA\20150602\20150602 2015-06-02 14-02-38\100-1000MS+3MIN- 1.5_(0.02%FA).M Last changed : 6/2/2015 2:02:38 PM by Li Shan(LCMS-02)Analysis Method : D:\AGLIENT 1260\METHOD\BL50-90A,220NM.M Last changed : 6/4/2015 11:08:28 AM by Li Shan(LCMS-02) (modified after loading)Catalog No : HY-B0653A Batch#16423 A-RP-132 Additional Info : Peak(s) manually integrated min
0.51 1.52 2.53mAU 0
200400
600
800
1000
DAD1 B, Sig=214,4 Ref=off (D:\AGLIENT...0\DATA\20150602\20150602 2015-06-02 14-02-38\BIZ2015-602-DJL13.D)
0.391 1.542 2.222 2.762 ===================================================================== Area Percent Report ===================================================================== Sorted By : Signal Multiplier : 1.0000Dilution : 1.0000Do not use Multiplier & Dilution Factor with ISTDs Signal 1: DAD1 B, Sig=214,4 Ref=off Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 0.391 MM 0.0357 61.19257 28.52801 1.3536 2 1.542 MM 0.0640 4454.27783 1160.72083 98.5293 3 2.222 MM 0.0663 2.76478 6.94781e-1 0.0612 4 2.762 MM 0.0551 2.52921 7.64703e-1 0.0559 Totals : 4520.76439 1190.70832 ===================================================================== *** End of Report ***
=====================================================================Acq. Operator : Li Shan(LCMS-02) Seq. Line : 53Acq. Instrument : HY-LCMS-02 Location : P2-E-02Injection Date : 6/2/2015 6:11:45 PM Inj : 1 Inj Volume : 3.000 µl Acq. Method : D:\AGLIENT 1260\DATA\20150602\20150602 2015-06-02 14-02-38\100-1000MS+3MIN- 1.5_(0.02%FA).M Last changed : 6/2/2015 2:02:38 PM by Li Shan(LCMS-02)Analysis Method : D:\AGLIENT 1260\METHOD\HM-046_7-H01P.M Last changed : 6/3/2015 3:09:39 PM by Li Shan(LCMS-02) (modified after loading)Method Info : MS:100-1000,Column ID:A-RP-98,30℃ Catalog No : HY-B0653A Batch#16423 A-RP-132 Additional Info : Peak(s) manually integrated min
0.51 1.52 2.530
200000
400000
600000
800000
1000000
1200000
MSD1 TIC, MS File (D:\AGLIENT 1260\DATA\20150602\20150602 2015-06-02 14-02-38\BIZ2015-602-DJL13.D) ES-API, Pos, Sca
1.553
MS Signal: MSD1 TIC, MS File, ES-API, Pos, Scan, Frag: 50 Spectra averaged over upper half of peaks. Noise Cutoff: 1000 counts. Reportable Ion Abundance: > 10%. Retention Mol. Weight Time (MS) MS Area or Ion 1.553 8708407 290.20 I 289.20 I
m/z 1002003004005006000
20
40
60
80
100
*MSD1 SPC, time=1.525:1.598 of D:\AGLIENT 1260\DATA\20150602\20150602 2015-06-02 14-02-38\BIZ2015-602-DJL13.D ES-API Max: 891315 290.2 289.2 *** End of Report ***。