Additives to biological substances. I. Effect of added carbohydrates on bovine serum albumin

J~vre~l o/Bt~/ogical Standardi~ti~ ( 198 l) 9. 12 l - | 29

Additives to ololog c substances. I E ct of added c bohydrates on bovine serum bumm

E. Ta

~ andJ. M.

The stability of freeze-dried solutions o l i v i n e ~r.~m a l b ~ i n (BSA) in the presence of~veral c a r ~ h y d ~ t e s (gltico~, ~actose, s u c ~ and ~ + ~ t ~ a l o s e ) w a s moni~or~ by ob~rving changes in chemical composition ~-~d biologic~ aetivi~y~ Over a F ~ r ~ of 6 months storage ~t 37 °C and 6 5 ~ ~ l a ¢ i ~ hurnidit¥ (r.h~) in tb.e p ~ e n c e of l ~ t o ~ or gh~cose, ehe p ~ t e l n exper~enc~:d conside~ble ( 7 ~ 8 5 ~ ) ~ c ¢ i o n s in its |)'sine coneent; s i m i | ~ t~.ough less p r o ~ o ~ c ~ red~ct l o ~ i~ h/st/dine ~ d arginir~e ~nee~t w e ~ g l ~ obse~'ed~ A~ay o f t h e ~ two predations a~iO~t BSA ant iserum show*:d that biolog kal { ~ i g e n ~¢)activlcy dee in a s~miiar ~ h i o ~ Under the , ~ e condltMns of tem~rat~re and ¢.h. t I > ~ p r e d a t i o n s c o n f i n i n g BSA with s~ctose or ~ - r ~ e h a l o ~ s b ~ w ~ no significant change in their chemicM c o m ~ i t ions ot in their ~tigenic p~Vmrtles. It is concl~d~J ¢F~t che~ last mentioned additives a~ tO ~ preferred o ~ r l~:¢O~* ~ ~inert" bulking agents for * ~ with pvoteinacet~ biMogical subst~ces, pa~iculady tht~se ~ith high lysine content.

INTRODUCTION T h e large scale ~tion oflnrernationM biological standards and reference predations usu~ly involves dis~nsing, iyophilizing, etc. ~iquots from a solution containi n g t h e a c t i v e m a t e r i a l t o g e t h e r w i t h a s o l u b l e s o l i d carrier~ U ~ a m p b e l l I 9 7 4 a , b L R e q u i r e m e n t s f o r t h e final p r e p a r a t i o n i n c l u d e l o n g - t e r m s t a b i l i t y o f b i o l o g i c a l ~ t i v i t y a n d t o t h i s e n d t h e S t a n d a r d s are p ~ v e n t e d , ~ Ear ~ is ~ s s i b l e , f r o m h a v i n g c o n t a c t with light, moisture and oxygen, any of which could cause chemica! r~aetions thereby altering biological properties~ In addgtion, the substances us~ ~ carrie~ should i n e r t ~h a v i n g n o h a r m f u l e f f c ~ t s o n ~ t i v i t y ~ d s h o w i n g n o i n t e f f e ~ n c e i n t h e ~ s a y o r t ~ t for w h i c h t k ~ p ~ p a w a t i o n is i n t e n d e d . A m o n g t k ~ s u b s r ~ c e s c o m m o n l y u s ~ f-or *R~celvoA ~ r publication 23 April |980~ ~Na¢ional |r~titute for Biologlca| S ~ d ~ s ~ d C o n t ~ l . Holly Hi|/~ H ~ p s t e ~ . ~ndon~ N ~ 3 6RB.

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E. TARELL! ANDJ~ M. WOOD this purF<~se are ' r e d u c i n g cartmhydrates (e.g. lactose) and in such c ~ e s the final preparation is a solid d i s l . r s i o n o f active material, often proteinaceoos (and therefore containing free a m i n o groups) which is in i n t i m a t e contact w i t h a potential alde/~ydo com|mu~xd. It is reasonable to ,~ssume that possible chemical reactions which could occur between proteins and c a t . h y d r a t e s in this e n v i r o n m e n t would be an imIx~rrant influence on the l o n g - t e r m stability of biological activity. This report describes the effect of a n u m b e r o f carbohydrates (reducing and n o n - r e d u c l a g ) on the chemical composition and antigenic activity o f a representative p r o t e i n ~ b o v i n e serum a l b u m i n . METHODS To solutions o f bovine serum a l b u m i n (BSA) (RIA Grade, Sigma Chemical Company, St~ ~ u i s , U . S . A . ) (0"5% w/v) in distilled water were added the following carbohydrates: (a) sucrose; (b) lactose; (c) glucose; (d) ~,~-rrel~alose (AnalaR gr~Je, B D H Chemicals Ltd., Poole, England), to give final solutions which were 0-023 M with respect to each carE<-~hydrate, g i v i n g an approximate 3: I molar ratio o f carbohydrate to free a m i n o groups present in the protein. T h e solutions (pH approximately 5" 3) were sterilized by p~s
ADD|TIVES

TO I~IOLOGICAL SUBSTANCES

v,'~ cons~rttcted and d~e ~ ¢ e t l c y of each BSA preparation was calculated by normal s l o ~ ratio m e t h o d s (Finney, 1952L RESULTS For aU four preparations, after several days ar 3 7 ° C and 6 5 % r~h,, considerable shri~kage occured o f the freeze-dried solid~ T h r o u g h o u t ~he t ~ r i o d of investigation, those preparations containing e i t h e r sucrose or ~ , o - r r e h a l o s e remained white, and the solids were readily dissolved ill distilled water. In contre~t:, mixtures o f BSA w i t h lactose or with glucose developed an initial pale brown CO|O~t ion which d a r k e n e d w i t h ¢in~e, and chelr solubility in distilled water became i n c r e ~ i n g ! y more difficult.

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F~g, I,(a) Variation o f t ~ : molar ~ t i o oflyslne: leucine with time o f s t o o g e ~e 37 ~C ~ d 65~, r.h, P r e ~ r a t i o n s containing g l u ¢ 0 ~ (o) lactose ( a ) sucrose fro) and t t e h a l o ~ (oL (b) Variation of ~he ~ A an~i:gen ~¢ti~'ity ~'~th t i ~ e 6 f ~ o r a g e a¢ 37 ¢C and 65% ¢ . h f-or p ~ g ~ r i o n s containing g l u ¢ o ~ (e) lacto~ (&) suceo~, (m) And cr~halo~ (OL Veer lea| [ ~ indlc~re t he 95 ~ confide~=e |imits for SR D ¢icta* i o ~ o f e ~ h BSA prep:~eaIion. For ~ t h (~)gI~] {b)the fi~t g~int (CO weeks) o n e ~ h g ~ # i is the ~:Mue o b t a i n ~ from t ~ initial a q u ~ S ~ i u r i o n which w ~ s not ~ b i e C ~ IO f ~ e - d r T f i o g .

123

E. T A R E L L I A N D Jo /~.L **;rOOD

T h e variation in molar ratio o f l y s i n e to leucine found for the BSA p r e d a t i o n s as function of storage t i m e at 37 ~C and 65% r.h. is shown in Ng. l(a). T h e e preparations con rain i ng sucrose or ~, c~-trehalose showed little or no alteration in this rat io t h r o u g h our the 26-week storage period, in the presence o f glucose, however, the p ~ t e i n showed a dramatic (*'~85%) reduction in its tysine: leucine ratio over the same ~ r i o d of t i m e , the major loss oflysine c~curring d a r i n g the first two weeks of storage. A similar, t h o u g h less pronounced, reduction w ~ also o b ~ in the BSA-lactose system, resulting, after 26 weeks, in a Ioss of approximately 7 0 % o f the lysine residues originally p r e ~ n t in the protein. In all cases o t h e r a m i n o acids m a k i n g up BSA (with t h e~exception o f h i s r i d l n e and arglnine in g l u c o ~ - and lactose-containing preparations) remained at a constant level t h r o u g h o u t the period o f investigation. For histidine and arginine a significant ( 4 0 - 4 5 % ) reduction w ~ obser~¢ed for the g l u c o s ~ B S A system (a~)er 26 weel.:s), a l t h o u g h little or no c h a n g e w ~ observed until three weeks had elapsed. A slight reduction in these two ~ s i c a m i n o acids for the lactose-BSA m i x t u r e was Mso indicated. Typical SRD zones obtained from the preparations, together w i t h their calculated a m o u n t s of a n t i g e n are illustrated in Plate 1. T h e relative a m o u n t s of BSA a n t i g e n in each preparation may be ~ e n more c l e ~ I y in Fig. t ( b ) w h e r e the change in a n t i g e n o~:tivity w i t h t i m e o f storage is shown. All o f the p r e ~ t i o n s w i t h s t o ~ freeze-drying w i t h o u t s h o w i n g m a r k e d loss in a n t i g e n activity and the preparations c o n t a i n i n g sucrose or ~ , ~ - t r e h a l o s e maintained the initial level o f activity t h r o u g h o u t 26 ,weeks o f storage. However, in the p r e ~ n c e o f g l u c o ~ a considerable ( ~ 8 0 % ) loss in ~ r i v i t y w ~ obser~,ed over this p e r i ~ of t i m e and the lactose-containing p e e ~ r a c i o n s also lost significant (~ 32 % ) activity. T h e result obtained from gel electrophoresls on sm,nples subjected to storage for 12 weeks is shown in Plate 2. In the presence o f sucrose or ~, ~-trehalose, the lowest m o l e c u l ~ w e i g h t c o m ~ n e n t cor~sF~gnde~ to BSA m o n o m e r (ca 66 0 0 0 daltons). For the preparations c o n t a i n i n g glucose or lactose the lowest m o l ~ u l a r w e i g h t c o m p o n e n t w ~ approximately 83 0 0 0 and 76 0 0 0 daltons respectively; no c o m ~ n e n t c o r r e s ~ n d i n g to BSA m o n o m e r b e i n g observed. DISCUSSION A variety o f additives are used ~ stabilizers or as b u l k i n g agents d u r i n g the manipulation and/or pr¢~cessing o f many materials o f biological origin. C a r ~ h y d ~ t e s , especially lactose, are c o m m o n l y used for this purpose since they are readily available in pure form and ~re considered ~ ~ (relatively) chemically inert. Substances of biological origin are however often proteins or peptides c o n t a i n i n g a n u m b e r o f free a m i n e func~ionM groups, and admixcure o f such materials with carbohydrates such as glucose or lactose may lead to incorporation of these c d ~ t e s into the protein p o l y m e r t4a Maillard reactions (for reviews see H ~ g e , 1955; H ~ r e l l & Carpenter, I 9 7 7 ) whereby the fiarm o f the c a r ~ h y d ~ t e undergoes condensation w i t h free a m i n o groups g i v i n g r i ~ to N~hiffw h i c h s u b s ~ u e n t l y u n d e r g o A m a d o r i rearrangements. A simple r e p r e ~ n t z t i o n o f this ~ , ~ of condensation b e t w ~ n glucose and a protein is shown in Fig. 2. T h e A m a d o r i - a d d u c t formed can u n d e r g o hydrolysis in acid to give products which may include the originM free a m i n e and hence the original a m i n o acid (Finot & M a u m n 1972L Therefore, in order to quanti@ the degree o f chemical reaction as s h o w n in Fig. 2, a ~ d u c t i o n (conveniently w i t h aqueoL~ boro~wdrlde f f h o m ~ , 1972; H u r ~ I I & C a r ~ n t e r , 1974) should ~ c ~ t i e d o u t (Fig. 3) prior to acid 124

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P | ~ e I. S i ~ t e ~di~| diffcsion y o f antigenic ~c¢i ~t~ of S A p~epa~ions aker 8 weeks s~o~ge at ~ ~7~C and 6 5 ~ r~h~ Oe! crmcalns 40 ~ t m| "~ anti-BSA ~¢~m~ I n ~ t s s re)~ive ~ o u n c s o f B 5A m t i g e , ~ t t v ~ty ° ' c ~ I c u i a ~ using a refere n~e ~ BSA p r e p a ~ i o n c o ~ a i a i n g 5"O mg mV i .

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4" Plate 2. S D ~ l y a c r y | a r a i d e g e l e l ~ t r o p h o ~ s i s at p H 8-5 ( t r i s ~ b i c i n e ) on s a m p l e s subiec*ed ~o 12 ~'¢eks sto~age at 37 0C and 6 5 ~ r . h . B . BSA re~-rence; M, Pharmacia m~rker m i x t u ~ : 1. ~ S A - s u c ~ m e ; 2. BSA~lactose; 3, B S A - ~ , o - t r e h a i o s e ; 4 , BSA~glucose.

ADDITIVES TO BIOLOGICAL SUBSTANCES

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hydrolysis and amino acid analysis. T h e reduced material is then acid stable w i t h resF~oct to regeneration o f the original amino acid. T h e interactions shown in Fig. 2 (early Maillard reactions) are k n o w n to occur in the initial stages o f the reaction ~ t w e e n proteins and reducing c a r ~ h y d r a t e s w h e r e ~ subsequen~ r r a n s ~ r m a t i o n s (advanced Maillard reactions) lead to a multitude of products such ~ ~ l y c a r ~ n y l . O N - and S- hererocyclic comp-ounds, etc., which car, themselves .everely degrade proteins. Final Maillard reactions lead to brown polymers o f complex structure. A l t h o u g h such reactions have. ~ r many y e ~ s , received considerable attention w i t h r e s t e r to browning reactions of food p ~ t e i n s , it is only recently that similar reactions have ~ e n described in detail for biological mareriMs. T h u s , non-enzymically glucosyfated haemoglobin (Bunn et a l . , 1 9 7 9 ) a n d a l b u m i n (Day, T h o r p e & BaThes, 1979), ~ i n g the result o f early Maillard reactions, have recently ~ e n characterized a~er isolation from h u r n ~ sources. Similarly insulin h ~ been shown to undergo subst~r~tlal chemical modification w h e n tyophilized in the presence o f glucose w i t h s u ~ u e n t storage. ( ~ h w a r t z & Lea. 1952; Amwya-F, I~e & Chichester, 1970; Clark & Tannen~ baum, 1974.) A n u m ~ r o f factors such ~ t e m p e r a t u ~ , water-activity and p H influence the ~ t e o f reaction ~ e e n a reducing sugar and a p ~ t e i n , Mthough it is p e that ~ c h 125

E. T A R E L L i A N D J .

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126

Str**ctureso f ( a ) # - g l u c o ~ , (b) ~.!~,ose. (¢) suc~m, and (d) a,~-rreh~lom.

ADDITIVES TO BIOLOGICAL SUBSTANCES In c o n t ~ t to th~ above ~ v o preparations, those containing the reducing sugars glucose and lactose exw:rienced considerable reduction in Iysine content o f BSA when stored under identical conditions. In tile p~sence o f glucose almost 8 5 % of lysine residues o f the protein had undergone modification after 26 weeks, the major loss ¢~curring in the first t~-o s o f storage, ~c¢ose, a l t h o u g h less reactive ~han glucose~ nevertheless c a n e d the loss o f half o f the lysine residues w i t h i n five weeks and less than 30f~ o f the original tesMues were present after 26 weeks. There w ~ also an indication from the gross amino acid comF~sition for modificatMns occurring ae other s l t ~ within BSA molecules. Notably the proportions o f the b a s k amino acids histidine and arginine were subs~antially reduced towards the end o f the sm ~ r i c ~ especially in the preparation containing glucose. These reductions were however only observed after considerable inte~c¢ion w i t h lysine had taken place and may ~ due to the onset of advanced Maillard reaction which is known to pa~icularly af~ct these two amino acids (Hurrell & Carw~nter, 1977). In addition it is probable that the N-terminaI residue ( ~ p a r t i c a c i d ) o f BSA also u n d e ~ o e s s l m i l ~ modification in the presence of ~ d u c i n g carbohydrates, however this would not ~ immediately apparent from the present resuks. T h e c h e m k a l alteration o f B S A w h k h o c c u r ~ d with glucose or Iacto~ resulted in a loss o f a n t i g e n k i t F as shown in Fig. |(b). in the c a ~ o f glucose less than 2 5 % o f the original activity of the remained after 26 weeks storage, the vMue for lactose-BSA t~iling by approximately 3 5 ~ . O v e ~ l l . adduce Formation between BSA and these reducing sugars, as she~vn from measurement of Iysine content o f the protein after borohydtide teduc¢ions~ ~ r e g ~ d c o r ~ i a t i o n to loss o f antigenic pr~o~rties. The incorporation o f c a r ~ h y d r a t e residues into the protein was fur:her demonsteated from gel electrophoresis (Plate 2) on samples which had been subiected m 9 0 days storage at 37 °C and 6 5 % r.h. T h e lowest afN~arent mol¢~:ular weight obse~,ed ma~ ca 83 0 0 0 dalmns (glu and 76 0 0 0 da!tons (lactose), values which correspond m the addition o f approximately 9 0 and 30 residues res~c¢ive!y to each BSA molecule. In the presence of sucrose or ~ , ~ - t r e h a l o s e the lowest apparent molecular weight corresponded to monomeric BSA ~a 66 0 0 0 dahons), a similar result being obtained from BSA in the absence o f any additives. Studies of the stability of this last mentioned pteparatMn also revealed no deterioration in amino acid content or antlgeniciiy of BSA w h e n stored at 37 QC and 6 5 % cA1. for several m o n t h s (Tarelli a M~, unpublishedk A 6archer ~ i n t of note from Figs I(a), (b) is that the freez~dD, ing prcmess itself had apparently little or no effect on chemical c o m [ . s i t i o n or on biological properties o f BSA. Lyophilization can, however, have q u i t e marked effects on some materials. A number of Fmptide-hormones ~ r example have been shown co lose almost h M f o f their biological activity after the £reeze-d~,ing of their aqueous solutions containing either glucose or l~ctose. This e f ~ c t has h ~ n accounted ~ r by the formatMn o f p r ~ u c t s via Maillard reaetionso (Ta~2||i a M . , unpublished). The results described here have indicated that lactose, in a similar manner to glucose, can ~ a h i g h l y reactive molecule when used ,as a b u l k i n g agent tbt proteinaceous materials of biological origin~ As a ~ s u h , caution should b~- exercised in its use e s ~ c i a l l y got the p ~ p a r a t i o n o f Standards and reference materials where long-term stability is r ~ u i r e d . Apace from the reactivity e n c o u n r e r ~ in this p r e ~ n t study where the intial p H x ~ s ~pproximately 5-3 it shoutd ~ n o t ~ t h a t in c o m m o n w i t h MI reducing s u g a r , lactose may undergo oxidation and/or d e g = d a t i o n under basic conditions givir~g rise t o a complex mixture o f ~active products ( H o u g h & R i c h a r d ~ n , (I967). Since b i o ! ~ i c M materials are o ~ e n stabilizcM by buffer solutions with p H > 7. 127

E. TARELLI A N D J. M. WOOD the usaz o f any r e d u c i n g c a r b o h y d ~ t e s u n d e r c o n d i t i o n s o f h i g h p H should ~ avoided. As stated previously, an ideal +ine~' carrier should have n o harmful effects o n activity nor should it interfere w i t h the ~ s a y or test for w h i c h the preparation is i n t e n d e d . In this rc.s~ct sucrose aplx~a~ to fulfill these criteria, a l t h o u g h because of th e h i g h acid lability (Capon, 1969) o f t h e sucrose m o l e c u l e , e q u i m o l a r a m o u n t s o f fructose and g l u c o ~ may be produced u n d e r conditions of acid p H ( w i t h obvious consequencesL O f the carriers used in this present study, ~ , ~ - t r e h a l o s c w o u l d a p ~ a r to be th e most satisfacto~ for general use since the molecule h ~ r e . u n a b l e stability (Cagx~n, 1969) towards ~ r h acids and b ~ e s . In conclusion it should be emphasized that th e c o n d i t i o n s o f storage used in this study were chosen for their probable adverse effects. International Standards and rel~erence materials are m a i n t a i n e d at ~ 2 0 ° C until use, and f u r t h e r m o r e they are extremely low in m o i s t u r e c o n t e n t ( C ~ p ~ e l l 1974a). It is k n o w n that th e rates o f Maillard reactions are substantially reduced at low levels o f water activity and therefore any such reactions t a k i n g place u n d e r the n o r m a l c o n d i t i o n s o f storage should extremely slow. Nevertheless, for long t e r m (20--25 years) usage o f International Standards, c h e m i c a l interactions of the t y ~ observed here could play an i m ~ r t a n r role in d e t e r m i n i n g stability over such a ~ r i o d o f time.

Acknou ts kWe thank Miss Sheila l~th~.el| (National Institute for ?,Iedical R e ~ h , ~ndon, NW7 1AA, U. K~) for the determination o f ~ i n o acid compositions ofhyd~ly~ates. Mrs Lesley Lloyd (NIBSC, U.K.) for ~ r f o r m i n g the el~ttophoresis experiment ~ d Miss Una Dunlea~,), (NIBSC, U . K . ) f o r ~ r f o r m i n g S R D t e s r s . Drs P. J. C a m p ~ l l , D. H. Calam and P. H. Cor=n (NIBSC, U . K . ) are thanked for help~l discussions.

REFERENCES Amaya, F. J , Lee, T-C. & Cbichester, C~ O° (I970L Biological inactivation of proteins by the ~ | a i | l a ~ react iota E ff.'_~tof mild heat on the tert ia~ structure of ir~u|in.Jour~lojCAgricu~u~ and F ~ Ctx~ai3try 24, 4 6 ~ 4 6 7 . Bunn, H. F., Shapiro, R., M c M ~ , M., ~ r r i c k , L., l',fcDonald, M, J . , Gallop, P. M. & y, K. H. (1979). S t r u c t u ~ heterogeneity of Human hemoglobin due to nonenzymarie glycosylation. Journal of Bi~q'ogical C ~ i s t o * 254, 3892-3898. C a m p ~ l l , P . J . ( I 9 7 ~ ) . International biological standards and refe~nce p r e d a t i o n s . I. Pre~ration and presentation of materials to ~rve ~ standards and reference preFm~tions. Journal o r b Stan~ n 2, 249-258. Camp~ql, P. J. ( 197~) , International biological standards ~-td refe~nce pre~rations I I. P r ~ e dures used for the production of biological standards mad ~ f e ~ n c e p~pararions.Journal of Biological Standardization 2, 2 5 ~ 2 6 7 . Capon, B. (t969). M~ha~qisms in c a r ~ h y d ~ t e chemistry. C ~ i c a l Reviews 69, 421-423. Clark, A. V. & Tannenbaum, S. R. (1974). | ~ l a t i o n and characrerlsarion of pigments from proreir~car~nyl browning systems, i~todels foe two insulin-glucose pigments. Journal of Agriculture and Food 22, I 0 8 ~ 1093. Day, Jo F., Thorpe, S. R. & BaynesJ. W. (1979). Nonenzymically glucosylated Mbumin. In 14tra pre~zarMn and isolation from normal b u m ~ ~ . l m of B 254, 59~597. Finney, D. J . , (1952L Sta~'stical Meth~s in Biological AssaLV. London: G r i ~ n . 128

A D D I T I V E S TO BIOLOGICAL SUBSTANCES

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