Antibody purification

Presentation on antibody purification

1. Antibody Purification Sulov Saha 30 Nov, 2015

2. Contents • Sou r c es of a n t i bodies • D if f e r e n t m e thods of p ur i f y i ng a n t i bodies • W h y “ p ur i fied a n t i bodie s ” a r e n ot all the s a me • H o w t o pur i f y a n t i bodies ea s il y w i thout s p ec i al i s t kn o w l e d g e or e q uipm e n t • Con s i d e r a t i ons f or c onj u g a t i ng a n t i bodies t o en z ymes, d y es and na n opart i cl es • Introduction

3. Introduction • The production and use of specific antibodies as detection probes and purification ligands – often called immunodetection or immunotechnology – has revolutionized bioresearch and diagnostic technologies . Animals immunized with prepared antigens will produce specific antibodies against the antigen. • Once they are purified (and possibly after labeling them with an enzyme or fluorescent tag), these antibodies can be used directly to probe the specific antigen in Western blotting, ELISA and other applications .

4. Sou r ces of a n ti b od i es • Animal s e rum – pol y cl onal a n t i bodies • Asc i t es – mono c l onal a n t i bodies • Cell c ul t u r e s u p e rn a t a n t – monoc l onal a n t i bodies • E g gs – a vian an t i bodies • Bac t er i al e xp r e s sion s y s t ems – r e c omb i na n t a n t i bodies E ach of the s e s ou r c es has c o n t ains the s p e ci fic a n t i body of i n t e r e s t amo n g s t other p r o t eins, li pids and other c ompo n e n ts.

5. Di f f e r e n t meth o ds of pu r i f ying a n tibodies • F r ac ti on a t i on b y globul i n p r ec i pi t a t i on • Ion e x c ha n g e ch r om a t og r ap h y • S i z e e x cl usion c h r om at og r ap h y • P r o t ein A a f fin i ty c h r om a t og r ap h y • P r o t ein G af fin i ty c h r om a t og r ap h y • P r o t ein L af fin i ty c h r om a t og r ap h y • A n t i g en af fin i ty c h r om a t og r ap h y Physicochemical fractionation   Class-specific affinity  

6. Glo b u l in p r ecip i t a tion • Fi r s t publ i s h ed i n 1899 b y Jam e s A t ki n s on in J . Exp. Med. – s e pa r a t i ng albumin f r om the a n ti- t o x i c c ompo n e n ts of a h o r se a n t i s e rum u s i ng magn e sium sulph a t e. • Am m onium s ulph a t e and s odium s u l ph a t e a r e m o r e c ommonly u s e d , t ak i ng a d v a n t a g e of the pr i nc i ple of “ s a l t i n g -ou t ” . A t the a p p r opr i a t e c once n t r a t i on the s e p r ec i pi t a t e globul i n s , wh il s t ma n y other p r o t eins, i nc l uding a l bumin, r emain i n solu t i on. • W hi l s t th i s i s a s i mple a n d g e n t l e p r o c e d u r e, i t only p r o vides a p ar ti al l y pu r e p r e p a r a t i on i f s t ar ti ng f r om a c ompl e x mixtu r e s u c h as s erum. • This s t ep i s of t en c ombined w i th f u r t h e r pur i fi c a t i on u s i ng one of the other m e thods discus s ed in th i s presentation.

7. Ion e x chan g e ch r om a t og r ap h y • Ion e x c ha n g e ch r om a t og r ap h y (I E C) u s es po s i t iv ely or n e g a t iv ely c ha r g ed r e s i ns t o bind p r o t eins ba s ed on th e i r n e t c ha r g es in a g i v en b u f f er s y s t em (p H ). • T ypi c al l y , a c ompl e x mix t u r e i s ad d ed t o the c o l umn i n a cer t ain set of bu f f er c ondi t i ons ( e . g. l o w sal t) , and the buf f er c ondi t i ons a r e then chan g ed ei t her s t e p -w i se or on a g r adie n t ba s i s. c o l umn i n di f f er i ng c ondit i on s . D i f f e r e n t p r o t eins a r e r elea s ed f r om the • I E C is p e rha p s mo r e o f t en u s ed t o p u r i f y pol y cl onal a n t i bodies than f or mono c l ona l s – no t e th a t ea c h mono c l onal i s u nique i n t erms of cha r g e, and w i l l the r e f o r e b e r elea s ed u n d e r a s p e ci fic set of c ondit i ons – so m e th i ng th a t i s u s e f u l f or r e p e a t ed pur i fi c a t i ons of the s ame a n t i bod y , but also m e ans th a t o p t i mis a t i on i s r e q ui r ed f or each a n t i bod y . • Th e r e a r e v ar i ous I E C r e s i ns av ai l able, w i th p e rha p s the mo s t w i d e l y u s ed being D E AE-Sep h a r os e .

8. Ion e x chan g e ch r om a t og r ap h y Star ti ng b u f f er c ounte r - i ons Su b sta n c es to be s e parated Gradie n t i ons

9. Si z e e x clus i on ch r om a t og r ap h y • Se p a r a t i on of c ompl e x mix t u r es on the ba s i s of si z e or molecu l ar s h ape c an be ach i ev ed b y si z e e x cl u s i on (S E C) or g el fi l t r a t i on c h r om a t og r ap h y . • The mole c ular si e ving p r o c e s s t a k es pla c e as a s o l u t e pa s s e s th r ou g h a pac k ed b e d s t a t i ona r y p h a s e. Se p a r a t i on d e p e nds on the di f f e r e n t abi li t i es of the v ar i ous mole c ules t o en t er the p o r es of the b e a d -ba s ed s t a t i ona r y p h a s e. • La r g e mole c ules, wh ic h c an n ot e n t er the p o r e s , a r e e x cl uded and pa s s th r ou g h the c o l umn qu ic k l y . Sm a ll er mole c ules th a t c an e n t er the p o r es a r e r e t a r ded and m o v e th r ough the c o l umn mo r e sl o w l y . V e r y small mole c ules, s u c h a s s a l t, a r e able t o f u ll y p e rme a t e and elu t e l a s t.

10. Si z e e x clus i on ch r om a t og r ap h y • S E C t e n ds not t o be u s ed f or pr i ma r y p u r i fi c a t i on of a n t i bodies other than I g Ms. H ow ev e r , i t m a y b e a v alua b l e “ p ol i s h i n g s t e p ” f ol lo w i n g pur i fi c a t i on b y other m e thod s . • Ran g es of ch r om a t og r ap h y r e s i ns a r e av ai l able th a t s e pa r a t e p r o t eins w i th i n di f f e r e n t r anges of si z e (e . g. AcA22, AcA34, AcA44 f r om U l t r o g el®, and S200, S300, S400 i n the Sep h ad e x r an g e) D E - S A L TING C O L UMNS • So c al l ed d e -s a l t i ng c o l um n s a r e u s e d t o u n d e r t a k e a b u f f er e x c ha n g e, or t o r em o v e s mall s i z e c o n t amina n ts. This is s i mply a s p ec i al c ase of S E C , w i th the mo s t c ommon l y us e d g el be i ng Se p ha d e x G -25, of t en i n p r e -p r e p a r ed P D10 c o l um n s.

11. Si z e e x clus i on ch r om a t og r ap h y Di f f e r e n t si z ed p r o t ei n s Mi x t u r e of d i f f e r e n t si z ed p r o t ei n s Gel f i lt r a tion c o l um n

12. P r o t ein A/ G /L ch r om a t o g r ap h y • In their n a t iv e f orm the s e p r o t eins a r e e xp r e s s e d b y bac t er i a as part of th e i r d e f e n se m e c h a n i s m s a g ain s t the mam m al i an i mm u ne r e s pon s e. • A l l bind t o mamma li an immunog l obul i ns via c on st a n t r e g i ons – P r o t ein A a n d G t o the F c r e g i on, and P r o t ein L t o the k ap p a l i ght c hain. • R e c ombina n t v e r sions a r e u s ed immobi l i s e d t o v ar i ous m a t r ic es t o pur i f y IgG b y a f o r m of af fin i ty c h r om a t og r ap h y .

13. P r o t ein A/ G /L ch r om a t o g r ap h y

14. P r o t ein A/ G /L ch r om a t o g r ap h y • Sa m ples a r e a dded t o the P r o t ein A/ G /L m a t ri x in a s u i t able binding bu f f er – pH and i onic s t r e n g th s hould be c on s i d e r ed • Co l umn i s w as h ed, wh i c h ef f ec ti v ely l e a v es on l y IgG bound • L o w pH elut i on i s c ar ri ed out, wh ic h m a y be w i th di f f e r e n t bu f f er f ormu l a t i ons a n d m a y di f f er d e p e nd i ng upon the i sotype of the a n t i body b e i ng pur i fied. It m a y be i mpor t a n t t o kn o w the ba s i c f o r mul a t i on of the elu ti on bu f f er f or d o wn s t r eam p r o c e s sing • L o w pH elut i on r e q ui r es n e ut r al i s a t i on t o mai n t ain a n t i body i n t e g r i t y . Of t en c ar ri ed out w i th T r i s bu f f e r , but i s th i s s u f fi c i e n t f or y our d o wn s t r eam n e e d s?

15. P r o t ein A/ G /L ch r om a t o g r ap h y R e co m bin a n t P r o te i n A R e co m bin a n t P r o te i n G R e co m bin a n t P r o te i n A /G R e co m bin a n t P r o te i n L R e co m bin a n t P r o te i n A /G/L N at iv e Sou r c e S ta ph yl ococc u s a u r eu s S t rep t ococc u s N /A Pe p t o s t rep t o - cocc u s mag nu s N /A B indin g Si tes f o r Ig 5 2 6 4 13 O p t i mal B indin g p H 8 - 9 5 5 to 8.2 7.5 7.5 T y pic al e lu t io n p H 3.0 – 7.0 ( i s o ty p e dependen t) 2.5 – 3.0 2.5 – 3.0 2.0 – 3.0 2.5 – 3.0 Ig B indin g Tar g et Fc Fc Fc VL -ka pp a F c + V L -ka pp a

16. P r o t ein A/ G /L ch r om a t o g r ap h y Sp e ci e s Im m un oglo bu lin Bin d i n g t o P r o tein A Bin d i n g t o P r o tein G Bin d i n g t o P r o tein L Hu m an Ig G 1 Str on g Str on g Str on g Ig G 2 Str on g Str on g Str on g Ig G 3 N e gligible Str on g Str on g Ig G 4 Str on g Str on g Str on g M ou s e lg G 1 Weak Str on g Str on g lg G 2a Str on g Str on g Str on g lg G 2b M e d i u m M e d i u m Str on g lg G 3 Wea k / me d i u m M e d i u m Str on g Rat lg G 1 N e gligible Weak Str on g lg G 2a N e gligible Str on g Str on g lg G 2b N e gligible M e d i u m Str on g lg G 2c N e gligible M e d i u m Str on g G oat lgG Weak M e d i u m N e gligible Rab b it lgG Str on g M e d i u m Weak Sh ee p lgG Weak M e d i u m N e gligible

17. A n ti g en a f fi n ity c h r om a t og r ap h y • In this c a s e a s p e ci fic c h r om a t og r ap h y r e s i n is p r e p a r ed t o whi c h the ac t ual a n t i g en th a t the a n t i bodies bind t o i s i mmo b ili s e d. • The s am p l e c o n t ain i ng the s p ec i fic a n t i bodies i s then p a s s ed ov er the c o l um n , al l o w i ng only s p ec i fic a n t i bodies t o bind, w i th all other c o n t amina n ts, i nc l ud i ng other a n t i bodie s , b e i ng w a s h e d a w a y . • B u f f er c ondit i ons a r e th e n c ha n g ed t o elu t e the s p ec i fic a n t i bodies f r om the c o l umn. • T ypi c al e x am p l es of the u s es of a f fin i ty c h r om a t og r ap h y a r e i n p r e p a r a t i on of s p ec i es s p e ci fic s e c ond ar y a n t i bodie s , i n pur i f y i ng a n t i bodies t o p e p t i de a n t i g e n s and f or pur i f y i ng p r o t eins w i th e p i t ope t a g s (e . g. M y c , H IS e t c).

18. A n ti g en a f fi n ity c h r om a t og r ap h y P r e p a r a t i on of gel m a t ri x M a t r i x L i g a n d I m m ob ili z ed L i g a n d Ap p li c a t i on of s a mple Samp l e Co m p l e x I mpuri ti es E l ut i on of p ur i fied a n t i body

19. W h y “ p urif i ed a n tibodie s ” a r e not a l l the same Ma n y a n t i bodies a r e s o l d b y c omp a nies as “ p ur i fied” – but wh a t does th a t mea n ? • Conce n t r a t i on • P r e s e r v a t iv es / s t abi li s e r s – h a v e a n y b e en ad d ed back i n t o the p r e p a r a t i on a f t er pur i fi c a t i on? • Buf f e r c om p o n e n ts – i n cl u d i n g c o n t am i n a n ts f r om e l u ti o n e t c. • Le v e l of pu rity - e . g. Ig f r acti o n vs. t o t al IgG v s. s pe cific IgG • U n e x p e c t e d c on t ami n a n ts – e . g. bo vi n e IgG • Endo t o x i n e t c .

20. W h y “ p ur i fied a n t i bodie s ” a r e n ot all the s a me Y our fi n al b u f f er…….. • The final bu f f er th a t y our a n t i body is in m a y h a v e a signifi c a n t ef f ect on wh a t y ou c an u s e y our a n t i body f o r . F or i n s t ance, the p r e s e n c e of g l y ci ne i n a bu f f er c an i nh i bit c onj u g a t i on r eact i on s , or so d i um az i de m a y k il l li v e cel l s. Some b u f f e r s m a y be s u i t able f or c onj u g a t i ng t o p r o t eins, but not t o n a nop a r ticl e s . • If y ou under t a k e a dia ly sis s t ep po s t -pur i fi c a t i on t o t r an s f er an a n t i body i n t o a p ar tic ular bu f f e r , y ou n e ed t o be s u r e th a t the dia l y sis h as b e en ef f ect iv e. • e . g. T o r em o v e ( i . e . t o 1nM l ev el) 100mM gl y ci ne f r om 10ml of a n t i body y ou n e e d t o dia l y se a g ain s t 1 mi l li on li t r es of P B S . Al t ern a t iv el y , c ha n ging the dia l y sis 4 t i mes w il l ach i ev e the s a me ef f ect wi th only 4 li t r es of PBS. • H ow ev e r , a s i n g l e dia l y sis i n t o 1 li t r e l e a v es gl y ci ne p r e s e n t a t 1mM.

21. W h y “ p urif i ed a n tibodie s ” a r e not a l l the same T o t al I g G v s. specific IgG • The s erum p r oduced when a n y pol y cl onal a n t i body i s r aised w il l c o n t ain only ap p r o x i m a t ely 10% of a n t i bodies th a t a r e s p e ci fic t o the a n t i g en of i n t e r e s t • Th e r e f o r e if y ou p ur i f y all of the I g G p r e s e n t, only 10% of th a t IgG w i l l be s p ec i fic f or t he a n t i g e n . • On l y a n t i g en af fin i ty c h r om a t og r ap h y e f f ect iv ely p r o vides I g G th a t i s 100% s p ec i fic f or a n t i g en – P r o t ein A/ G /L c h r om a t og r ap h y p r o vides t o t al Ig G , albe i t appa r e n t l y 10 % pu r e when anal y s e d b y SD S - P A GE. • Th e se di f f e r e n c es lead t o sig n i fi c a n t di f f e r e n c es in ac ti vi t y – a w or ki ng di l ut i on of an a n t i g en af fin i ty pur i fied a n t i body i s li k ely t o be 1 - f o l d high e r than th a t of a P r o t ein A/G p u r i fied a n t i bod y .

22. W h y “ p ur i fied a n t i bodie s ” a r e n ot all the s a me Un e xpec t ed c o n t am i na n ts – e . g. b o vine IgG • B y d e fin i t i on, y our pur i fied monoc l onal a n t i body m u s t be 100% pu r e. Is th a t r i g h t? • Be a w a r e th a t i f y our c ell c ul t u r e me d i a c o n t ains f o e t al / n e wborn b o vine s e rum th e r e m a y be s i g n i fi c a n t l ev els of b o vine I g G p r e s e n t – wh ic h m a y c o-pur i f y w i th y our monoc l onal a n t i body when u s i ng m e dia s u c h a s P r o t ein A or G. • A v o i d th e se either b y u sing a m o u se I g G s p ec i fic pu r i fi c a t i on s y s t em or c o n v ert i ng all of y our h ybr i doma c ul t u r e t o s e rum f r ee me d i a. • If y ou u s e a s ci t es y ou a v o i d b o vine Ig G , but y ou w i l l h a v e normal mou s e I gG p r e s e n t wh ic h also c o -pur i fies.

23. Conjugation consider a tions Y ou n e e d t o k n ow s o m e t h in g s a b out y our r ea g e n t. C onj u g a tions a r e r e a l l y s i mple bu t y ou n e e d p r o t ein in t h e rig h t f o r m a t t o w o r k e f f e c ti v el y . C on c e n t r a tion – 1m g / m l or h ig h e r is p r e f e r r e d Pu rity – e n su r e o t h er p r o t ei n s h a v e b e en r e m o v e d , a n d also m a k e s u r e t h e y h a v e n ’t b e e n pu t b a c k a g ain a f t e r w a r d s! B u f f er f o r mu l a tion – m o s t c om m on f o r mu l a tions a r e s u i t a b le, bu t e n su r e th a t a m in e s su c h as gl y c ine a r e truly a b se n t, as w ell as t h i o ls su c h as D T T or m e r c a p t o e t h a n o l . T ris is O K u p t o 2 m M P r e f er r ed bu f f er f o r mu l a tions f or t h e a n ti b odies t o b e l a b elled w i th t h e v ari o u s k its d i f f er ( es p e c ially f or n a n oparti c les ) , a n d pu ri f i ca tion m e t h ods a r e i mp o r t a n t t o und e r s t a n d

24. Future of Antibody Purification Strategies include : decreasing the number of steps expanded and simulated moving beds disposable and process analytical technology membrane chromatography non-chromatographic methods such as flocculation, precipitation, crystallization and aqueous two-phase systems.