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SUMMARY

I. Title

Development of refolding process for rhG-CSF using size-exclusion chromatography

II. Purpose of the research

The aim of this research is to develop and commercialize a high quality rhG-CSF by applying refolding process using size-exclusion chromatography. Human granulocyte colony-stimulating factor(hG-CSF) is a hematopoiesis agent that principally affects the differentiation of neutrophils in the bone marrow. At present, rhG-CSF is used successfully in the treatment of chemotherapy-induced neutropenia and its indication has been expanded to bone marrow transplantation and aplastic anemia. In 2000, the world market for rhG-CSF was 2.12 billion U.S$ and 12.0 billion won was spent on importing rhG-CSF as final product or raw material in the domestic market. Therefore, the development of G-CSF production technology will benefit the domestic market by substituting foreign rhG-CSF and, subsequently, by exporting the product. In addition, the development of G-CSF refolding process can provide an opportunity to explore more efficient and cost effective technology.

III. Scope of the research

1. Large-scale production of rhG-CSF

• Development of large-scale cell culture

• Development of large-scale purification process

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2. rhG-CSF production and characterization

• Production of pre-clinical and clinical trials.

• Physicochemical and biological characterization

3. Efficacy and toxicity

• Efficacy in normal, neutropenia induced mice.

• Acute toxicity, subacute toxicity, subchronic toxicity, genotoxicity and immunotoxicity

4. Clinical trials

• INO

• Preparation of clinical protocol.

IV. Results of the research

1. Large-scale production of rhG-CSF

A large scale rhG-CSF manufacturing process has been established. The working cell bank, which contains E.coli producing recombinant human G-CSF, is seeded and expanded to 20 L large scale culture.

The composition of media has been optimized and the culture condition has been determined to maximize rhG-CSF production. The optimized large scale manufacturing process involves isolation and solubilization of inclusion body, and refolding by column chromatography of the insoluble protein followed by 4 step column chromatography with an overall yield of 20% and more than 99% purity.

2. Preparation and characterization of rhG-CSF

More than 2g of rhG-CSF was produced and used for its characterization, preclinical and clinical trial. The physicochemical properties of rhG-CSF were analyzed by amino acid composition, N-terminal amino acid sequences, C-terminal amino acid sequences, disulfide bond linkage, CD-spectrum, isoelectric focusing, SOS-PAGE and western blotting. The analyses confirmed that the rhG-CSF is identical to native G-CSF. The in vitro and in vivo activity of rhG-CSF was compared to that of the international standard. The

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specification and dosage of rhG-CSF were determined, and the long-term and severe condition stability has been tested according to the specification and quality control procedure.

3. Efficacy and toxicity test

The efficacy of rhG-CSF was studied on normal mice and neutropenia induced mice.

Administration of rhG-CSF resulted in the increased hemoglobin concentration and neutrophil number in a dosage dependent manner when compared to the control.

4. Clinical trial

The safety/efficacy data and the specification/quality control procedure had been submited to KFOA according to the guideline and INO approval was obtained from ministry of Health and Welfare. For clinical trial of rhG-CSF, 5 clinical centers were selected and the clinical protocol was established. Currently, the clinical trials are in process in Asan Medical Center, Samsung Medical Center, Korea Cancer Center Hospital, Gachon Medical School/Gil Medical Center, Seoul National University Hospital to test the safety and efficacy on chemotherapy induced neutropenia patients.

V. Plan for application of results

In completion of phase III clinical trial on chemotherapy induced neutropenia patients, NOA will be filed and the pilot production and commercialization process will be followed. For industrialization of rhG-CSF production, studies will be conducted to enhance productivity, to reduce cost and to adapt present technology for manufacturing scale. The key technologies established in this research such as large scale cell culture and development of efficient refolding process using size-exclusion chromatography will be further utilized for development of other valuable drugs.

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CONTENTS

Chapter 1 Introduction 23

Chapter 2 Current features of technical development

in domestic and foreign country 29

Section 1 Current features of technical development in domestic country 31

Section 2 Current features of technical development in foreign country 33

Chapter 3 Methods and results of research .. 35

Section 1 Methods of research 37

1. Manufacturing process . 37

2. Measurement of content . 38

3. In vivo activity of rhG-CSF 38

a. Manufacturing of standard solution .. 38

b. Manufacturing of test solution 38

c. Test 38

d. Calculation of biological activity . 39

4. Physico-chemical properties of rhG-CSF 39

a. Electrophoresis 39

b. Isoelectric focusing 39

c. Molecular weight 40

d. Reversed-phase HPLC 40

e. CD(Circular Dichroism) spectrum 41

f. Immunodiffusion test 41

g. Endotoxin test 42

h. Residual peptide analysis 42

i. Residual DNA analysis 43

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5. Characterization of rhG-CSF 45

a. Composition of amino acid . 45

b. N-teminal amino acid sequence analysis 46

c. C-teminal amino acid sequence analysis 46

d. Location of disulfide bond 47

e. Peptide analysis 49

6. Pre-clinical toxicity 49

a. Acute toxicity 49

b. Subchronic toxicity 49

c. Genotoxicity 50

d. Local irritation test 50

7. Efficacy 51

a. Efficacy in repeated injection 51

b. Efficacy in neutropenia induced mice 51

c. Equivalence test 52

Section 2 Details of Research 54

1. Establishment of large-scale culture process 54

a. Establishment of cell bank 54

b. Fermentation process 57

2. Establishment of large-scale purification process 58

a. Purification process 58

b. Isolation and washing of inclusion body...... 59

c. Refolding of inclusion body .. 60

d. Purification 60

e. Development of test method 60

3. Stability 62

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4. Pre-clinical toxicity 63

5. IND 63

6. Clinical trials 63

Section 3 Results of research 64

1. Large-scale serum-free culture process 64

a. Establishment of cell bank 64

b. Fermentation process 65

2. Large-scale purification process 66

a. Cell disruption 66

b. Purification 66

c. Physico-chemical properties of rhG-CSF 68

d. Specification 73

3. Characterization of rhG-CSF 74

a. Composition of amino acid .. 74

b. N-teminal amino acid sequence analysis 74

c. C-teminal amino acid sequence analysis 74

d. Location of disulfide bond 77

e. Peptide analysis 83

f. UV spectrum 84

g. Molecular weight 84

h. Size exclusion-HPLC 86

i. Ion exchange-HPLC 86

i. CD(Circular Dichroism) spectrum 87

4. Stability 87

a. Stability of raw material.......... 87

b. Stability of finished product .. 92

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5. Pre-clinical toxicity 96

a. Safety 96

(1) Acute toxicity 96

(2) Subchronic toxiclty . .. ·· ·· .. ·· .. ······ .. ··· .. ······· .. ·· .. · · .. ·· · · · · ··.... 96

(a) 4 week subchronic toxicity of YHB6211 on rats by intravenous

administration 96

(3) Genotoxicity 98

(4) Local irritation test 99

b. Efficacy 100

(1) Efficacy in repeated injection · .. · ·· .. ···· .. ·· .. ··· .. ···· · .. · .. · · ·· .. ·100

(2) Efficacy in neutropenia induced mouse 104

(3) Equivalence test ····.····· ·· .. ····· 110

Section 4 Conclusion of research 115

Chapter 4 Evaluation and contribution of research 117

Section Annual objectives of research 119

Section 2 Degree of achievement 120

Section 3 Contribution · · ·121

Chapter 5 Plan for application of results of research 123

Section Field and plan for application 126

Section 2 Plan for Industrialization · ·127

Chapter 6 References 129

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Chapter 7 Subprojects·· 139

I . Subproject 1 141

II. Subproject 2 ·····147

III. Subproject 3 ·····153

IV. Subproject 4 161

V. Subproject 5 · ·167

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%~At .AR3:.~ G-CSF(~.:lctAtg)~ AJ719.l- ~ol ~~l l:lJ:>g-~ ;.-11'£(5637) %~R~ mRNA§. -¥- E-l ~~<>i ~ eDNA 1 i braryOil),~ ~ §. 'cJ ~ .:g-Al ~ ~ z! .2}ifJ -=jL ~ §. 1-1 38 AJ At~ ~At %~At2.j <fI71),~~Oil 271"6"1-0:1 %~At .AH3:.t[ l:lJ~.2.§_ t:H~irOil),~ A~{!~ .AR3:.t[ ~z! .2}ifJ-=jL ~§_q 38AJ At~~A1-§.),~ t:H~irOil),~ ~{!~ 71-e~ ~z! .2}ifJ-=r- ~§.q ~AJ At~~AH ~.:lctA tg (.AR3:.~))9.l- ~ol otulx w1t!Oil un)i;l..2..'tl(methionine)O] ~~ 1757R~ otu]x{!.2.§. -=r-AJ~ td1V1~§. 1V1~-=r- ).-1].£% ~%-=jL~ ~~. -%~),]:1] ~ ~At 0] ct.

%~Oi]),~ .A-J]3:.~ G-CSF~ ~Zl .2}ifJ-=jL ~§.q ~AJ At~~At9.l- ~o] 1V1~-=r- ).-1].£% ~%-=r-~ ~~ . -%~),]:1] ~ ~AtO]nf ., %~At~ ~~] l:lJ:>g-~ ).-1].£(5637) %~R~ mRNA §.-¥-E-l ~~<>i~ eDNA libraryOil),~ ~§.~~ .:g-Al~ ~z! .2}ifJ-=r- ~§.q ~AJ At~~At %~At~ <fI7]),~ ~ Oi] -2-71 "6"to:1 .A-J]3:.~ ~ 0] ct.

%~Oi]),~ ~ %~At .AR3:.tt l:lJ~.2.§' {l-it .AR3:.tt ~Q.{%~ ~-=jL7Rl:lJ7I]~~ 4-ifJ"6"t 2, ~)i;t~.2.§_ ~~] l:lJ:>g-~ ).-1]£( 5637) %~R~ mRNA§.-¥-E-l Jt!~<>i ~ eDNA 1 ibraryOi]),~ ~§.~~ ~Zl 2J-ifJ-=r- ~§.q ~AJ At~~At %~At~ <fI71),~~Oil -2-71"6"to:1 t:R~ir ).-11£ Z!%-(periplasm).2.§. ~t8(folding)~ 38cR~ ~z! .2}ifJ-=jL ~§_q At~~At~ ~\:J] • ~ {!'Y 4- 11 ~ t:R ~ir( YRCl)~ .A-J]3:."6"t91 ct. o].:f 0] o:Jl .A-J]3:.~ ~E-l (pYRCl)~ 0] -§-i>to:1 ~z! .2}ifJ-=jL ~§.q 38AJ At~~At %~At~ <fI71),~~%- 5'-Wtd ~71),~~ -¥-~ol t:R~

- 26 -

-rr0n).~ ~~Oll !i!.:ct ~~i5t.£~ ~~~AJi5tO:} AJl~~ ~Z! 2}ifJ-l'- ~.£q ~AJ At-9-~At %~At~ t:fl;g.-rr ~~ 1!!l'BfOll ~~i5tO:} t:fl;g.-rr ~~~17l10l1 ~~i5t2 ~~~ol oH~ ~~

~-?~ 1!!l'Bf(pYRC621-1)~ AJl~i5t~2, °l~ t:fl;g.-rr K-12 ).~l.£f- %cfl -rrf-Oll .£~i5tO:} G-CSF~ A~{!'~f-~ YRC621-1 ~ ~ 9j'Ct.

~lfl, G-CSF~ ~H~~ ~~~~.£ 7fl~i5t71 ~li5to:} 1) A~{!'~f-~ ~i!l~ ~l~ Dt~ 'Bf 11 l'I~3.(master cell bank) tM AJl~% 11 l'I~3.(working cell bank Is] AJl~, 2) AJl~

% 11 l'I~3.~ llfloJ~ ~~ G-CSF~ ~~, 3) ~~l.£-¥-BJ ~~P8~(inclusion body)~ G-CSF ~-?, 4) It''8~~.£~ ~flA~(refolding), tM 5) ~~ 3..£Dt~ -1i~fl!!l l:IJ~~ 01%

G-CSFOll t:fl~ ~i!l tM ~~AJ %~ ~ ~AJ '?:!-l'-~ -?"8'~"O"tO:} G-CSF~ in vivo .Ee:t, ~ll..H %cfl tM ~~AJ~ ~7ti5t~~~ ::1. ~2} 71.e-~ filgrastim(Grasin@ in]. )2} %~~ in vivo.Ee:t tM ~ll..fl%cfl~ ..5!.~2, ~~AJ 1i10l1).~.£ ~AJol ~~ ~~.£ Jlitg~9j'Ct.

~ know-howz] ~..fL~ 211{!, G-CSF ~fl~~ ).~l.£f- AJl~71~. t:flcJllfloJ tM ~AJ171~ % ~ At~17fl~~ ~~ 2~~ A~7t~ ~fl~~ G-CSF AJl~~ 7fl~i5t2At i5t~'Ct.

211{!' ~fl~~ G-CSF ).-ll.£f-~ AJl~. t:HcJ llHoJ.:g-~~ 7H~. 2.E~~ t:flcJ iE-i!1 • ~ AJl.:g-~~ 7fl~ tM G-CSF ~-llAJl 7fl~ %~ AJ.:g-~~.£ -9'-"8'~i5t~~~. 7fl~~ ~H~~ ~Z! G-CSF~1~l<YHB6211) ~ ~<g~).1~0l1).~ ~-?~ <!{.E~ ~~ ~~AJ~ 'l-f-ell..floJ ~~fl ~13

~ ~AJ).l~ ~~ ~ol'Ct.

~H~~ G-CSF AJl~~ 7fl~~ AJ-cJ~ -9'-~t:H~l.E2} tM -?~71t:H.E2}~ ~~~H ~ -9'- 9J.~~. :Etl- ~ 2}~10l1).~ 7H~~ 71~~1l71~~~ 'Ct~ 2.!f-7t7tj:l~ %% ~1il}j:l.li~l %~ 7H~0l1.£ It%~ -? 9J.~ ~~.£. 71t:fl~'Ct.

-- 27 -

- 29 -

%% ~~~ A~{!~ ~Jt;fl A}%~ ~ 4ffA'il~~ t:fl~-tr~ 18--o-}~ %~A} ~fl~~ 1t!~ ~~ ~~Al~ 7J~ inclusion body-.£ ~al~ ~%Acl %{l~l~ tsaAcl-o-}!::.-.£ ~~"&J~-.£~ ~t± 2f1I~ ~R~(refolding)2flIol ~~~olt:} . .:lcl"t.+ ~%Aa ~~~~ ~fl~~~~ 1t! ~~u}t:} t@"c.}~ ~ 9101 ~-o-}~ ~~~~ J:fl~~ ~lii}~~ ~~~ ;.H-.£ ~~t;fl0l=-o-}~ 01 al%o] 9lJl 01 2f1I <>ilA~ ~{!1f_~£ '5!"o}Al ~ ~All{}~ ~2 91 01 1f_2f~ Ctl ~RA~~ 0]Jf-7] ~]t;flA~~ 71-e~ ~al~ zt~ ~R~l:IJ'tJOil t:R~ ~"li~ Ctl ~-=r7} ..Q..-=r~o:1 ...., 7} -{f:-t:~1 7}~ 0] AJ~ Ctl l:IJ'tJ z] &~ ~ ~~ -o-}~ ~ 01 ~..Q..-o-}t:}. n:}C.}A~ 2-¥-7}7}~] ~ ~~l %~R cytokine %~ %~A} AR~~ ~ ~%~ ~~$!l A~{!1f_~-.£ t:fl;;g--rr~-.£-¥-E-f ~{!-o-} 2A} ~ 7J~<>il ~ A»A~-¥-JIl ~ -%7}<>il n:}~ ~1I2.l l:f]1f_~AJ z] tl]2~ '5!"~ J:fl~~~~ ..!i!_0l ~ 71-e~ !.l~l:IJ'tJ2.l t:fl~J ~1I7H~01 ..Q....;z.~01~t:} .

..;z.~J~~3:.. ~o:j2f ~AJ(size-exclusion chromatography)~ 0J%~ AflA~l:IJ'tJ ~ 2~ £3:.. %t;R~ ~%Acl ~~~~ !.l~~ol i!-l:f}3:.. columnoJl ~%AJ7'j ~ff~~ %.£~ ~ 91 01 ~~ ~R~~ Al.li2.l -¥-JIJ~ 71-e2.l !.l~'tJOil ~~ ~..!i!_t:} ~± 1013RoJ1A~ ~t:fl 10013R 7J}AJ ~~ ~ 9l~o:1 E:.~, 50%01AJ2.l i¥-~ ~flA~~~3:.. 1f_~~ ~ A~{! ~1I 7Rl:IJ 7}.!g-Acl ~ ..!i!_oJJl 9101 ~ l:IJ~2.l {!~i:f 7}.!g-~01 uRJf- i¥-t:}.

~~RoJIA~ *~A} ~fl~~ 71~~ ~%-o-}o:j ~t-oJl. %o}AlI~, AlI<MAllcJ %<>iIA~ %~A} ~fl~~ G-CSF A~{!ol AI'£~2 9l~o:1 %o}All~~ filgrastim~ A}~1~{!-o-}2 9l~o:1 ~ .2.lAll~~ ~~2.l Chugai A}oJIA~ %~A~l~ %~R *~A} AR&~ G-CSF~! l enogr-as t im-S- ~ All ~~l{}uflii}291t:}(Jf 1).

- 31 -

Filgrastim

CD ~ ~ A~ tJ-AJ {:! 3: ~ ~·n.£_ ~-? l}l 71 'E} 71 =t!( oJl : spl een) 611 A~ ~ ~ %-=j1-~ ~~ t3J ~~6fl ~~J~ %. %~A} ;;(H3:tJ-_2_.£_ human G-CSF % ~ A}~ cH ~it6fl AJ~ -o-}o:J ;;(H 3: tJ- human G-CSF ~ l:lJ~ -o-}.£ ~ 2 ~~L

(2) Chemistry

single chain polypeptide: 1757H o}ol~~ ~A}eJ ~ ~A}~ : 18,800, C84sH133gH2230243Sg

@ Fi Igrast im-j, 1..H<cl.AJ human G-CSF.2f o}ol~~ lIH~;: ~_2_q, N-terminal methionine.s] ~7l- t3J B-?~~oJ ~r.l-~ {I (non-glycosylated)ol r.}~.

* N-terminal methionine : cH~~6flA~~ %~Al- l:lJ~AJ ~AJ~ ~ o}oJ ~~

@ All3: : human G-CSF %~A}~ ~%t! cH~it~ master seed All3: -~All -- ;;(HA~(refolding) -- ~~ ~All

® Stabi Ii ty( fA}Al!) : pH 3. 8-4. 26flA~ <d~

5% dextrose(with or without 0.2% albuimn human)_2_.£_ ~~ 7}~.

0.9% NaCl.£_ ~~AJ ~~ ~AJ~_2_.£_ %~~ :lIl-o-}2 10'C 0l-o-}6flA~ .!i!.~

CD ~~ : 3!:%%~).·n3!: %cH ;;(H3:tJ- human G-CSF.£_A~ human G-CSF %~A}~ chinese hamster ovary ).~l3!:(CHO cell)6flA~ l:lJ~Al7-l A~~t! 1747H o}ol~~ ~71.£_ -=jL-'8{:! t;J1(!1:ll}~

Lenograstim (2) Filgrastim2f r.}~{1 : glycosylated. 1..H<cl.AJ G-CSF~ -=jL3:6fl r.~ % A}

@ Chemistry @ Stability

1747H o}ol.x.~, ~A}eJ ~ 20,000

10'C 0 l-o-}, %~ ~ :lIl-o-}o:J .!i!. ~ . pH 6 -7. 5611 A~ ~ ~

CD ~ ~ : human G-CSF %~ A}.£_ ~ ~ ~ ~ {:! cH ~it6fl A~ casset te ~ 0 1 ~ ~ 71~~ ol%-o-}o:J human G-CSF 1..H~ ~-¥- o}ol.x.~~ *l:t!Al:"{l ~ol~l ~.£_-¥-Bl ~~%6flA~ <d~-o-}2 ~~~.!i!.r.l- ~AJol 311H ~;: ~_2_.£_ -'j~ t! ~.

Nartograstim (2) Filgrastim2f~ *}ol{l : 1, 3, 4, 5, 17 ~l*l~ o}ol.x.~ *l:t! @ Chemistry : 175~ o}ol.x.~, -¥-~.lf-tg~ Q]f~l

@ Stabi n ty : 5'C ol-o-}, *}:;g--o-l-o:J 3\1 n % ~

~.£, :;g-6fl cHt! ~~AJ : 5'C6flA~ 4-,?-, 10001ux6flA~ 3-,?- ~2fAl ~"5l ~AJ6flA~~ ~~-o-}q pH 7 0lAJ6flA~ ~"5H~ ~7}

- 32 -

~H~~ ~Z! G-CSF~ 27tAl ~*2.l ~1~17t ~l~£l<>l l£!0H£l2 91cL ol~ ~~,.q9.J~~ KirinAt7t A~17-l1~~..£. .:g.%7H~~ %~At~H~~ ~Z! ~.g--=t- %~ ~At~ 1991 \1 01 ~ FDA..£.-¥-Bl %~% ~2 1997\1 ~~H A~17-l1 347H~ojlA~ All£!£l2 9lcL nil2r..i!l~~-E -711Bl eJ ~A~ 1!!Bl (Memorial Sloan-Kettering Cancer Center)2.l ~c~ %(1985) ~ ~ l:lJ :;g.~ A~l.£2.l 13HoJ~ojlA~ Ct-%AJ ~..£.q tgAJ At-9-~AHpluripotent CSF) ~AJ% ..5!.ol ~ ~At~ ~~l-o-t9i.Q..l.-t, 01 ~At7t 01-'f2.l ~-=t-~ 18--o-to:J 2.J-~-=t- ~..£.q tgAJ% ~01~.Q.. ..£. ~~Al~ol mt>:j~ ~ l:lJ:;g~ A~l.£ 13HOJ~oJlA~ ~~l~ CSF7t ~ 2.J-~-=t- ~..£.q tgAJ At-9-~AHhG-CSF : human G-CSF)~Ol ~~£l~Ct. AmgenojlA~ ~ <il klJ:;g~ A~l.£-? 56372.l 13HoJ~ojlA~ ~~~l~ ~n 2.J-~-=t- ~..£.q At-9-<ilAHhG-CSF) N W~~ °tOlx.{}A~~% 7J~ -o-to:J 01 0tOlx.{} 13H~2.l ~..5!.ojl 71~-O-to:J f-~~ mRNA~ AJ..5!.-o-t~ o l i gonucl eot.i dejj~AJ-o-t2 5637%~H2.l mRNA~ At%-o-to:J tt!"~<>l~ libraryojlA~ hG-CSF %~At~ ~..£.'-6-o-t 9icL 01 hG-CSF %~At~ t:H~~ l:lJ~~Blojl ~7J-o-to:J t:H~~ojlA~ A~{}~ rhG-CSF2.l ~ ~~~ tl"AJ% ~<il ~ 7J2.J- t:H~~ojlA~ ~~l~ rhG-CSF~ WEHr -382.l -{E-~%5:.tl"1:i 2.J- 2.J~-=t- ~..£.q tgAJ% ~01~.Q..~ ~~~ol ~~£l~ 5637 A~l.£-?%~»2.l ~Z! 2.J-~-=t- ~..£. t.] At-9-~AHrhG-CSF)9..j- %%~ tl"AJ% ;Q2 91%01 ~~£l ~c:t. 01 rhG-CSF2.l O-glycosidetg ~ cJj:jj~ ;Q~ cJ~~olAltt!" 01 cJj:jj-=t-~~ A~~~~ tl"AJojl ~?~~ Jl ~7t 0t'd ~% <M-711£l~.Q..nl 0l~ BFe:J.Q..~ ~~:Q:~ol -j£~ t:H~~% 4-?..£. -o-t~ A~ {}7-Il~ 7H~-o-t7l1 £1~.Q..o:j, t:»~~ojlA~2.l ~~ojl ..5!.Ct ~~~ %~A~~ AJ7-Il~ -f- ~~ ~AJ-O-to:J At%-o-t7l1£l~ct. ~~~1:i %~At~ ~~ ~Blojl ~7J-O-t2 t:H~~ K-12 7-Il-?ojl 5:. ~-o-to:J ~{}A~l.£-?~ 7HklJ-o-t9ict. tttctAl ~.:J..i!t.A.l£J (rhG-CSF)~ t:H ~~ojlA~ ~{}~ ~Z! 2.J-~-=t- ~..£.q At-9-~AtOlnl hG-CSF2.l °tOlx.{} w~ojl meth i oni neo] c~ ~~ 175 7H2.l 0tOl~{}..Q....£. -=t-AJ~ ~~~OlCt.

3:~, ~~ ChugaiAt~ 1983\1 ~~ Chugai ~-=t-~ojl ~l.:g.~ nude mouse °l~ At'iJ -=t- 7JA~~ A~1£7t ~~Hoj]~ ~tt! G-CSF A~{}-%~% 7tA]2 9l~ ~~ ~7.!-o-t9iCt. 01 ~oJ

- 33 -

~(~1frtl5 ; lenograstim) ~ 0to).x.-t!- 174.7H~ polypetide9.f 4% O-glycoside tg sugar

7-11 ~~§.. -&j.7H!ot lJJuH-o-t9i..Q..o:j ~~H AlI{!- %<li G-CSF A-jl%~ Jf. 2<>11 q~-o-t9it:t. ~ -2-<>11:::: ol~, ~~, *~ % ~Jn7-l1<>llA~ ~AJ~..Q..§" ~cl At%~T?1A~ ~Jn7-l) A)~ -Tt5:.

CSFs Developer/Manufacturer Brand name/Generic name Marketer
U.S.A Amgen Neupogen/Filgrastim Amgen
G-CSF
Europe Amgen Neupogen, Amgen/Roche
G-CSF Chugai/Rhone-Poulenc Granulokine/Filgrastim Rhone-Poulenc
Rorer(RP Rorer) Granocyte/Lenograstim Rorer(PR-Rorer)
Japan Chugai/Rhone-Poulenc Neutrogin/Lenograstim Chugai
G-CSF Kirin/Amgen Gran/Filgrastim Kirin/Sankyo
Kyowa Hakko Neu-Up/Nartograstim* Kyowa Hakko
Kirin/Amgen Grasin/Filgrastim ~;l~~%
Korea Chugai Neutrogin/Lenograstim %~A-jl~
G-CSF %o}A-jl~
Dong-A Leucostim/Filgrastim - 34 -

- 35 -

YHB621l(rhG-CSF)~ %~At 1i~1 ~

YHB6211~ %~At ~~~AJ ~

~~ ~"Bf0i1 ~~ ~

t:H~~ K-12 tg~~:t!(transformation) ~

YHB6211 ~ l3J~ tg ~ ~ :t!~1 4-~ ~

otA"Bf 11 l3~3..(Master Cell Bank) AlIa:

~

AlIa:% 11 l:l~3..(Working Cell Bank) AlIa:

~

~ A·n~ :tl4- ~

~%~ tg EH ~ YHB6211 ~ :tl4- 13J A·n ~ ~

;(HA~ (Refolding)

~ ~Tff3...£.ot~.:lcHJ!1 ~All

- 37 -

2{ ~£~ Jf~~ 9J {l ~ 50 td~ -tj"8}0'1 C4 {f~Oij f'-'?J -t- r:~g ~z!_Q_§_ ~~j3. §_u~.£.:lcflnj (RP-HPLC)~ 4-~~"8}~r:t.

{fW : 300 A, C4, Vydac, 214TP54, uj~

OJ%AJ : 0.1% E cj~f..2.§_ii':~ + 50% otJ.il.£Ll E~ - A %~ 0.1% Ecl~f..2.§_ii':~ + 70% 0tJ.ij.£Lj§.~ - B %~ iF'-J4f£ : 1. 0 ~/min

nt-?l" : 280 nm

7t. Jf~~ A-Jl~

NIBSC Jf~ rhG-CSF~ "Srr-J- 1 ~on ~<l!:if- <[!4f~~-o-to'l ~1:Jj-o-t~r:t.

Lt. {l~~l~

YHB6211(I mg/~)~ ~7-Il~~-o-to'l 8.0Xl04, 1.6xl05, 3.2X105, 6.4xI05 1:Ifl§_ ~~Al{! {l~~ ~l::lj-o-t~r:t.

c], Al ~

(I) ~-t- 8-10f'-£l ~ Balb/c ut.!f-6~ t:fl~~§_-¥-Ei ~4-;'ij.£~ ~fl-tl-o-t~t:t. (2) 4·COllA~ 10~Z! l:lJ~I~ :if- AJ*~~ +l-o-t~r:t.

(3) 400XgOllA~ 10if:-Z! ~1J~cl-o-to'l ~4-;'il.£~ !l4--o-t~t:t.

(4) ~4-;'il¥.~ nj~(percoll) %~Oil t!~Al{! -t- 15 ~ 37..Ll{f-if.!!.OlI ~<lir:t. (5) 2,000XgOilA~ 10~Z! ~1J~cl~ -t-.5:.<l! ;.il.£~ +l-o-t~r:t.

(6) ~ol~-~nt 1:IflAl 10 ~Oll t!~Al{! :if- 400Xg5?. ~1J~cl-o-t<>i !l4--o-t~r:t.

- 38 -

(7) All~{l z.t ~.£~ NIBse Jt€- rhG-eSF9.t ~qJj 50 td~~ z.t welloJl 'l].2 €-l:fl {l ).il~%qJj~ 1 me~ 7t~ -f- %-If:--o-l ~oF~·~t:t.

(8) 5% C02, 37·C BHOJ71oJl).1 5-7~Zl" BHOJ -f- 20-507H~ ).il~.£. _:;LAJ{l ~.:s?.'-l~ 1 7H.:s?. -o-tO:f ~ AJ {l ~.:s?. '-l -?~ 7Il-? -o-t ~ t:t.

ct. ~~~~ ~7t 711-8:

z.t NIBse Jt€- rhG-eSF~ Uni tlmeoJl t:»~ ~.:s?.'-l -?~ _:;L-o-tO:f ;~ ~~:t-l ~~ _:;L-o-t2 ~BJ~~ :&P8-o-t~t:t. z.t ~qJjoJl).l ~~ ~.:s?.'-l -?~ ~BJ~oJl t:»~-o-tO:f ~qJj~ Unitlml ~ _:;L-o-t2 z] ~ qJj~ ~~ l:I»-?~ -jg-~ -f- ~it--o-tO:f ~~~~ ~ 7t~ _:;L-o-t~t:t.

4. ~cl~~~ ~Aa 7t. ~71~%(SDS-PAGE)

(1) YHB6211(1 mg/me) , filgrastim(Grasin@ inj.)2.j- ~-"o'" ~71~%).1 ).t%~~ ).lli

%qJj (2X)~ 1: 1.:s?. ~~-o-tO:f ).lli~ €-1:Jl-o-t~t:t.

(2) ).lli 15 td~ 10-20% .::I.c)1t:l~E.1101 ;g.~{l ~71~%;g.~1~ ).lli -?~71oJl-?~-o-t~ c].

(3) ~71~%;g.~1~ ~-9-~ ~~;g-il-;g.~loJl ~~).l{l -f- 125 V~ ~1]~oJ-o-toJl).l Q{ 90-lf:- 1].£ ~7»).1 ~ t:t.

(4) ~7»{l 11-£ .}l-o-tO:f -ll-ot).l.E!.~c]~E.~f- R250 ~~qJjoJl ~2 AJ~oJl).l ~ 60-lf:%~ 't!%).]{l -f- t:t).] \!:~~qJj~.:s?. \!:~~).l~t:t.

~t. ~~~ ~~(IEF : Isoelectricfocusing)

(1) YHB621l, filgrastim(Grasin@ inj.)2.j- rEF ).lli%qJj(2X)~ 1:1.:s?. ~~-o-tO:f ).]li~ €-1:Jl-o-t~ r].

- 39 -

(2) ~71OJ%~:j.10i1 pH 3-10 .:1cJlt:l~§ ~alot3.~otD1E. ~~ 1t<u • :J..1J~ -f- "0'-

~Oil~ g~-&-t~%~(cathode buffer)~ ~HJf-:J.. -&-t~Oil~ °J~-&-t~*~(anode

buff er ) -£ ~H ~ r},

(3) ~l:Jl{! .Alli t3J Jf.~ pI Dt7l~ ztzt 20 t-d 13J 3 td~ ~71 OJ%~:j.l£.l .Alli -?-<U 71 Oil -?- <U -&-t.:;1. ~ ~ .:g--8-~:j.l Oil ~ -9--£ <B ~ A 1 ~ c],

(4) 7;j g 1Al Z!%«t ~ 100V-&-tOilA-l ~7HA171.:;1. t:tAl 200 V -&-tOilA-l 1Al Z! %~ ~7HAI ~ -l- 500 v-&-tOilA-l 30{tZ! ~7HAI ~t:t.

(5) ~7H{! ~-£ ?l-&-to:J fixing %~Oil If:t7t 1.A1Z! 1fr%Al~ -l- -=t-DtA]~if- .!:!.~al~ E R250 ~~~Oil ~.:;1. AJ~OilA-l Q.{ 30{tZ! 1fr%Al~t:t. 01"'" 0l~ ~~~~Oil ~.:;1. ~~~ Al~t:t.

r}, {tAtaJ ~<U

A] 'tJ~ LC/MS system~ o]%-&-to:J c] g £.l ~<!~§_ ~~g~ -l- YHB6211£.l °l~:j.l {tAtaJ ~ ~ 1J -&-t9i r}

(l)RP-HPLC

~~ : Vydac C4. 300 A

0l%AJ : A %~ 0.1% ~~Eal~if-.2.E..

B %~ 0.1% ~~§al~if-.2.E.. + 90% 0t)."ll.£1.-j§~

~~ 0.7 ml/min

at. ~AJ ~~l3.§_Dt.£., eHnl

(1) ~ o:J:z.t ~ ~13.§_ 0 t .£.:1 eH n]( size exc1 us i on - HPLC)

YHB6211 (1 rug/me) 50 td~ .}l-&-to:J t:l-& ~<!~ Q]j~l3.§..Dt'£.:1eHn](HPLC)~ ~~g-&-t 9ir:t.

- 40 -

{t~ : 7.5 mmX60 em, TSK-Gel G3000 SW, TOSOH, 05103 ol%AJ : £t:.1l1l ~-8"-q§-H. ~-8" !H%~(pH 5.4)

20 mM ~-8"-q§-H + 100 roM ~~l....P=-H + 0.03% £t:iIAJ ~-8"l....P=-H, pH 5.4 %eJ4j-£ : 1 me/iE-

u] . .!t!lfl~ 01-!:.lfAJ ~~§l:l(Circular Dichroism Spectrum)

At71.!t! ol-!:.lf"ci iE--8"J.ll(Jasco, Model J-715)~ At%~tO:i et% 3':.z!'£'£' ~~ ~t~et. l!t~ : 200 - 250 nm

,g.£ : 25t

:;g-£~ol : O.lem

Band width: 1.0 nm

-{f£ : 10 medg

Response : 2~

A 7fl t6 4j- £ : 50 nml iE-

1:lt. ~ ~ 1tf~ "ci ~

(1) YHB62112} filgrastim(Grasin® inj.) 100 tdOiI 1 M Ecl~llt ~]o]~ %~ 5 td~ 7t~tO:i pH~ ~"ci.£.£. 3':. ~ ~t~ r},

(2) ~1:l]!H Alli 10 td~ +]~tO:i ~~7Jl~ .2l~ &::.:g.0i] -?-~~t2 ~1J &::.:g.0i] ~&:: ~J ~ 2}ifFjL ~.£.q ~AJ At.;r~At ~J~](Goat anti hG-CSF Antibody: 1. 0 mg/me) 50 td~ -?-~ ~ .:r- ll,g.OiIA~ ~t*~ ~ j;l A] ~et.

(3) 7Jl~ +1~tO:i ~cl ~~4-(0.85% NaCl) 100 meOi] ~7t ~,g.Oi]A~ J.i]~(2~, 5:tl J.i] ~~ .2.~)~t2 etAI ~-ff-4-.£. ).il~(1~, 5:tl ~-ff--? ~~) ~.:r- .:r-lltAI.!:!.'tJcl~ §~Jf- R250 ~-!:.lf~0i1 '42 30iE- %<?l Jt!%AI:tl et% etAI ~~-!:.lf~.£.£. ~~-!:.lf Al ~et.

- 41 -

).t. ~s.:~~

(1) {l ~ 2l 3:All

YHB6211(I mg/me) 200 t.te~ ~l~ -=f 1 M .:§.cl~ot Bilol~ %~ 10 t.teit 7t-o-]-o:f Jilit %-"-a~ 3:~-o-t9ir:t.

(2) Jf.~ ~s:.~~2l 3:All

?'l.:§.<>Il .::g-.g-Xl ~ .R~ ~s:.~~ % ~ ).1 ~ ~*~~ ).t%-o-tO:1 ~71 .R).l ~s:.(X)2l 10/X BH~_£ ~ ~ -o-tO:1 10 EU/IDt ~s:.2l .R~ ~s:.~~% ~l:ll-o-t2 r:t).l ~ ).1 ~ ~*~_£ 10 BH ~~-o-tO:11. 0 EU/me2l .R~ ~s:.~~% All3:~ -l- ~4f ~~-o-tO:1 .R~ ~s:.~~% 3:All-o-t 9i t.],

(3) ).l~

(7t) ~A~ otol~~;;Jl0l'=2l z.t ~(well)<>Il 0JAat:H3:~. .R~ ~s:.~~ tM ~ ).l~ -s IT 1"( gAJt:H3:) 50 t.te~% ~ ~-o-t2 0:171<>11 ~(LAL) 50 t.te~ 7t-o-t9ir:t.

(q) z.t ~2l Jt!%£% J!tol~~_£ 2-3 ~~).l{l -l- 37±1.0°C<>ll).~ 10~n ~J*l-o-t9i c],

(r:t) 71~%~ 100 t.te~ z.t ~<>Il 7t-o-t2 5-6!l ~~<>l -'?-<>l ~tl").l{l -l- 37±1.0"C <>Il).~ 6~n ~J*l-o-t9ir:t.

(ct) Jt!%~li ).1 ~ 100 t.teit Zf ~<>Il 7t-o-tO:1 Jt!%% T?:I-€- -l- ~;;Jlol.:§. clr:~ (Plate Reader)_£ Zf ~2l -a-~s:.it 410 nm<>ll).~ ~~-o-t9ir:t.

0t. ~-,?-*cH ~E1E. -¥-~).l~

(1) ~7Jl ~J 0l~l(!lfll~ ~J~l %~% 5iEd %~~ ~A~ otol~~;;Jlol.:§. ~(well )<>11 200 t.te~ ~ ~-o-t2 4 'C<>Il).~ -o-l*~ ~*l-o-t9ir:t.

(2) Zf ~<>Il).~ ~J~l %~% All71-o-t2 A"ll~~~_£ 3!l A"ll~-o-t9ir:t.

- 42 -

(3) 25·C Oil.J..' 1.J..1 Z! ~ ;i:l-o-t~ 'Ct. (4) (2) 2}~..Q_§_ "~l ~ -o-t~ 'Ct.

( 5) €- B 1 ~ 0 l-!-li:! ~ ~ J! €- Q}j . {l Q}j "'ll OJAJ t:H s: Q}j -£ 200 td ~ ~-? -o-t.:11. 25"C Oil.J.."l 2 .J..IZ! ~;i:l-o-t~'Ct. g~t:H~Q}j~ <tl~~ %Q}j..Q_§.. -o-t~'Ct.

(6) (2) 2}~..Q_§_ "~l ~ -o-t~ 'Ct.

(7) oJ4l-A ~J °l-!-l(!~~ ~J~l %Q}j-£ <tl{!~ %Q}j..Q_§.. 50013H ~~~ -:f Zf ~Oil 200 td ~ ~-? -o-t.:11. 25"C Oil.J..' 1. 5.J..l Z! ~ ;i:j-o-} ~ 'Ct.

(8) (2)2}~..Q_§_ "·n~-o-t~'Ct.

(9) ~± ~J oJ4l-A IgG ~J~l-nl~.J..j'CtAil %~-£ <tl{!~ %~..Q_§.. lOO013H ~~l! ..:f Zf ~Oil 200 td~ ~-?-o-t.:11. 25·C Oil.J..' l.J..l Z! ~ ;i:l-o-t9i 'Ct.

(IO) "-11 ~ ~..Q_§.. 4 ~ "-11 ~ -o-t ~ 'Ct.

(II) €-Bl~ 71~%Q}j-£ z] ~Oil 200 td~ ~-?~ -:f ll..g.Oil.J..' 3~Z! tl"%.J..j=d .:v- tl" % -!-.It.J..l ~-£ lOa td~ 7t-o-to:J tl"%~ -!-.It.J..l ~'Ct.

(I2) ~~101~ cj'C,§.. 410 nmOil.J.., Zf ~~ ~~.£. {I:-£ -=jL-o-t9i'Ct.

At. ~-?%~H DNA Jf-~.J..l~ (I) .if€-Q}j Ail s:

t:H~i!:- .if€- DNA(IOO jig/m€)-£ DNA !.l~~*~~ !.l~-o-t~'Ct.

(2) {l~ "'ll °J~t:H~~ ~ cj

(7t) {l~ 600 td9.t 0JAJt:H~~~ ZfZf 500 td~ -}j-o-}~'Ct.

(q) ~1~/~§..§..~~/oj±ot~<tf~(24:1:1)-£ ZfZf %~ Jf-:lIl ~7t-o-to:J 2~ ~~-o-t ~'C}.

('Ct) ~§..§_~ ~ 101 ±otn,J <tf~( 24: I)~ %~Jf-:lIl ~ 7t-o-to:J I~ ~~-o-t9i'Ct.

(ct) TE ~*~..Q_§.. ~~~-£ 500td§.. ~~ l! 'Ct% 213H Jf-:lIl~ -¥--?OiltE!~2} 0.1

- 43 -

-¥-nl~ 4 M <fl.tfel%(LiCI)~ 7t-a'tO:f -70'C ~A~~7lOll 30~Z! tlJj;l~t~:c.t.

(nt) 4'C, 12,OOOrpmOll}.~ 3~Z! -<t!ll~el~ r:J-% ~AJ~ ~~@i~ 7(}>A> OllB~§.. ;.~l~~l.:iL °J5ir:t.

(}.t) INA ~~~QJ} 10 J.d~ 7t~tO:f ~~~l ~~ r:J-% ION ~J.tf1-P=:it 1 J.d~ 7t~t~r:t. (ot) 95'C OlAJOll}.~ 10~Z! HO:f ~AJ}.l~ -f- ~ gOll tlJj;l~t~r:t.

(3) DNA ~ ~ (Dot Blotting)

(7t) q~~ ~.!:!..cjl~~ ~~~ .::vl§.. ~t zx sse %QJ}Olll~Z! ~H:lr:J-% ~El{!-(Whatman) O:f2l-Al ~lOll \=-2 ~~~l <!~}.l~r:t.

(q) °r~fl~ ~~ tlJ~Oll n:rcr zt Jt-€C!Jl ~ 0JAJt:R~C!Jll, 2~ 1 J.d~ lc12 {lC!Jl 4 J.d~ ztzt ~~ift~r:t.

( 4) DNA ~AJ.tf Jti%

(7r) ~~~ ~.!:!..cjl~~ ~"8.tf710l1 ~2 DIG Easy Hyb %C!Jl 10 me~ ~7t~ -f- 42 tOll }.~ 2}.1Z! °lAJ Jti%}.l~r:t.

(q) tg-~At INA~ 95'C 0lAJOll}.~ 1~Z! 7r~~tO:f ~"8}.1~ -f- ~%Oil tlJj;l~ r:J-% ~~J~ ~ .}lifrO:f ~~J~J~ DIG Easy Hyb %~Oll ~ttift~r:t.

(r:t) (q)~ DIG Easy Hyb %~ ~~J~J~ q~~ ~.!:!..cjl~ol ~~91~ ~"8 .tf710l1 ~ 7tift2 42·COll}.~ ~t*~%<{!-(16-24 }.1Z!) Jti%}.l~r:r.

(el-) Jti%ol ~\l-f- DIG Easy Hyb %~~ J:Jl cl2 2 XSSC/0.1% SOS %~ 50 me§.. ~~Oll}.~ 5~ 2~ ;.~l ~ ift~ c],

(Dr) 0.1 x SSC/0.1% SDS %~ 50 me~ 7tiftO:f 68·COll}.~ 15~Z! 2~ ;.~l~~t~r:r.

(5) {l~

(7r) 0.1 x SSC/0.1% SDS %~..Q..§" ;.~l~~ ~.!:!..cll~~ r:t}.l DIG-;.~l~~..Q..§" 2~Z! .2.

- 44 -

li!~t\1).~ ;.·u ~ ~t~ 'Ct.

(q) DIG -:j;t l{! ~ ..2.. .£. 30~Z!: :j;tl{!).l ~'Ct.

('Ct) ~J 'Cl~).l7il'd ~J~l-~7tet~ :Y=.A.:Y=c:t.A-fli} DIG-:j;tl{!~Oil 1: 5, 000..2...£. ~ ~~t~ 'Ct(2 t-d Anti - digoxigenin Ab-AP + 10 me DIG-:j;tl{!~).

(et) (q)Oil).~ :j;tl{!Jt~%ol ~td ~l!.cfl~-£ ('Ct)Oil).~ €-tll~ %~..2..'£' 30~Z!: li!%).l ~'Ct.

(ot) (et)li!%.:f %~~ ale].:;:L DIG-;.~1~~..2..'£'15~Z!: 2~ ;.~]~~t~'Ct.

(tit) Dig-;.·n~~~ alel.:;:L DIG-{tAl~Oi]).~ 2~Z!: li!%).l~ 'C]-% ~l!.cfl~~ :!jeJt! 3.7]~

<Mli! tll'el ~(bag)Oil ~9i'Ct.

().t) (tlt)Oil ~~).l~ %~ 2 me~ ~7tt! .:f *~~t.:;:L 5iE-Z!: li!%).l~'Ct. (ot) <?:flIOil).~ X-ray l€l~Oil <M~).lZ!: .x.~).l~'Ct

(At) Densitometer.£. z.t li!%~Hspot)~ {t£(density)~ ~~~t~'Ct.

7t. -=rz-AJ 0tol.x.-t! ~AJ

(1r?:I~~ .A-fl~(Prep. & Hydrolysis)

Cysteine2} tryptophan-£ .A-fl~t! 0tol.x.-t!~ general hydrolysis \:IJ~Oil ~~to:J Cyseine~ 5% formic acid, tryptophan~ 4M methylsulfonic aciOil ~~to:J 44 7t?~ t;R~to:J {l ~~ €-tll~t~'Ct.

(2) .£€- °tol.x.-t! .A-fl~

.£€- otolx-t!(2.5tlmoles/me) 10 t-d 4z.t~- -if-l!.Oil ~.:;:L~..:g. z!~).l~'Ct.

- 45 -

(3) PITC - oto]x-8- %s:.:~:n A-j]~

(7t) Zf ).]lic>J] u-J]B% : %-ff--? : Triethylamine(2 2 I, v/v) %~ 10 p.e~~

7t"6"tJl ~oj €: -V- r:t).] ~.:g. -,?!~).]~r:t.

(q) u-J]B-& : %-ff--? : Triethylamine: Phenylisolthiocyanate(7: 1 : 1 : I, v/v)~ PITC %s:.%~~ Zf A]lic>J] 20 p.e~ ~7t"6"tJl AJ~c>J]).-I 10-20~n Jt!%).]'tl -V- ~.:g. -'?!~).] ~r:t.

(r:t) Zf ).]lic>Jl A%~ 400 p.e~~ ~ 7t"6"tO:f -'?!~).] 'tl ).]li~ %t;H).] ~r:t. *A%~ : 140 mM ~-8-~t~it(pH 6.0) + 0.002% EDTA + 6% Acetonitrle

(4) :{l ~ 13J 1t€: 0to]x~ %~~ 50 p.e~ +]"6"tO:f r:t% ~-'?!..2.'£ ~;lf]3..£ot~.:LcHJI] ~(HPLC)~ -?~~"6"t~r:L

~~ : Pico-Tag Free Amino Acid Analysis Column

O]~AJ : A %~ - 140 mM ~~~t~it(pH 6.0) + 0.002% EDTA + 6% Acetonitrile B %~ - 0.002% EDTA + 60% Acetonitrile

JIt;g : 254 nm

q. N-~tt! 0to]x~

Edman degradation l:IJ~..2.'£ At~ tt!~~ ).-1 ~~~ 7j(protein sequencerf All.l-s], Model 476A)~ o]%"6"tO:f 107H~ N~tt! ~7]~~ ~~"6"t~r:t.

c], C-~tt! °to]x~

(1) YHB6211~ ~AteJ A-j]~ 3.7]7t 10, OOO~ ~l:i~]~..2.'£ ~~"6"t~r:t.

(2) ~~{! ).lli 1 mec>Jl pepsin-agarose gel 0.4 me~ 7t~ -V- %~"6"1 ~Q ).]~r:t. (3) 37"C i ncubator-oll-s] Q.{ 1,800 rpm..2..£ 2Jt!t;tnl l).]n Jt!%).l~r:t.

- 46 -

(4) (3)~ ~%.Alli~ "Cl-%~ a:-Cl.~ RP-HPLC~ LC/MS(API -electrospray LC/MS

system) {f-~~ -?~~'6"r'li"Cr.

(5) RP-HPLC

~Tf:f : Vydac C4, 300A

01%-~ : A %~ 0.1% J:.~.§.cl~.!f-..2.. e

B %~ 0.1% J:.~.§.c1~.!f-..2.. e + 90% 0rJ.~l~1-l.§.ifl

%4;- o. 7 me; {f-

nr~ 215 nm, 280 nm

(6) Post column addition

J:.~Ecl~.!f-..2..eon ~t! LC/MS~ {f-~ ~~R~ '9-~'6"r71 ~t! a:-Cl.~ 0r~~ %nR ~ HPLC .Al~2l- lt~l mixing tee ~l31~ ol%'6"ro:t 0.1 me/{f-~.£. It:1Jll -i-a1~9i"Cr. ol%-AJ : ~.£.n1..2..\9ojl.AIE./ol~~~~ = 20/80

%4;- : 0.7 me/{f-

(7) LC/MS

Source: API-ES(Electrospray)

(7r) RP-HPLC Acr<>l1.A1 ~~!tl .£~ nl3.~<>l1 cRt! LC/MS {f-~~ -?~c5"ro:t zt n13.~<>l1 cRt! ~aJ(mass)~ Tc5"r 'li "Cr.

(~r) LC/MS system~ peptide tools(Protein & Peptide analysis software, Hewlett Packard, USA)~ ol%c5"ro:t zt ~EIE.~ ~aJ(mass)<>l1 ~Rt;Jc5"r~ 0rtll.x.~ a:AJ ~ ~~~ 4- ~~ mass-matching~ -?fs~t! .:f C-oJl(!-¥-~l~ 0rtll.x.~ a:~~ .!i!.01~ ~E1E. nl3.~ {f-+lc5"ro:t Edman degradation ~J~~§_ Ar%- ~~~ .A1 ~{f-~ 71~ ol%'6"r of °rtll.x.~ .A1 ~~ ~~c5"r'li"Cr.

cr. "Cl~nrolE. ~"U(disulfide bond)

(I) YHB6211 ~ {f-AraJ Ai1t! 3.717r 10, OOO~ ~.!:!..~1~~ ~~c5"r'li"Cr.

- 47 -

(2) ~~{! }..l.li. 1 me Oil ~{l-Or7t§...6. ~ 0.4 me-£ 7tt! .:f %if;-irl ~~ }..l~'Cr. (3) 37·C incubatorcll-s] Q..j= 1. BOOrpm_Q_§.. ~~irto:j p·1Zl ~%}..1~'Cr.

(4) (3)~ ~%}..1.li.0i1 ~% 0.1 M5:l7i1 Ec1/-..-<g{l-(pH B.O)~ 7tirto:J pH~ 7. 5-B. 001 5:l s.~ ~~irt9i'Ct.

(5) Di thiothrei tol(DTT)~ ~% 20 mM5:l7i1 1I7tt! -f- 37"COil}..~ 1}..1 Zl ~%}..l~'Ct. (6) DTT~ ~ c1irtA1 ?J ~ }..1.li.( 1:11~~ ~)9.}- ~ c1 t! ;..1.li.(~~ ~)~ ztzt 'Ctg ~ ~{! _Q_§.. LC/MS(API -electrospray LC/MS system) if;-~% -?wgirt9i'Ct.

(7) RP-HPLC

~r& : Vydac C4. 300A

01%-"'J : A %QJ} : 0.1% ~{l-,§_c1~.if-..2...s

B %QJ} : 0.1% ~{l-,§_c1~.if-..2...s + 90% 0t;'i1.£.t..],§_~

%4 O. 7 me/if;-

nt~ 215 nm. 2BO nm

(8) Post column addition

~{l-Ec1~1f-..2...sOil ~t! LC/MS~ if;-~ ~~H~ -9-~irt71 $f1~ ~.c!_Q_§.. 0t~H~ %oH ~ HPLC }..1~2f tI"~n mixing tee ~1:11~ ol%irto:J 0.1 me!if;-_Q_§.. tI"~l ~aif-~'Ct. 01%-"'J : n§..nl..2..1::l0i1;..1E../old::~§"~~ = 20/BO

%4 : O. 7 me/if;-

(9) LC/MS system~ peptide tools(Protein & Peptide analysis software. Hewlett Packard. USA)~ 0l%irto:J zt ~E1E..~ ~~0i1 ~HcJirt!: oto1x{l- ~~-£ ~~~ -? 91 ~ mass- matching% -?t>g~ .:f ;..l.6.E-ll~% tI"%~ -? ~!: ~E1E.. nl3.~-&- if;-.}lirro:J Edman degradation l:IJ~_Q_§.. At%- ~~~ ;..~~if;-~71~ ol%irto:J °rolx{l- ;..~~-£ if;-~ irt9i 'Ct.

(10) ;..1 /-..E-ll~% tI"%irt2 9l!: ~EIE.. nl3.~Oil t:H~ ~~~% ~-f-~ ~~.£ ~ ~irto:J Zf ~E1E..Oil}..~ -?d::~Ar 27HOil ~HcJ5:l!: ~eJ .*tol~ ~'ll~ -f- zt ~E1E..Oil}..~~

- 48 -

n], ~El~ ~~5:.(Peptide Map)

(1) YHB6211 1 mtOil ~{! -ot7t_£~ ~ O.lmt~ 7ttl' -'f *~til ~ ~/q ~J:ct. (2) 37t incubator-ell-s] ~ 1800 rpm..Q._£ 2Jtititni 1 A1z! Jti%Al~t:t.

(3) Jti%Al {l Alli ~ t:t g_ 2l ~~..Q._£ ~;\·Il3._£ot~..:L eQ lll( RP-HPLC)~ 4-~~tit9i c],

(4) RP-HPLC

~~ : Vydac C4, 300 A

01l8-~ : A %~ 0.1% s.1!.§..t!1{i-.!f-..2....s

B %~ 0.1% s.1!.§..t!1{i-.!f-..2....s + 90% 0tAil~q.§..~

%4r O. 7 mt/~

llt~ 215 nm, 280 nm

7L ~~.!f-o:l~AJA1~

(1) ~nJf,_n ~~.!f-o:l~AJA1~

YHB6211~ ot-)?-A(Crj:CD-I), '9!~(Crj:CD) lJJ 7Q(beagle)Oil ZfZf 0, 10000 ltg/kg %~..Q._£, 1~ ~nJf,_n .!f-o:}tit9it:t. Al~7-ll~ AJ*l-lf-2l ~-)?- z] ~~ ~. 4- Zf lOott!l ~, 7n ~ ~. 4- Zf 2ott!1~ 13n~tit9it:t. A1~~~ Jf-o:l -'f ~AJ%AJ~1l", ~1*~~ ~ -¥-~~ 1JAltito:} A1~~~2l ~~"'8~ ~7ttit9it:t.

q. Jti~.!f-o:l ~AJ A1 ~

(1) '9!~0i1A-l 4-,?-Zl ~nJf,_n Jti~Jf-o:}~AJA1~

- 49 -

YHB6211~ Crj:CD(SD)IGSJl1 <;J!.soll 0, 10, 100, 1000 pg/kg~ %aJ..2..§.. 4-f-Z! DR~ li.l ~tlJll.-R Jti.J!}Jf-o:l-o-t9ir:t. ),1~Jl1~ zt ~cJ crJ-.4- ztzt 14utc1~ llR~-o-t9itl .EE~, i.l.J!}~..2..§"),~ crJ-.4- ztzt 14utc1~~ .£.~ ),ltl~Oll ~cJ-o-t.:lL, Jf-o:l71Z! ~li .:1f- 2-f~ 4-f-~R0l1 crJ-.4- ztzt 7utc1~ ~e.f),t),l~r:t. ),ltl71Z!%~ ),tllJ~ ~ <HAJ~~=t!1f, ~n-%, ),tli 13J ~Aj-}laJ~~, ~2}~~~),t, ~~~ 13J ~~:£}~~~),t, .k~),t, ~71-% aJ~~, -¥-~ 13J ~c1~~~~~),t~ AJ)'l-o-t9ir:t.

r:t. *~ ~AJ )'1 tl

(1) Salmonella typhimuriu~ ol%t!' .J!}:H~~~ol),ltl

YHB6211~ 0, 6.25, 12.5, 25, 50, 100 ~ 200 pg/plate ~~§.. Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 ~-f-0l1 ~%-o-to:l .J!}f-1~~~ol*l:IJAJ~ ~ 7t-o-t9ir:t.

(2) ut~6 ~4-).-l1.¥.~ ol%t!' ::±.~),1~

YHB6211~ 7-f-~~ Crj:CD-1(ICR) 4-~ ut~6011 0, 100, 1000 ~ 10000 pg/kg~ % aJ..2..§.. 1!.l ~~l.-R Jf-o:l-o-t9ir:t. %DRt:H~~Oll~ 10mM sodium acetate ~*%~(0.004% Tween 80, 5% D-mannitol, pH 4.0)~ 10 me/kg~ ~~..2..§" 1!.l ~tlJll.-R Jf-o:l-o-t9i..2..o:l, oJ AJt:H~~Oll~ mitomycin C~ 2 mg/kg~ %aJ..2..§.. .J!}7Jl.-R Jf-o:l-o-t9ir:t. ~4-).-l1.¥.~ 4-7i~ )'1 ~ ~~ Jf-o:l .:1f- 24)'1 Z!0l1 -o-t9i c],

ct. ~~~AJ),ltl

(1) ~7J10l1),~ JI1-¥-At-9-),1~

YHB6211~ ~ 37H~~ ~7Jl(New Zealand White, NZW)~ JI1-¥-0l1 0.03%(300 pg/me)~ ~ ~ 1i.l ~~ .::if- JI1-¥-AP,AJ o:l~ ~7t-o-t9ir:t.

- 50 -

(2) £~loflAi <ll~llf;x.t-9-Alt1

YHB6211% ~ 37H~~ £~l(NZW)~ ~~llfofl 0.03%(300 pg/me)~ ~£§.. 1~ ~<ll'U- -Y<ll~ llf;x.t'9-AJ 0:] ~~ ~ 7ttlt~ .. ct.

7t. ttl-~lf-O:]Al~

AJ ~%¥~ %AJ ICR mouse~ At%tlt~.Q.r:r:j, YHB6211 l_M fi 19rastim~ 5mg/lOmllkg 01 ~5:.~ 3:.~~l%~of] .ar~ :f 100pg/l0ml/kg~ %eJo] ~5:.~ !l~tltO:] At%tlt~"Ct. A] ~~~ CD Control (Vehicle, S.C.), ~ YHB6211 If-o:]~ (100pg/kg, S.C.), aD Fi l gras t im .!f-O:]~ (lOOpg/kg, s. c. ).Q..s:.. 1il~tlt~"Ct.

YHB6211 l_M fi 19rastim% 0.lml/10g~ -¥-nl.s:.. nltlt-?At tltO:] 1 ~ 1~ ~"CH 7~Z! ttl~.!f-O:] tlt~"Ct. "CH3:.~ofl~ 3:.AJI %~~% nltlt-?At tlt 9i "Ct.

~H~~ ).]~Q.{¥ If-O:] ~ l3J -f- 4,6, 8, 15~~Hof] ~-=j1-~H~~_2_.s:.. +ltlt9i"Ct. ~~ 5:.w{l~] ~~l~~ ~H~ -f- ~).l hematocrit 4~% capillary tubeof ~~eJ~ ~~% +1 t! -f- slide glass -?-]ofl £ Wtlt 9i"Ct. £W -f- Wright-Giemsa(Sigma, Cat. No. WG-32, USA) !fl~~ofl n:tet !fl~tlt2, ~~-=j1- ~~7-I14(di fferential counting) ~ ~).]tltO:] -8- 1007R~ ~~-=j1-..g. :Q:_..g.-=j1-~ ~~~% -=j1-t! -f- 0:]71011 ~-=j1-;x.t%4~7].s:.. 4~t! %-~~-=j1- -?~ _:g.tltO:] ~~~ tj~1-¥-n](mm3)t:.J :Q:_..g.-=j1--?~ -8:~tlt~"Ct. t-tnJ;x.] ~~~ ~ ~~'U-

-f- ~-=j1-;x.t%4~7] (Minos Vet, Roche).s:.. %~~-=j1--?(WBC)~ 4~tlt~"Ct.

t-t. Neutropenia .2.t:Jlof]).i~ Q{~).l~

AJ~%¥~ %AJ ICR mouse~ ).t%tlt~.Q.r:r:j Neutropenia~ %~).17171 -?-l~H).i ~ 3~ ~ ~J<?JAJ][CD Cyclophosphamide (CPA, 78mg/kg), ~ Vincristine (VCR, O.15mg/kg), aD Doxorubicin (DXR, 5. 2mg/kg)]~ ~~ ).t%tlt9i"Ct.

- 51 -

~l 3~£.l neutropenia agents-£ 313H£.l %eJ.Q_§. ~At% ~*? :g:~ ~At% ~al~ ~?Of] .arO:J ~AJlt!.:f ZfZf -n-%t! -¥-lIl~ +]-a-tO:J -a-t1-}-£.l vial e] ~tI"-a-tO:J At%-a-t~r:t.

A]~~~ CD Control (vehicle), ~ Neutropenia ~~~, ~ Neutropenia + YHB6211 If-O:J~ (IOOJ,tg/kg, s. c. ).Q_§. "'"J~-a-t~r:t.

Neutropenia agents-£ 0.lml/10g£.l -¥-1I]§' -Jit7Jt..H§. 1il ~Att! ~ 24A1Z!~H-¥-"Bf YHB6211~ 0.lml/10g£.l -¥-311§. 311-a-t~At -a-tO:J ~t:H 5~Z! lfr-Jitlf-O:J -a-t~r:t. t:H~~£.l ~ ~ neutropenia agents t:H~ ~At% "'".gal ~ ~?~ -Jit7J~At t>t~2, Al ~~~ t:H~ YHB6211 ~Ail %~1i!~ 311t>t~At t>t~r:t.

~H1fI ~ neutropenia agents If-O:J ~ 13J .:f 2, 3, 4, 5, 6, 8, lO~~H Ofl <tl".:;z.~H1fI~.Q_ §. +It>t~r:t. ~~£w{i~n AJl~~ ~H1fI ~ 4Al hematocrit ~~% capillary tubeof ~~eJ£.l 1fI~~ 4-1t! ~ slide glass ~lOfl £W-a-t~r:t. £oJ ~ Wright-Giemsa(Sigma, Cat. No. WG-32, USA) ~ ~ ~ Ofl u:tat ~ ~t>t2, ~~-=t- {f~71]~(di fferential counting) ~ "'"J A] t>tO:J %- 1 007H£.l ~ 1fI.:;z.-8- ~-8-.:;z.£.l ~iE-~~ .:;z.t! ~ O:J 7] Of] 1fI.:;z.~P8- ~~7]§. ~~t! %-~1fI.:;z.?-£ .:g.t>tO:J ~1fI£.l 1(!~1-¥-31I(mm3)"CJ :2:-8--=t-?-£ 1!~t>t~r:t. 1-}-uf~] 1fI ~ ~ ~ ~'Ut! ~ ~-=t-~t%~~ 7] (Minos Vet, Roche)§. %-~1fI.:;z.?(WBC)-£ ~~t>t~r:t.

c], Q{~ %%"'"3 A]~

"'"J ~%~~ %"'"3 ICR mouse-£ At%t>t~.Q_tr:j, YHB6211 tM fi 19rastim~ 5mg/10ml/kg 0] s:I£~ ~AJ]%~Of] ~~ ~ 500, 100, 20, 4J,tg/10mllkg£.l %eJo] s:I£~ ~~t>tO:J At %-a-t9ir:t. A] ~~~ CD Control (Vehicle, i. v , ), ~ YHB6211 4J,tg/kg If-O:J~ (i. v, ), ~ YHB6211 20J,tg/kg If-O:J~ (i. v.), @D YHB6211 100pg/kg If-O:J~ (i.v.), av YHB6211 500J,tg/kg ~O:J~ (i. v.), aD YHB6211 2000J,tg/kg ~O:J~ (i.v.), CD Filgrastim 4J.tg/kg If-O:J~ (i.v.), ® Filgrastim 20J.tg/kg If-O:J~ (Lv). ® Filgrastim 100J.tg/kg If-O:J ~ (t.v.). @) Filgrastim 500J.tg/kg If-O:J~ (I.v.). @ Filgrastim 2000J.tg/kg If-O:J~ (L, v, ).Q_§. "'"J~t>t9ir:t.

- 52 -

YHB6211 13J filgrastim~ O.lml!lOg~ .!f-1I]§' 12.c] ~1lJft..jf§. l!l f-).}-g-}~r:}. t:Ha:. ~Oi] ~ a:.Ai] %~ttl~ ~1lJff-).} -g-}~r:}. ~H~~ ).]~ ~~ Jf-of .:f 12).] ~~HOi] <cl".:;z.~H~ ~~§. -l-]%}9ir:t. ~~_£~{:}~l Ail~~ ~H~ .:f ~).l hematocrit ~1}% capillary tube Oil <tJ1}~~ ~~~ +]~ -:f slide glass ~loll _£oJ%r9ir:r. _£oJ .:IfWright-Giemsa(Sigma, Cat. No. WG-32, USA) ~~~Oil tq.2.t ~~%t.:;]_, ~~.:;z. ~~71I~ (differential counting)~ 1J).]%tof %- 1007H~ ~~.:;z.~ ~~.:;z.~ ~~-i-~ .:;z.~ -:f of 71 Oi] ~.:;z.Ar%4~ 7]§. ~~~ %-~~.:;z.?~ ~%tof ~~~ l(!~].!f-1I] (mm3)"CJ ~~.:;z.? ~ {!-~%t~r:r. qufA] ~ ~ ~ ~ ~tt~ .:f ~.:;z.Ar%4-~ 7](Minos Vet, Roche)§. %-~ ~.:;z.?(WBC)~ ~~%t~r:r.

- 53 -

l acl", laePL8, ampC:: Ptrp cI, merA, merB 01 ct.

( 2) 1l tl~ 3.. ( Ce 11 Bank) All3:.

(7t) otAEl 1l tl~3..(Master Cell Bank) ~ifJ .2..~5:.

eij ~-tr YRC621-1 % {} -'&-&to:f 30 "C c>Jp.~ 12).1 z! llij oJ ~

lj-~ ~.£.q~ ?l-&to:f RM llijA1 200 mec>Jl {}-'&-&to:f 12).1 z! llijOJ (30·C, 250±50 rpm)

~1Hf.-c1 (lO,OOOXg, 15~)

~ %~z!3:. t Ji!.-t!"( 4 ·C )

- 54 -

RM llR;;tl ~1a:(200 me) ~

tltAEl ;.~l.£ 13~a.ofl.A~ 17H~ ~~~ 7Jft.Hof 0.8 me RM llHA1~ ~~ ~

RM llH:Al 200 meofl ~~.:f llHoJ ( 30 DC, 250 rpm ± 50 )

~

RM llH;;tl (15% 1tcl ;.·n~) ~1:il

~

1 mt llHoJ~ + 1 mt RM llH;;tl(15% 1tcl;.~l~)~ %~<:!a: ~~ofl ~~

~

~~( -70'C ~;;t~~71)

(3) ~ 13~a.~ ~AJ lJJ ~~;;tt~ ~~AJ a:.At

zt ~ 13~a.~ ~AJ ~ ~tlp~ ~~, 11 13~a.~ ..2.~o:f-¥-, A~-e-£, ~ctAtll.s~ ~ ;;tl-£ %~ ~~~to:f a:.At~t~'Ct. !E~ ~1~~6::: ~~ofl ~~ ~~At~ ~~AJ a:.At lJJ ~71.A~~ ~~(DNA sequencing)ofl ~~ YHB6211 ~~At~ ~~AJ~ ~.At~t~'Ct.

%~<:!a: t}l %~~ tltAEl ~ 13~a.~ ~la:% 11 13~a.~ ~o:f .=Lifj-~-!Jf~ ~~t~! .:f 7FT ~ 3JEH t}l a.71~ ~tll7J.Q..5:. ~~~t~'Ct.

- 55 -

(l-t) 11 l:lg 3..2.l _Q_ ~ o:J-¥-

Zf 11 l:lg3..~ ~'li t:tg, ~-tr ~cl ~~-?-_£ 10-8 7JtAl !.l~tt .:f Tryptic soy agar llRAIOlI ZfZf .£~"8"to:J ~Al.'{! llR6J"8"h2 01ttJ{ _Q_~!f:! oll1~2.l Age o:J ~ ~ :eHt"8"t91t:t.

(t:t) Age~

Zf 11 l:lg3..~ ~'li t:t g, ~ -tr Ag cl ~ ~ -?- _£ 1O-87JtAl !.l ~ ~ .:f Zf !.l ~ ~7-I12.l Al li~ .£~t2 30·COlIA; 2~{! llR6J~ .:f Ag~!f:! ~_£q~ 7-Il-?-"8"to:J ~_£q "dAJ 7-Il-?(Colony forming uni t , CFU/me)~ -=jl-"8"t91t:t.

(ct) ~ct~olE..2.l 'li"~AJ

Age~ Al~OlIA; 6J:2:."8"t7il ~AJ!f:! ~_£q ..g. Q{ 3007H~ ?1"8"to:J RMG-AP llHAIOll ~~ "8"t2 30·COlIA; 2~z! llH6J~ .:f RMG-AP llRAIOlIA; "d~!f:! ~_£q~ 7-Il-?-"8"t91t:t.

(on Ail~~± ~~ {E-~

Zf 11 ~3..~ ~1J{E-cl"8"t09 -tr*ll~ ~-?-"8"t2 ~ct.AOIE.. DNA~ {E-cl~ -f HindIII JS- 2.l Ail~~±_£ ZfZf ~~"8"t91t:t. ~~!f:! ~ct.AOIE.. ~lfl~ °t7t_£_Q__6 ~Oll ~7108% "8"t2 OlIa:lt:l% .!:!__£0tolE..(EtBr) %~.£..£ ~~"8"t91t:t. ~~Xloi L-}ElJd Zf ~lfl~ pYRC62l-l ~ctAolE.. DNA2.l ::<il1(! fi.{- ~lfl:zj- Bl~"8"t09 %~~ ~lfl~ li!..%"8"H=AI~ :it'li "8"t91 c],

(Bt) ~~~ {E-~

Ail~% 11 l:lJ3.. 17R~ l:lJ1f_ f-llHAl 100 meOll {j~tt c] g 30·C, 250±50 rpm.£._£ 600 nmOllA;2.l .:g.:;g..£7t ~ 1. 001 ~ ttJ{7JtAl llR6Jtt ..if- E~r~lt!~ ~% 100 uM Xl7l1 7t "8"to:J YHB62112.l ~~~ %.£"8"t91t:r. %.£ -f Q{ 4).1 z! llR6Jtt t:tg ).il~~ ?l"8"to:J ~71 08%% Alli%~OlI ~'li ..if- SDS-PAGE~ -?-~"8"t91t:t. Densi tometer~ °1%"8"to:J *~~il Oll t:R~ YHB62112.l ~{E-%~ -=jl-"8"to:J ~~~~ {E-~"8"t91t:t.

- 56 -

q. ~~ ~~

J:.A~~710il ~-?-a-to:f li!..:t!~ ~~-rr-?(Ail~% 1l l:l~3. : working cell bank)~ 200 me2.} RM lIRA10il ~~-a-to:f ~-rr(seed) lIRoJ~~§.. At%-a-t~r:t.

J:.7j l:lJ1f_OilA~~ ~~~ ~AilAl7'12 -rr~lAJ~~ ~t:R§_ Al4fAl7'171 ~l-a-to:f -rr~l AJ ~Oil %%~ yeast extract~ t:R"'T'- ~~7J(exponential phase)'ltAl ~-E--a-t~r:t.

-rr~1~.£7t ~~"'T'-€- °lAJol Xl~ ~7Hfeeding) lIRA1§_A~ glucosez] tt~l l(!~~2.} tp·J oJl ~ ~H! -£c12~l:qol.:c=.(ol igopeptides) AJ~ 13J %.£~~ ~ .§.ifJ~Jt! ~~~ tt %-a-t2 9l~ 7tAtulxOilAl.:c=.(casamino acrds l.z] EifJ~(tryptone)~ ~-E--a-to:f -rr~l~~ 2j- YHB62112.} ~~~ -9-t:Ri:tAl ~r:t.

EE~. lIRoJ ~.liAl'ltAl pH~ 7.3-£ %Al-a-to:f l:lRoJ% 1J~Xl ~ ~~(acetate)ol -rr AJ~Oil ~~J-£ ul~lAl U.£~ -a-t7l1-a-tu:f. %.e ~~eJ(OO level)~ 10% olAJ-£ %Al-a-t.£ ~ -a-to:f -rr~lAJ~ol -9-t:Ri:t~ t:R"'T'- ~~710ilA~.£ {}~ ~~(oxygen limitation) ~ER7t Xl x] U.£~ -a-t~r:t.

(1) ~l:lRoJ( ~-rrllROJ)

Ail~% 1l l:l~3. 17R~ -}l-a-to:f 40 me2.} RM lIRA10ilA~ 6Al<1' %~ 150 rpm~§_ ~~t lIR 0J~ ~ r:rAl 200 me2.} RM lIRAl 57ROil 2%(4 me)§.. ~~-a-r2 14Al<1'%~ 150 rpm~§_ l:lR °J-a-to:f ~-rr lIRoJ~~§_ At%-a-r~r:t.

(2) l:lJ~

30"C2.} lIRoJ~.£§.. ~~~ ~~710il ~-rr l:lRoJ~~ ~~~ r:t% l:lRoJ yeast extract AJ~ol tt%~ ~7t l:lRAlI~ 2Al<1'%~ ~-E--a-t~r:t. lIRoJ~.£~ 37·C§.. jrr0l~A~ %.£~ ~ ~ .§.~~Jt!~ ~~ ~'£7t 50 lJg/meol Xl.£~ ~7t-a-t2 EE~ glucose. casamino aci ds s] tryptone %01 tt%~ ~7t l:lRA1II~ ~-E--a-t~r:t. lIRoJ~2.} pH~ 5 N NaOH~ At %-a-to:f 7.301 %Aj Xl.£~ ~~-a-tu:f ..nLJt!4r.£~ %.e {}~eJ (DO level )01 10% 01"'0'" % AIXl.£~ -%7tAl~r:t.

- 57 -

(3) l'Ifl oJQ!f.Q.j ~~ (7t) -tr~l~

-tr~lcJ~ Uv-vi s ibl e spect rophotometer-jj- ol%"6"tof 600 nm<>llJ-~.Q.j ..:a-~s:..£. ~~"6"t91

ct.

(-q) YHB6211.Q.j ~~cJ ~~

-tr~llIt:tllQ!f~ 12% ~t!l ot3.~otol~ ~<>ll ~71 ~%~ .:f scanning densi tometer~ ol%"6"tof ~ efl;g~ B~ll<>ll eflt!- YHB6211~ J:fl~ -;z."6"t2 °l~ ~ B~~ ~£<>ll eN ~ "6"tof YHB6211.Q.j 1:3J~ cJ~ 711 -8-"6"t91 ct.

(ct) ~-cJcJ ~~

~-cJcJ.Q.j ~~ ~ DNS 1:3J~~_£ "6"t91ct.

( t!t) <l!".£.1-] oP-J ~~~~

<l!".£.1-]°tAJ 1l~~ ~~~ ~~71~ J-t%"6"tof l'IfloJQ!f ~<>l1 ~*"6"t!::: ~~~ 1t-€-% ~(500, 2,500 ppm)2} B12iit9 ~cJ iit 91 ct.

(ot) ~-8-~£ ~~

Boehringer Mannheim J-t.Q.j ~-8- ~cJ 71§.(cat. 176303)~ J-t%"6"tof acetyl CoA synthetase(ACS)<>ll .Q.j"6"t9 %7t!H NADH.Q.j 0J~ ~7tiit9 l'IN°JQ!f~~ ~-8-~ ~cJiit91ct.

2. -1} ~1%-1} 71-. ~An.:g.~

YHB6211.Q.j ~~l.:g.~ ~ 1) ~%AJ ~Efl(inc1usion body).Q.j YHB6211~ !l1=-1jl ).il~, 2) AflA~(refolding) ljl 3) ~~l 2}~~_£ ~~~~ c;tlct.

- 58 -

<YHB6211~ ~~l ~ ~£>

~%"8 ~EH~ YHB6211 ~

%~H(solubilization) ~ ~H~(Refolding)

~

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- 59 -

t::t. Inclusion bodys] ;.{flA~

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(2) ~~AJ~ YHB6211-£ ~%~ %tsflt1!l-£ §cl~ut llilol~ %-£ ~%~ ;.{fl~%t1!l~§. ~ ~ :£.tAl {l ~ ~.2} .=:L£.ut~.:::lcflnlOlI .!8-.2}Al:11 ~A-l ~AJ~~ YHB6211-£ ~9it::t.

ct. ~All

(1) ;.{flA~~ YHB6211 %t1!l-£ Bio-Had 5M2 ~-3l' 3§.ut~.:::lcflJIloJl .!8-.2}Al7'l ;.{fl~Al A%~ 7l1~~AJAll~ ~-?).~l.£ %cfl~ ~~~~ AlI7-li;t~t::t.

(2) !l-?~ %t1!loJl %c~pf-nl~ %-ff--?~ 7ti;t~ ~~AI{l -V- ol~ %~ -¥-nl~ d:.{!% t1!l-£ ~<Ui;}~ ti],

(3) %~5:l~ Alli~ Macro-Prep High Q ~~oJl .!8-.2}AI7'l !l-?i;}~t::}.

(4) ~* ~AlI~ YHB6211 ~All ~3~ ~.£~ 4~~ -V- ~cl~.§_llilol§ 80-£ ~%~ ~ %%t1!l~§. ~~i;}~ YHB6211 ~*~t1!l-£ AllS:.i;}~t::}.

tit. ~ AlI~~ .2}~ ~ AJ ~ ~ (1) ~:;g.£ (AZ80)

~~ 3§.ut~.:::lcflnl .2}~~~ It!l!!f~ ~ ~~oJIA-l %~5:l ~ %~t1!l01 UV-moni torOlI l.-R;.{R~ Zl'.;r 1 cm~ Tr~-£ .!8-.2}i;t~Ai 4~5:l~ 280 nmOllA-l~ -a::;g.£§. :tt~i;t~t::t. Zf ~~lt!711oJIA-l 4~5:l~ ~:;g.£~ {l'.£(sensitivity)~ %~5:l~ It!l!!f~ ~.£Oll ~tsfl ~ ~5:lu:j Uv-aoni tor s] .2..x...:::lcH.!£AJ~ n13 Tt!~ ~ %~5:l ~ It!l!!f~~ 0J~ qE}~t::t. tt} crA-l 0l~ 71d:.§. Zf ~All!l-?f:-Zl'~ -?~~ %AAJ-£ 1:!1..2~ -? 9l~ -2-7'f§. i;r~t::t.

- 60 -

(2) ~7108%(SDS-PAGE)

~71 08% ... q~ ~ t:H11"-tr A·n~1..H~ %U lfJ.:1ct.6'fH~H~~) ~~ °Jc>JIA~-¥-"&f ~~. ~R~. 1I Ail c>Jl °l~~ ~ .:g.1Ic>JIA~ t:Ref~<{! ~.£~ ~1I~ -? 9l~ :AtJi.§. ol%-o-t~t:t. zt .:g.~1j-711c>JIA~ ~~l~ A1Ji.~ :t!~ EE.~ l:Jl:t!~ ~z!c>J1A~ ~7108%~ ..,. densitometer ~ ol%-o-to:f scanning-o-t2 O8"cJ".2.§. ~e:f~ zt eil<{!c>J1Al ~~n 1j-~-1J.~ ~.s\1~c>Jl t:HU YHB6211 ~.s\1~~ l:Jl~§. ~.£~ 71l1!-o-t~t:t.

(3) ct4t~~{!q.§.-it ~~ ~<{!Al~

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Bio-Bead SM2 ~~~ -!-2}~ %~~c>JIA-l~ ~~-o-t!::: ct4t~~{!q.§.-it 0J~ ~.Aa~ .2..£ ~1Iirt71 ~lirto:f -=t-ot1-]1tl ~{!(Guanidium-HCl)~ 01%~ ~~~%~ -?~~-o-t~t:t. ~.Aa~Z!c>J1A-l -=t-ot1-]1tl ~{!~ ct4t~ ~{!1-P::-it2} ~%-o-to:f t!~~ %~~l~ "d~-o-t o:f A1Ji.% ct4t~~{!1-P:-it~ ~~o:f-¥-~ ~<{!~ -? 9l!::: Z!{j~<{! -c717t ~t:t.

(4) ~.£~{l Al~

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- 61 -

~¥!'Ct. .:g.:;g-£9.} ~£~{! i5-s:.~ 0.01-1.0 EU/ml ~~IOil).-l %.E~ til~I:t!711~ l·let 1...Ho:l ).1.li.1...H ~£~{!2.l \t~ ~ :11.§.1...HOil .¥.~¥! l:H~ ~s::~.{! JE~ ~~(CSE)2.l ~£ ~ .2.§.. Jf- 'Bi 711 {} ¥! 'Ct.

(5) ol~~ ).ltl

~AlI2f~2.l zt i}~tt!7110il).-l ~ olA~~Oil 2.l~ ).1.li.2.l ..2..<el~ ~±~~t71 ~l~tO:j ).t %£1 ~ zt %~ ~ ).I.li.~ 0.45 fiID2.l i}.;;r~ ~~ ~'Bi§. All~ O:j2f~t~..Q..o:j ~if} ~ ~ if} %~~Oil).-l oIA~~ ..2..<elO:jJf-~ ~c:u~t71 ~l~tO:j 2.3% Nutrient agar ~Jt!Oil).-l ol~~ ~~ol 3JAJ£f ~AI2.l 0:jJf-~ ~c:u~t~'Ct. 3:.~ ~All .:f zt ~if} ~ %~~I J.·n~~ .:f ~ ~~ .!8-~tO:j l-h~: %~~ ~71.2.} ~ol ~~!(agar) ~li!-l3HAlOil £~~tO:j ~~ 3JAJ%4'-~ :t!-~~t~ 'Ct.

(6) tt!~~ ~eJ

zt i}~tt!7110i1).-l2.l 4--£ ~ ;{H~AJ~ ~c:u~t71 ~1~tO:j tt!~~ i5-£~ ~~~to:j til.;!. ~ '(!lfl~ tt!~~ ~eJ~C:U .!:!.~H'£.¥.'£(Bradford) ~J~OiI tt}-ct ±~~<M-Jf-~~ Jf.€-~~ §. ~tO:f 4-~~~t~'Ct. ~ BJ~OiI rr}c} ~~~ ~~ -2-71§. oH §. Eot'Ct2.l i}~2.l ;{H~AJ ~ ~c:u~t~'C}. ~% ~All%Oil t:H~tO:f~ 1I~~~ All All 71€- l3J ).ltl~J~2.l tt!~~ ~eJ ~-% §.cl(Lowry)~, 3:.~ t:H~C1{~2.l ~~l.3.§.ot~.:L;~IJJ!I~ofl tttct :J. i5-.s:.~ ~eJ~tO:f ~% lI{}eJ~ ~7t~t~'Ct.

3. ~1l Aa).l <"t:l

;{H~~ c:uz! G-CSFAlIAll(YHB6211)2.l -ii-Z4 ~ All3J%~ ~~~ .:f 1t71li!.~).ltl~ .!8-~t O:f AlIAlI2.l AJ ~¥! %.E7J Z! %2.l %~li!.~~ ~J~ ttl~ ~).ltl~ -?~~t~..Q..o:l .g.£, :;g~ ~ pH %Oil rrt;: 7t~).1 ~~ .!8-~tO:f ~tsH{}~ lIAJ 0:fJf-~ ~~~t~ G-CSF AlIAll2.l ttl ~~).ltl£ 4-~~~t~'Ct.

- 62 -

4 ~ 01 A1- £A4 Al;i;-l . '\...:. P 0 -, 0 0

~Ra:.~ ~{!- G-CSF ~1~l(YHB6211)~ ).1~%£~ 7Ro.I1 t:R~ 4-?lfr~ ~.AJ).l~, %~ lJJ \:! ~ ~.AJ).l ~, .Ag ~ ~.AJ ).1 ~ %~ jf~~ T~ %<>11 -9-1 ~-o-toi -? ~g-o-t~ 'Ct.

- 63 -

z}. 11l:l~3.~ :tt~

(1) rhG-CSF ~ %~At

YHB6211~ %~At~ ~~ll:lJ:;g-~ ).·ll3£§....!f-sJ ~~1~ mRNA~ cDNA§....!f-sJ :tt~~ ~71A~~ Oil-2-7li>to:1 ~~~AJi>toi ~l~i>t~t:t. °to1x.~l(!~ ~71A~~~ "CH~~ ~t!~17l1oJ1 ~~i>t x~ t:toJ~ %-ft-~ %~At l(!lfl~ ~AJi>toi "CH~~ l:lJt! ~17l1oJ1 ~~i>t2 ~t!°1 4L4-~ YHB6211 %~At~ ~l~i>t~t:t.

(2) 11 l:l~3.~ ~AJ 13J %~At~ <tl;~PJ ~At (7t) t! 0178 :t!~

~~~ ~-?~ ~EH~ z!"~ ~EHo1oL Gram gAJ ).·n~.2..§" "*~~-£ trl9it:t.

(q) 11 l:l~3.~ .2. ~ oi-¥-

.2.~~ 01~%~ ~-e oi~ :t!~ ~2} .2.~£lA1 ~9lt:t.

( t:t) A~ -e-i"

~§..q ~AJ 7114-(Colony forming uni t , CFU/JDt) :tt~ ~2} 1. 0 X l09CFU/JDt 0lAJ.2.. §.. :t!~£l9i c],

(~t) ~1~~± ~l(! ~~

zt 11 l:l~3.§..-¥-sJ llHoJ~ ).-I13£-? llHoJQ!}~ ~ll~~li>toi ).-113£1' ~ ~4-i>t2 ~~t601~ ~ ~~l~ -f- HindIII %~ ~l~-g..f. S; ztzt ~l(!i>t~t:t. ~l(!~ ~~tAolE ~lfl~ ot7t§.. 6 ~oJ1 ~71~%i>t.:J.. EtBr %Q!}.2..§.. ~~i>t~t:t. ~~£l<>l qEt\:!: zt ~lfl~ pYRC621-1 ~

- 64 -

GF-0002, GF-0003, GF-0004 lot21 ~~ -%1f-£ 4-tr~~ ~2.j- -, 'lJ l-A9.}- ~ ~ ~2.j-~

A.

M 1 234

-

....... , ....

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4''- .i!~''''

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...... 14 - ....

" ~H'·:;';"<4I· ~

B.

10

o

4

12

14

2

6

8

10

.::l~ I-A. SDS-PAGE M: Molecular weight standard, 1-4 After 3, 6,

9, 12 hr culture, cell extract preparation

.::l~ I-B. Growth curve

- 65 -

7}. ~~l n}±ll l}l ).·n~

*Aa t§Efl~ inclusion body~ ~~ln}±ll(sonication) l}l ).~l~ 2J-1h~- 1fr~ii}o:l inclusion bodyttl-~ ~l?~ 3f- ~71OJH 4~g~ ~~ 2~ 2eil .£~~ii}O:j 1-}E}t..fl9;it:l-.

1 234

2~ 2. ~~!f:! inclusion body s] SDS-PAGE .!l.~.£

1-}. 1J ~l

~flS:.<U <tlzt G-CSF 20L \lfloJQJ}~ nl-±ll. ).11~ 1_M ~fl~2J-1J~ 7l~ 3f- 4~7-Il column chromatography ~7-Il~ 7l;i;f 2~'£~ <tlzt G-CSF~ 1J~lii}~t:t. l;f.} Biobead SM2 col umneil fOJ ii}o:l rhG-CSF ~ .!8-2J-.Al ~ t:}. .!8-2J-!f:! rhG-CSF %~QJ} ~ 20mM sodi urn acetate buffer(pH 7.5)-.£ ~t§£lol91~ Macro S/Q co lumno] f~ii}O:j ..:g.~-,q{l 3fsodium acet.ate s] ~.£~ %7}.A17'l ..:g.~!f:! rhG-CSF~ %~ii}~t:}. ~%~.2.-'£ Sephardex G-75 gel filtration column~ 0l%ii}o:l Macro S/Q %~QJ}~ 1J~lii}~t:}. ~ ~-;z-~ .!8- ii}O:j ~ ll!_!f:! rhG-CSF 1J ~l .:g-1J ~ ~ %4~ ~ 20%0 19;i.2. o:f 1J ~1!f:! rhG-CSF ~ ~.£~ 99% olAJol9;it:}. Gel-filtration column% .!8-2J-iitO:J ~% 1J~1!f:! ~QJ}(1J~1 Bulk)~ ~.£~

- 66 -

RP-HPLC~.Q..s;_ -;z.~ .:f ~~iito:} .!t!..li~ Ail~iit~t:t. ~~!H GF-0002, 0003, 0004 batchs] ~71l ~ % 2038 mgol ~.Q.o:f 2f ~A-jlt(!711~ 2J-~ ~ ~71 ~%£ -'T-3giito:} ~A-jl~

A.

M 1

2 3

4

-

1M

'lit "-.I... _ ....

~

.,

c.

1

5

2

B.

M 1

2

5

3

4

..

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.~

3

4

5

.:I. T{J 3. ~ Ail t(!7-Il ~ ).l.li~ SDS-PAGE. A. GF -0002, B. GF -0003, C.

GF-0004. M; Molecular weight standard, 1 ; rhG-CSF standard, 2 inclusion body, 3 ; column chromatography<>ll ~~ refolding, 4

Bio-bead eluent, 5 ; Macro S/Q eluent.

- 67 -

(1) ~AJ

YHB6211 ~li~ AJAJ 7]-€-~ .!f-t~Hl Ql}~]AJEHo]t:1-. A~{}{:! z.j- GF-0002, 0003, 0004 lot ~ .£.¥ .!f-tg ~ Ql}~]AJEHo] nl, lXj-ej-A"i 7]-€-oJ] ~ tl"iij-~t:j-.

(2) pH

YHB6211 ~li~ pH A]t} 7]~~ 3.7 - 4.3 0]t:1-. GF-0002~ 3.91, GF-0003~ 3.96, GF-0004~ 3.95.£ .£.¥ 7]~OfI ~tl"iit~r:l-.

(3) .!f-it- A] ~

t:H ~ <lJ'~ Ofl ttj-.s. \1, -¥-it-"'] t} ~ Ail it--¥-~ AJ t} 2} ~ it--¥-~ A] tiE...£. -T~ ~ o-l ~ t:j-. Ai]it--¥-~Altl : <lJ'~c>fllXj-~ AJ~~J~E..'£' AJtliij-~~ u:lf A-llit-0l Aj-etAI ~oj-o]:

~t:j-. z.j- Afl3':~~(GF-0002, 0003, 0004) .£.¥ A-llit-0] ~~ ~~.£.

l-j-Ej-L-t 7J-€-Ofl ~~iij-~t:j-.

<lJ'~oJ]lXj-~ AJtI~J~~.£ AJ~iij-~~ u:lf ~it-0l Aj-ej-Al ~oj-o]:

~t:j-. z.j- Afl3':~~(GF-0002, 0003, 0004) .£.¥ ~it-0] ~~ ~~.£.

L-tEj-L-t 7l~oJ] ~tl"iij-~t:j-.

YHB6211 ~li~ 71€-~]~ 4.0 EU/ml O]iij-.£. Bi owhi t taker-xj s] 1.-H~~ ~~ 7]E~ Aj-

(5) ~7]oa%Altl

.::l~ 4c>fIA"i.2.J- {[Ol YHB6211~ ~e]oj-3..~Oj-llto]E. ~~ o]%~ \:I]~~ ~7]oa%Of]Al

filgrastim(Grasin@ inj.)2} %<Mt! ~]~]c>fl"'l lX]~ li!.O:j {[~ ~At~~ ~JL ~%~ Z! 1:1 ~~.s:_ ~ -? ~ ~ c],

- 68 -

.=!.~ 4. ~.=!.ct~1hl(fiR~~)on t:R~ l:Il~"ci ~71~%(non-reducing SDS-PAGE) A: YHB6211

B : Filgrastim(Grasin@ inj. )

(6) tt1 <4 ~~Al ~

tt1<4~~Al~~ 71iC-*1 ~ ~J~l.2.J-~ AtolOil ~li~ ~7J~01 ~AJXl ~ ~olt:t. zt ~l ~~~(GF-0002, 0003, 0004) _£.!f- goat anti-hG-CSF ~J~l.2.J- ~%).l~~ c:l] I~ 5.2.J- ~ 01 ~li~ ~7J~~ 2i!..O:f 71iCOil ~~irt~t:t.

A B C
1 2 1 2 1 2
0 0 i I 0 0 I ~

.=!.~ 5. zt ~l~'tl~~ tt1<4~~Al~ ~:4 . A. GF-0002, B.
GF-0003, C. GF-0004. 1 <€I ; liiC% (GF -0052), 2 <€I ; {l ~l - 69 -

(7) %~1;}4j-).l~

Isoelectricfocusing ~oJ1).~ YHB6211~ I~ 6oJ1).~9.}- ~01 pH 6.59.}- 6.0 ).to1-% 6.0-¥-2-oJl).1 t8~~ ~E~ 2i!.0}2 ~_2_o:J filgrastim(Grasin@ inj., %~1:l : 6.1 )2}£ %~ ~ Jf1ji:1oJ1).~ ~E~ 2i!.~ %~1:l(pI {!:)ol 6.l<?J~ ~ -? 9J.~"Ct.

!if 6. S-pH 6.0---+

I ~ 6. 3€l1 ct~~ (~R~tf)oJ1 t:R~ isoelectricfocusing A : pI marker

B : YHB6211

C : Filgrastim(Grasin@ inj.)

(8) ~-?%cH ~E1E -¥-~).l~

~-?%cH ~E1E -¥-~).1~2.j ~li 71~ji:1 ~ ~-?%cH ~EIE2.j ..2..~ ~£7t 100 ng/ml 01 -o-to1 "Ct. zt Ai1~~~( GF -0002, 0003, 0004)2.j ~f%cH ~E1 E2.j ..2.. ~ aJ~ ~~ ~ ~:z.t, 2;tl2.j ~.J!t).1~oJ1).~ zt Ai1~~:2:.:s:..!f- N.D.2.j ~2}~ t.tEt~~"Ct.

(9) ~f%cH DNA -¥-~).1~

~f%cH DNA -¥-~).l~2.j ~li 71~ji:1~ ~f%cH DNA2.j ..2..~ ~£7t 20 pg/ml °l-o-t

- 70 -

oJr:t. zJ- AiJ~~~(GF-0002, 0003, 0004)~ ~"'r%~ DNA~ ~~~-£ ~~~ ~2}, 2!l ~ Jti~AJ~oJJA~ zJ- AiJ~~~ .£.Jf N. D. ~ ~2}~ 4Ett.H9ir:t.

(0) 0J~ ~EJE.. {t.¥. AJ~

op.J ~EJE.. {t.¥. AJ~~ ~.li 7J€-~J~ oJ~ ~EJE..~ {t.¥.~oJ 2% oJ-B"toJr:t.

TSK-SW column-£ 0J%~ size exclusion-HPLC§.. zJ- AiJ~~~(GF-0002, 0003, 0004)~ oj AJ~EJE.. {t.¥.~ {t~~ ~2}, oJ~ ~EJE..~ ~.¥.i!JoJ O%§" 4EtXl:r:t. oJ ~2}OiJA~ dimer, trimer%~ oJAJ ~i!l:31 .. n1(oligomer)7t ~9i..Q..n:j, ., 0J~~ oF~' 1(!~~.s:. ~oJ monomer ~EH~ YHB6211 ~~Jt!-£ tt%'6"t2 ~ g-£ ~ -? ~r:t. tttctA~ zJ- AiJ~~~ .£. f 7J€-Oil ~~'6"t9ir:t.

(II) ~'6"toJAJ~1 Al~

~'6"tOJAJ~J AJ~~ ~.li 7J€-~J~ 0J~ ~'6"toJAJ~I~ {t.¥.i!JoJ 3% oJ '6"toJr:t. TSK

SP-NPR column-£ 0J%~ cation-exchange HPLC§.. ~~~ ~2t. GF-0002.2..f- GF-0003~ ()% ~ l.lEtt.H~ pI 6.1~ %<tl~ rhG-CSFJt!-£ tt%'6"t~ ~-£ ~ -? ~9i2. GF-0004~ 0.6% ~ ~2t~ l..}"Ett.H9ir:t. tttctA~ zJ- Ail~~~ YHB6211 5:..f 71€-Oil ~~'6"t9ir:t.

(12) l:Ir~'AJ Al ~

otJj?-;6. ~-?).~l.¥.~ 0l%~ in vitro assay(CFU-GM colony assay: Colony Forming Uni t.-Gr-anul ocyte Macrophage colony assay)OilA~ YHB6211 ~ 0J~~ ~§..y ~AJif~ ..!i!.. 9i..Q..n:j (.::LTfJ 7). 0lttil~ ~~~~ ~ 7t~ NIBSC .It€- rhG-CSF~ oJ%~ {l ~~..Q..§..-¥-"Bl ~~'6"t9i-£ ttil 9.52 X 107 u/me(l:IJ~AJ ~7t~ 9.75 X 107 U/mg)..Q..§.. ~~£l9ir:t. oJCi~ A~~~~ ~ 7t.2..f- l:Il~AJ ~ 7t~ ~ 2}i£J.:r- ~§..y ~AJ At.;;r~At~ ~-?).~I.¥.Oil t:H~ ~

- 71 -

.s;.1-} ~-'8-1g--£ ~~~t~ ~ ~7t~~~0l1).~ 18-AJ ~ai~ ~7H1.0±0.6 x 108 U/me).2.flfr~'Aa ~7t(1.0±0.6 X 108 U/mg)0l1 -¥-~~t~_Q_n:l, ~~1 lfl~Aa ~7t \1Oll).~~ ~~13. .s;.ot~ -, cRn1 ~~ Oll).~ ~o:j ~ ~£~2}.2.f- <M~1~-£ ~ 4- 9l<);l:c.t.

A

B

~~ 7. YHB62110l1 cR~ in vitro assay(CFU-GM colony assay)).l ~Aa~ ~4-).·n~ -R-cR ~.s;.1-}.

A : NIBSC Jf~ rhG-CSF : 31.3 U/well B : YHB6211 : 0.625 ng/well

(13) ~eJ).1~

RP-HPLC~0l1 ~t;R ).1~~ 01 ~~ ~~).1~ 7l~~l ~ 0.9 - 1.1 mg/mlolct. .£.Jf- 6 ~ ).l~~to:j ~-tr-£ .:;z-~ ~_Q_'£', GF-0002~ 1.04 mg/ml012, GF-0003~ 1.00 mg/ml 01 n:l, GF-0004~ 1. 01 mg/ml.£. 71~0l1 .£.Jf- ~~~tc;rtct.

- 72 -

(14) ~~~~ ~7t ).l~

t!--?A·n.£~ %AJ tlt-4l-~<>Il).1 ~R-}l~t~. ~-thymidine uptake~<>Il ~iiR ).l~~t~ 01 ~~ ~~~~ ~7t~ 71€-~1~ 0.7 - 1.4Xl08 U/mlolrl 211~.J!t ).l~~t~ ~-tr~ -=t-t! {t..2..§.. GF-0002~ 1.24xl08 U/mlol9i..2..oi, GF-0003~ 1.34xl08 U/mlol9i.:il. GF-0004~ i.rsxio" U/mlol9ir:t. a:tct).1 Zf fiil~~~ YHB6211 .£..!f- 71€-<>Il ~"U~t~ r},

t!l G-CSF -Ttz:t

0J~t! ;tR"~AJ tM 'l!~""8~ .!t""8~ ~~ ~fiil *~~ ~~t~ .:;::U~·.£.§' ~fiil~ YHB6211~ ~~ -? 9l9ir:t. ~-tr l:Illl"""8 ~7t~ ot-4l-~ t!--?A-il.£~ 0l%t! in vitro bioassay AJ<>Il),1 1. O( ±0.1) X 108 U/mg 0l9ir:t. uH §..!§..0tr:t~ YHB6211~ ~.£. tM %~ AJ~ SDS ~ ~~, ~~~~ ~~. %~~ ~~ . .:;r.£.),1~:H~.£.~~!. 4-?%cH ~E1E. 4-?%cH DNA. -£cl.:ilu1. ~~tol~~]) %~ ~).l~~ -"'8-~~ ~~ ~t~..2..o:f ::L ~2} uH §. Eotr:t %<Mt! ~cl·~~~ ~""8(~71~%~ AJ~. tr!~~~ ~7J{!. %~~)~ ~~ YHB6211~ ~~ -? 9l9i2. ~.£.~~. 4-<r%cH ~E1E. DNA tM -£c12u1. ~~tol""8~l %~ YHB6211 %cH~ ~tg~l~ 7f~ {l~5:lAl ~~r:t.

- 73 -

7}. -=jLAa O}U1,x-8" 3:Aa

YHB6211~ Pico-Tag(Waters) J;JJ~o{llLtc} :1. 3:Aa% o}~R9.} ~ol ~~-o-t'r! /qAE·Il<tl (cysteine)~ 4.0-6.0, o}AJ!}c:}Zl-8"(asparticacid) rM o}AJ!}c:}Zl(asparagine)~ 3.0- 5. 0, ~~E}tj -8"(glutamic acid) rM ~~E}ttl (glutamine) ~ 24. 0-2B. 0, ).il i{! (serine) ~ 12.0-15.0, ~c:l-8(glycine)~ 13.0- 15.0, -o-lAEl~(histidine)~ 4.0-6.0, 0}~71\:! (arginine)~ 4. 0-6. 0, .:§.~j..2..\:l( threonine) ~ 6. a-B. 0, ~c:}\:l(alanine)~ 19.0-21. 0, E:.~i{!(proline)~ 12.0- 14.0, J;JJi{!(valine)~ 6.0-B.0, njj:*l..2..\:l(methionine)~ 3.0- 5. 0, 01~5r..01-8 (isoleucine) ~ 3.0-5. 0, 5r..01-8 (leucine) ~ 31. 0-35. 0, lljl ~ ~c:}\:! (phenylalanine) ~ 5.0-7. 0, E ~.£J{!( tryptophan)~ 1. 0-3.0, E)£.-8 (tyrosin)~ 2.0- 4.0, c:j-8(1ysine)~ 3.0-5.0 oj9ic}.

q. N- tlJ~ o}ulx-8"

Edman degradation J;JJ~..Q..£. A}%- ~\i!f~ A1 ~~~ 7j(protein sequencerf Alil x], Model 476A)~ 0l%-o-}O:j 107H~ NW~ -z!71~% ~~~ ~2J- 10~717J}Al DNA~ ~7113R~ o{jA1 .!i!..0l~ o}u)x-8" 13fl~2J- ~:*l-o-}~ njl:*l..2..\:l-E~l..2..\:l-E:.~i{!-5r..0l-8-~c:l-8-n~i{! - ~c:) \:l-.Ail i{! -.Ail i{! - 5r..0l-8..Q..5r.. -=jLAa 5:l <>l YHB6211 ~ N- tlJ~ o}ul x-8"0l -efl ;g.-n-o{l A1 J;JJ~ Al A~Zl njl:*]..2..\:l~ Ail9.l-o-}'r! E~l..2..\:l ~%:if<tl ~ -? 9l9ic}.

Ct. C-tlJ~ o}u]x-8"

~-8..Q..5r.. ~t;fl~ YHB6211~ ~El.s~ 28~t:fl J!]3.~ LC/MS5r.. ~~~ ~2J-(:1.~ 9) n~\:!% ~%~ C-tlJ~ -¥-~]~ o}ulx-8" 97H~ -z!7]5r.. -=jLAa~ ~El.s5r.. :if<tl5:l9ic}' 01 o{l t:fl~ o}ulx-8"~ A-l~% ~~~ ~2J- DNA~ ~71A1~OllA1 C-~~ -¥-~lo{lA1 .!i!..0l ~ oj n] x-8"~ A1 ~2J- ~:*]-o-}~ -~7l \:l- ~itl-*-8 -~71 \:l--o-16El~ -*-8-~c}\:l-~~E}tj -E:.~ \:l0l9ic}. 01 Cl~ ~2J-5r.. YHB6211~ c- W~ o}ulx-8" ~ E:.~\:! ~% :if1!-o-}~c}.

- 74 -

mAU

3SO

200

150

100

50

DI>D1 ,I., Sig-215,10Ref-o!f (G-CSF'GCSf0114D)

- 75 -

'MSOl SPC, tine=28.263 of G-CSfilCSFOl16D API-ES Posiive
l00j III
III
" Hax: 105351
£()
60 Q
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40
20 IJ)
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0 ,I j 1
400 BOO RM 1nm mh A

100000 A 1
60000
60000 A:3
40000
20000
A:l
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,nn onn Ann 1nim B

160000 - A: 089
140000 -
120000 -
100000 -
80000 -
60000 -
40000 -
20000 -
0- ...
1000 1n<:;n -i -r rm 11-",n
C (API-electrospray spectrometric analysis)

A 28~t:fl nj3.oJj t:flt!- MS .A.l!!1.§.~ (mass spectrum) B 28~t:fl nj3.oJj t:flt!- deconvoluted ion set

C 28~t:fl nj3.oJl t:flt!- deconvoluted mass(1089)

- 76 -

ct. t:ll111tolE ~~(disulfide bond)~ -?-l~l:if't!

~'t! ofl ~~to:j ~i>R~ YHB6211~ ~E1E~ DTT.£ :t±~).l '{! ~-f-~ uv 3...£ot~, 1!I (,ifJ 10)ofl).; %Al).l{!,ol ~7t~ 11l3..~ofl cRt! LC/MS ~~~~ :t±~'li% ~~ 34~cR.2} 56~cR 1113..7t 2 ~At~~ ).l~Eil't!-£ -Y%t! 0tcR~ °tolx.~..2..'£ .:;z.~~ ~E1E'£ O:flAJ 5:l9i t:t( , ifJ 11, 12 ) .

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< 57~t:R 11l3..ofl t:fltt ofl-'cj otolx.~~ ).~ ~ > -Leu-Gly-His-Ser-Leu-Gly-Ile-Phe-Trp-Ala-Phe-Leu-Ser-

-Ser-Cys64-Phe-Ser-Glu-Ala-Leu-Glu-Leu-Ala-Gly-Cys74-Leu-

AJAfl f- ~E1Eofl cRt! °tolx.~ ).;~ ~~~2} LC/MS ~~ofl).i O:fl-'cj~ otolx.~).i ~-£ Jl!.o:j 34~2} 56~t:R~ f- ~E1Eofl ztzt 2 ~At~~ ).lAEil't!-£ ~Jl ~g-£ :if't! ~t~t:t .

.EEt!, .:LifJ 13, 14ofl).;.2} ~ol :t±~ ~% -f-~ f- ~El E~ ~ eJol ¥ 7R~ -?±'~At ofl t>RcJ5:l ~ ~eJ 2~ol ztzt ~7t~to:j(34~cR ~E1E : 1790 - 1792, 56~t:R ~E1E : 2618 - 2620) f- ~El.sofl).; zt ).lAEil't! ~At~ t:ll111tolE ~~-£ o]Jfl·Jl ~g-£ :if't!~t~t:t.

olclt! ~2j-~ %~~P~ YHB6211~ 0tolx.~ ).i<€lAJ ).l~Eil't! 362} ).l~Eil't! 42, ).l~Eil't! 64.2} ).l~Eil't! 74ofl).; t:1AJntol.s ~~-£ ol-y-Jl ~%-£ 9J -? ~t:t.

- 77 -

DAD1 .6., Sig=215,1 0 Ref=o1f (G-CSF'GCSF0114 .D)

350

300

2SO

200

150 0)
..
0) 0)
CD N CD ;: 0)
100 ~ q "- ....
CD
50 A

DAD1 A, Sig=215,10 Ref=off (G-CSF'oGCSF011S.D)

mAU

350

(I) .. (I)

300

250

"-
III ~
'" '"
"- •
N
0)
'"
'"
"-
(')
" 200

B

B

- 78 -

'MSOI SPC, 1ine-3482Q of G·CSf'(lCSf0114.D API.£S!'me
"!
"-
OJ !lax: 16367
100
00
"!
III
OJ
.,
60
40
III
.,;
•
•
20 OJ ~
~ .,
0 oj
1 .,
"-
0 I
500 1000 1500 1M A

1600J A 3
14lXXJ
12lXXJ
A:Z
IIJCOO
8000
600J
A:4
4lXXJ
2lXXJ
A:l
0 I

500 Itm 1500 B

30000 -

25000 -

20000 -

15000 -

10000 -

5000 -

0-

A: 790

170a

-r ann

1A~n

spectrometric analysis)

c

A 34~ t:.H 1I13.. on t:.H ~ MS C:. ~p=: ~H mass spec trum) B 34~t:.H 1I13..~1 t:.H~ deconvoluted ion set

C 34~t:.H 1I13..~1 t:.H~ deconvoluted mass(1790)

- 79 -

'MSD1 SPC,tirJe056.931 ofG-CSF'OCSf()114D .aJlI.ESPoslive
III
'"
"- !lax: 14262
100
'"
iii
'"
'"
00
&I
'"
40 oi
0
~
20 ~
0 IL tIl
BOO IOL teOO_ 12m mil A

14!XX1 A 3
1:4
12000
10000
S!XXI
6!XXI
A:Z
4!XXI
2000
0

BOO 800 1000 1200 B

30000 -

25000 -

20000 -

15000 -

10000 -

5000 -

0-

A: 618

J \.

?~5C'1

2snn

2650

2700

.::l~ 12. ~~ l:{l-% ~~ 56~t:H nl3..0U t:H~ LC/MS .6.1E1.§.~ ~~ (API -electrospray

spectrometric analysis)

C

A 56~t:H nl3..Oil t:H ~ MS AlE1.§. ~ (mass spectrum) B 56~t:H nl3..Oil t:H~ deconvoluted ion set

C 56~t:R nl3..Oil t:R~ deconvoluted mass(2618)

- 80 -

50000 A 3
4!XXl1
som A:Z
2!XXl1
A:4
10000
A:l
0
sin 1nrY1 iSOO. A

'IAS01 $PC, 1iIIe>35.149 01 G-CSF'GCSf0116D APl-fS Posiive
'"
iii
0> Hax: 51896
100
IrI
&J "
iii
0)
"
40
q
0>
..
•
20
'"
q ..;
III '"
" ....
CD ~
0 1~
_500_ .um mil B

80000 -

60000 -

40000 -

20000 -

0-

A: .. 792

1700

1850

1AOO

C

.:l~ 13. ~H:! ~% .:fE-l 35-lE-t:R n!30i! t:R~ LC/MS ~~~~ -lE-~ (API -electrospray

spectrometric analysis)

A 35-lE-t:R nJ3.Oi! t:R~ MS ~~ E ~ (mass spectrum) B 35-lE-t:R nl3.Oil t:H~ deconvoluted ion set

C 35-lE-t:R nl3.Oil t:R~ deconvoluted mass(1792)

- 81 -

'MS01 SPC, tiIle=58.ffi9 of G-CSf1DCSf0116D APf.fS Posiive
'"
..
" Hax: 22400
100
00
0
<Ii
'"
ro <D
40 CD
ci
;;;
20 ~
0 IlL
600 800 1000 1?M nI' A

22500
1 3
20000
17500
15000 1:4
12500
10000
7500 1:2
5000
2500
a

sao 800 ~ 1,;,0 B

40000 -

35000 -

30000 -

25000 -

20000 -

15000 -

10000 -

5000 -

A: :; 620

C

.:J.~ 14. t!~~%.:f.Q.J 58~t:ff nJ3.OiJ t:ff~ LC/MS c~E'i:I ~~(API-electrospray

spectrometric analysis)

A 58{f:- t:H nl3. Oil t:H ~ MS c ~ §.'i:I (mass spec t rum ) B 58{f:-t:H nl3.Oil t:H~ deconvoluted ion set

C 58~t:H nJ3.OiJ t:H~ deconvoluted mass(2620)

- 82 -

u], ~EIE.. {f-~'£(Peptide Map)

DAD1 8, Sig·2IIJ,10 Ret'off (G-CSF'OCSF0123D)
rMU
35
30
2S
20 ~
.,
., "- '"
~ '" ~
15 ..;
'" ~i
III
" "-
10 0 '" ;: Ol "
'" '" '" "
(0 '" III ~! " '"
(.0 "": "- III ..
'"
ID (0 '" J ~I 1
5 11 ~ ~
0 J _A

·5 ri
10 _10_ 30 40 _5C _m ...IIIi: A
DADI A, Slg=215,10Ret."ff (O-CSF'OCSf0123D)
rMU
350
300
250 8
"
~ 0>
:il "
200 0>
"- .., ~
~ ..
150 &1 .. i
(!)
ID ~ "-
(0 ::: "
$ ..
100 '"
50 B

.,~ 15. Peps inej £.ltt YHB6211£.l ~-cIE.. {f-~.£(peptide map), A 215 nmOiIA1£.l UV 3..5:.nt!?.., ~

B : 280 nm Oil A1 £.l UV 3..5:. u t !?..:2 ~

- 83 -

1._

YHB6211~ ~;;tteJ~ API-electrospray LC/MS system..Q...£_ ~~~ tt\l .::J..~ 17on).~~

- 84 -

'MSD1 SPC, tlme-46.436 of G-CSF1GCSF0138.D API-ES PosKlve ~ ~

'"

100

Hax: 1.9640

~
80 ~
§;
~
~ ~
60 ;::
"!
40 ~ ,._
,..;
;!l;
20
0 A

A: JI.9 .1.9

20000

17500

A:: .1.7

A: ZC

15000 -

12500

A:Z

10000 -

A:Z A:1S
A: Z A:.L4
A:Z ~ A: .1.3 A:.1.Z
I I I A:.1..1.
I B
.,..';n 1nnn 1?",n 1 "inn 17:"n 7500

5000

2500

o

120000

A.:

c

..::::L'iJ 17. Ion-spray MS .A.~ E ~Hmass spectrum) ~~~ .!8-~ YHB6211~ ~AteJ ~~ A MS .A.~E~(mass spectrum)

B Deconvoluted ion set

C: Deconvoluted mass(18, 799)

- 85 -

o t. ~ oi 2J- ~ ~13_'£ tl t .£.:L ;;H nl( size exc1 US i on - HPLC)

YHB6211 ~ .:L'iJ 18oJl).~9.} :Qol 15-16iE-oJl).~ %Al).l Z!~ ~~ ~eJ~I~ ? nl3_~ .!i!_0lu:f YHB6211 %;;H~ ~~~l(mul timer) ~ ::t!~5:lAl ?J~t:t.

15.07

ze=

At. ol.g. 2~ ~~I3_.£.tlt'£.:L;;Hnl (Ion exchange - HPLC)

1'iJ 19oJk'~9.} :Qol YHB6211 ~ 8-10iE-oJl).~ %Al).lZ!~ ~~ ? nl3_~ ~~ .!i!_~~ u:f YHB6211 %;;H~ ;j "Gtol AJ ~'ll ~ ::t!~5:l:Al U~t:t.

9.13

,
.s:
-
19- r
-
15-
.... - 86 -

~t. ~lfl:.g- 0l~~ ........ ~E~(Circular Dichroism Spectrum)

.:::z.TfJ 200l1J.;~ ~ol YHB6211 ~ 200 run - 250 run2.l far UV Al~(region)OllJ.; llt~ l:il~2.l

(Circular Dichroism Spectrum)~ ..!i!.c;a_Q.o:J 203-210 nm.2.} 220-225 nmOiJJ.; -¥-2.l -9-~}(negative

220 230

W....c.noU\{nmJ

2'0

240

250

- 87 -

Jf.3. YHB62112.l ~7]li!.€:-).] tl ~ 2l-
7]z! ).]tl~J~ ~la:.~~
GF-9702 GF-9703 GF-9704
AJAJ If-~Jf-~~ ~ t:I~em~~ ~ If-~Jf-~~ ~
T To c-
).] tl pH 4.03 4.07 4.04
7H).] 0] AJ ~~]E.(%) 0 0 0
~ihl:(%) 103.4 96.7 98.3
A~~~~ ~7HU/me) 1. 080 X 108 0.977 X 108 1. 009 X 108
AJAJ ~~~g ~~~g ~~~g
pH 4.07 4.05 4.04
67H~ 0] AJ ~~]E.(%) 0 0 0
~cJ(%) 103.2 97.0 98.6
A~~~~ ~7HU/me) 1. 033 X 108 0.954 X 108 0.960 X 108
A-lA). ~~~g ~~~% ~:2:}~g
o 0
pH 4.07 4.02 4.05
127H~ O]AJ ~~]E.(%) 0 0 0
~cJ(%) 104.5 98.0 98.4
A~~~~ ~7HU/me) 1. 013 X 108 0.949 X 108 0.974 X 108
AJAJ ~~~g ~~~g ~~~g
pH 4.10 4.07 4.01
187H~ O]AJ ~~]E.(%) 0 0 0
~cJ(%) 102.3 95.8 98. 7
A~~~~ ~7t(U/me) O. 957X 108 O. 905X 108 0.921 X 108
AJAJ ~~~g ~~~g tt!~~g
pH 4.10 4.08 4.04
247H~ O]Ad" ~~Jt::(%) 0 0 0
~cJ(%) 101. 4 95.9 96.6
A~~~~ ~ 7HU/me) 0.936 X 108 0.914 X 108 0.915 X 108 - 88 -

Jf. 4. ..!l!.~~Z!, 71 {! tM %71
..!l!.~~Z! ..!l!.~71{! ).1 ~ {!:z:] ..!i!.e%71
.g..s:. 40"C 60~ 20~ ~ cj ~.£ l€1 'C! if-.!:!.
:;g- ~ 1,300,000 ~6 10~ OIPJ:
~).t (5, 500~.A. • ).1 {!) l:1E
pH 2.0, 7.0, 10.0 60~ 30~ -£ c I ~.£ l€1 'C! if.!:!. ~-£ ~~ 7H:! ~ 0.2 fJlD ~&j.£ o:j.:tt()"t~ .:tt~0l1).~~ -e-1J..£ ).ltl7R).1 ttJl~ ~7t7t pH ~~ ~oll lJl~to:j ~ 10% ~.s:. ;q~()"t~.Q_4 0l.:f~ pHOll 1:Hl 7t~).ltlOll).~~ ).ltl7H ).1 ttJl~ ~7t7} '1:R5:.. %AJ~oJ ~ ~z!oll).~~ ~~~~ ~7} e~-g. ~9i'Ct.

- 89 -

Jf 5. YHB6211~ 7t~"q~ 7J2l-
All£Jt!~
li!.:e£<i 717.1 AI~~d"~
GF-9702 GF-9703 GF-9704
pH 4.11 4.03 4.06
AI~7HAI OIAo" ~EI£(%) 0 0 0
A~~~~~7Hu/me) 1. 000 X lOB 0.968 X 108 0.952 X lOB
pH 3.94 4.04 4.03
20<M 0l~ ~EI£(%) 10 9.7 9.2
~£ A~~~~~7HU/me) 0.888 X lOB 0.885 X 108 0.856 X 108
(40·C) pH 4.03 4.13 4.04
40<M olAo" ~EI£(%) 14.0 14.6 14.6
A~~~~~7HU/me) 0.857 X 108 0.840 X 108 0.833 X 108
pH 4.11 4.10 4.01
60<M OIAo" ~EI£(%) 22.9 23.9 23.4
~~~~~7HU/me) O. 773 X lOB O. 750xI08 0.753X108
pH 4.04 4.04 4.01
:»J AI~7H).1 0IAo" ~EI£(%) 0 0 0
~~~~~7HU/me) 1. 050 X 108 0.986 X 108 0.992 X 108
10<M pH 3.99 4.03 4.13
(~ 1, 300. 000 0IAo" ~El£(%) 1.6 2.1 3.0
~A £At) A~~~~~7HU/me) 0.990 X 108 0.954 X lOB 0.946 X 108
t:H£).I~ pH 4.02 4.03 4.13
0IAo" ~EI£(%) 0 0 0
( :7.t:»J) ~~~~~7HUlme) 0.990 X 108 0.984 X lOB 0.976 X lOB
AI~7HAI 0IAo" ~EI£(%) 0 0 0
A~~~~~7HU/me) 0.921 X 108 0.886 X 108 0.900 X 108
pH 2.0 30<M 0IAo" ~EI£(%) 0.9 1.1 1.1
A~~~~~7HUlme) 0.877 X 108 0.900 X 108 0.916 X 108
60<M 0IAo" ~EI£(%) 1.2 1.3 1.4
~~~~~7HUlme) B.89X107 9.05X107 B.98X107
AI~7H).1 OIAo" ~EI£(%) 0 0 0
A~~~~ ~ 7HU/me) 0.872 X 108 0.910 X lOB 0.893 X 108
30<M olAo" ~El£(%) 0 1.4 0
pH 7.0 A~~~~~7HU/me) 0.908 X 108 0.931 X lOB 0.845 X 108
60<M 0IAo" ~EI£(%) 1.1 1.3 1.5
A~~~~~7HUlme) 0.920 X 108 0.911 X 108 O. 90B X lOB
).1~7HAJ 01Ao" ~E1£(%) 0 0 0
A~~~~~7HU/me) 0.900 X lOB 0.896 X 108 0.907 X 108
30<M 0IAd" ~EJ£(%) 0 0 0
pH 10.0 ~~~~ ~ 7HU/me) 0.916 X 108 0.951 X 108 0.905 X 108
60<M 0IAo" ~EI£(%) 0 1.6 1.7
A~~~~~7HU/me) O. 90B X 108 0.901 X lOB 0.911 X lOB - 90 -

-~

5=

.1(.~=

-

15=

110. 1 2 :3

TOTAL

RT 9.06 9. 54 15. 54

AREA 151.134 269293 1398776

HEIGH'or Be

64:35 BV 7339 VB 3.g255 an

~. NAME

B.217

1.5.729

76.063 G-CSF

1.C.300.000

1639203

48029

M

B

c

D

A

..:J.'fJ 22. 40°COil).1 7F~·).1~ -f-(60~)2.l ~71~%(non-reducing SDS-PAGE)

M ~AraJ ur~(250, 148, 50, 42, 30, 22, 17, 6, 4 kda}

A Jf~ YHB621 1 , B : GF-9703, C : GF-9704, D : GF-9705

- 91 -

01~ ~ ~z!<>l1).~ YHB621101 ~-¥- ~Aa~<>l tj~~ l...H-¥-~ ~-?-"8 ~~6:101 7Jt! 0t ol_lr_~ol Jf\:!.Q.:£ _lr_~~\:!).~ ~-?Aa ~~01 opr-<>l;;r;J ~H-¥-~ -£i!12u}~ ~Aat! ~.Q. :£ ~tj~u:f, Jtl\:!, ~ ~z!<>l1).~ ~ YHB6211~ ~i5HA~Aa~~ ~~~;;I:1 ~~t:t. Q.{ 25%~ -£i!1.2.~7t A~Aa{! Jt!.liQ.{%<>l1 ~Htsto:f ~~-Tt-:cj ~.liQ.{%~ 71€- l}l ).l~l:IJ~<>ll n:ti!t % ~~4r).1~2} ~tsto1-"8~1).1~~ -?1fgt! ~2} .=l~ 21, 22<>l1).~.2} ~01 .£-¥- ).1~7»).1 u:Jl~ ~ 2}9.} %).ttsto:f ~ 7t~~z! <>l1).~ ~-"8 {! -£i!1.2.~ 7t Q:j AJ ~~l3..:£0t~.=l ~HlIl <>l1).~ 9.} ~ 01 tjeJ~l~ YHB62112} %~t! %~1:l l}l %).tt! Jf\:! ~tst ~.£ %~ ~Aa~ ~~ ~.Q. :£ ~).t~ 9l c].

L-t. YHB6211 .:r).t~l~ ~~-"8 (1) ;G71li!.-e).1 ~

YHB6211 .:r).t~l 300~ ~t:;g-tsto:f 4ot<>l1).~ li!.:t:!t! .:f ).1~7H).1 l}l tlH 67H~ ott:t YHB6211 .:r).t~l 300~ 71€- l}l ).l~l:IJ~-% -"8AJ, pH, *-"8°1~{l).t, 01~ ~E1E(01igomer) ).1~ l}l ~eJ).l~ H ~ ).1~~<>l1 n:ti!t -?1f~tst~.Q.u:f .=l ~* Jf 62} ~01 247H~ %~ ~ li!.:C~z!<>l1).~ ~01t! ~~~01 ~~AJ~ %Altst~r:.t. 4·C li!.-e~z!<>l1).~ 247H~<! li!.-etst~ ~ u:Jl YHB6211 ?).t~l~ li!.-e 24 7H~ .:f<>l1s:. ).1~7H).1~ ).1~~2}9.} ~01 %).tt! ~~ JiLo:f ~ 71 <!%~ ~~tst9it:t.

- 92 -

.if. 6. YHB6211 9).t~1~ ~711i!..~).1~ ~2}
~l~lti~
71.:ct ).l~~J~
GF9703-3 GF9704-3 GF9705-3
AJAJ t:l -Yl e ~ ~~ ~ Jf--Yl Jf- ~ ~ ~ Jf--YlJf-~ ~ ~
T To'l..:.
).1~ pH 4.03 4.03 4.03
l::i-§-A.:j 010 ~-§-AJ01~ ~g ~%AJ01~ ~g l::i%A-l01 0 ~ 0
7R).1 '20'2 '2 0 '2 0
0lAJ ~El.E..(%) 0 0 0
~cJ(%) 103.5 95.1 95.7
AJAJ ~J:}~g ~:§:}~g ~J:}~g
pH 4.05 4.02 4.04
67R~ l::i%A-l 010 ~% ~g ~%
'20'2
0lAJ ~El.E..(%) 0 0 0
~eJ(%) 102.9 94.9 95.3
AJAJ ~:§:}~% ~J:}~g ~:9:tcaig
pH 4.07 4.06 4.05
127R~ ~%-'8 °1~ ~g ~g cai%
olAJ ~El.E..(%) 0 0 0
~eJ(%) 101.6 94.1 94.4
AJAJ ~:§:}~g ~J:}~g ~J:}caig
pH 4.03 4.04 4.02
187R~ ~%AJ 0l~ ~g ~% ~g
0lAJ ~El.E..(%) 0 0 0
~eJ(%) 101.3 92.5 94.2
AJ~ ~J:}~% ~J:}~g ~J:}~g
pH 4.07 4.06 4.04
247R~ ~-§-Aa 0l~ ~% ~g ~g
0lAJ ~El.E..(%) 0 0 0
~eJ(%) 102.9 92.7 94.1 - 93 -

40'C

60<M 10<M

(5, 500 ~A • Al Z!)

20<M

1,300,000 ~~ ~At

- 94 -

Jf 8. YHB6211 -?At~l 300~ 7~A1~ ~:z.t
~l~ttl~
~e~~ 71z! Al~'$J~
GF9702-3 GF9703-3 GF9704-3
pH 3.99 4.03 4.01
Al~7»Al op.J ~El~(%) 0 0 0
11%~~ ~7Hu/me) 0.241 x 108 O. 228X 108 0.237 X 108
pH 4.03 4.03 3.94
20~ ol~ ~El~(%) 0 0 0
.g.£ 11~~~ ~7HU/me) 0.235 X108 O. 223X 108 O. 236 X 108
(40t) pH 4.11 4.11 4.03
40~ op.J ~El~(%) 0 0 0
11~~~ ~7HU/me) 0.231 X108 0.228 X 108 0.241 X 108
pH 4.14 4.13 4.11
60~ olAJ ~El~(%) 6.6 6.8 7.0
A~%~~ ~ 7t(U/me) O.217X108 0.217X108 0.215 X 108
pH 4.02 4.04 4.02
Al~7»A1 olAJ ~El~(%) 0 0 0
A~%~~ ~7HU/me) 0.248 xI08 0.240 X 108 0.225 X 108
10~
pH 4.11 4.11 4.03
(<Lf
:;g. olAJ ~El~(%) 0 0 0
1, 200, 000 A~%~~ ~7HU/me) 0.234 X 108 O. 226X 108 0.231 X 108
~A ~At)
pH 4.13 4.03 4.04
t:H~Al~ olAJ ~El~(%)
0 0 0
(*t:;g. ) A~%~~ ~7HU/me) 0.235 X 108 O. 230 X 108 0.224 X 108 - 95 -

ct.

- 96 -

it§..~ ~E.J ~7}7} {!~.afE..§" :t!~5:.l9iE..4, -, ~%~ol .afJl %eJE.J-e.af 7J ~Jol ~ 9ic}. 2-9- :!J~71 Z! .:fE.J {lA}~2}-, -??! 1000 jig/kg .!f-oi~OlIA~ %-~~.:r-? l3J ~~~ -?7} {f~~}~r:}. 4-9- :!J~71Z! -f-E.J {lA}~2j-, -??! 1000 jig/kg .!f-oi~OlIA~ 1if1.£.it§.. ~-W-.£ l3J .af ~-=j1-%~ 01 ~7}~t~E..4, Al ~~~Jf-oi ~ :t!~¥! ~:!J-~ ~9ict.

~ ~:!J-~~ {lA}~2j-, ~.-? Aj ~~~.!f-oi~OljA~ alkal ine phosphatasaz] %eJE.J-e .afE..5r. ~7t~t9.tr:}. Creatine kinase.s] potassium~ 4-?! 1000 jig/kg .!f-oi~OllA~ {f~ ~t9.tE..uj, tr-i gl ycer i de-l= ~?! 1000 jig/kg .!f-oi~OllA~ {f~~t9.tr:L .::z.c-11..}, 2-9- l3J 4 -9- :!J~71 Z! -"fE.J {lAt~2j-, °l~ ~:!J-~ .£.f :!J~5:l9ir:}.

_k?lA}~:zt, .£.~ Al~~OljA~ j;toj7t ~9ir:t.

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