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US006858206B2

( 1 2 )

United S t a t e s

Patent

Kakkis

( 1 0 ) P a t e n t

N 0 . :

( 4 5 ) Date

o f P a t e n t :

US

6 , 8 5 8 , 2 0 6 B2

F e b .

2 2 , 2 0 0 5

( 5 4 ) METHODS

FOR

TREATING DISEASES

CAUSED BY

EFICIENCIES

OF

RECOMBINANT

ALPHA-L-IDURONIDASE

( 7 6 )

I n v e n t o r :

E m i l D . K a k k i s , 2 5 1 2 L a g u n a V i s t a

D r . , N o v a t o , CA

U S ) 9 4 9 4 9

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*

) N o t i c e : S u b j e c t

t o

a n y

d i s c l a i m e r , t h e t e r m

o f t h i s

p a t e n t i s e x t e n d e d

o r

a d j u s t e d u n d e r 3 5

U . S . C . 1 5 4 ( b ) b y 2 0 3 d a y s .

( 2 1 )

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( 2 2 ) F i l e d : N o v . 1 3 , 2 0 0 1

( 6 5 ) P r i o r P u b l i c a t i o n D a t a

US 2002/0164758 A1

N o v . 7 , 2002

R e l a t e d US. A p p l i c a t i o n

Data

( 6 3 )

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o f a p p l i c a t i o n N o .

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o n

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6 , 5 8 5 , 9 7 1 , which i s

a

c o n t i n u

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o f

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. .

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8 / 4 7 ;

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( 5 2 ) US. l . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . 424/9 41;

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4 3 5 / 6 ;

4 3 5 / 6 9 . 1 ; 4 3 5 / 1 8 3 ;

4 3 5 / 2 5 2 3 ,

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of

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. . 4 3 5 / 4 , 6 ,

6 9 . 1 ,

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1 8 3 T ; 5 3 6 / 2 3 2 2 3 7 ;

4 2 4 / 9 4 1

T , 9 4 . 5 , 9 4 . 6 , 9 4 . 6 1 ; 5 3 0 / 3 5 0 ,

412

( 5 6 )

R e f e r e n c e s

C i t e d

U . S . PATENT DOCUMENTS

3 , 4 7 2 , 9 3 1

A

1 0 / 1 9 6 9 S t o u g h t o n

3 , 8 9 1 , 7 5 7

A

6 / 1 9 7 5 H i g u c h i

5 , 2 7 0 , 0 5 1 A 1 2 / 1 9 9 3 H a r r i s

6,149,909 A 11/2000

Scott

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a l .

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mac/mas MGMEYECC emcctm

smcsmct

A B T A C A A T C 7027mm ccncmsn

s o v 0 1 0 0 1 1 0

1 2 0

1 3 0 1 4 0

A A G L E A G Y A Y

neurons meme en-menu

E A G I A G T G C E E G A G C A A A A Y mAscmA

1 5 0 mu no no 1 9 0

mm 210

nowssme GCTTGACEGA cum-ma nmcmc msusmvs

eusmmcs

r s r v v c e c e

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mmcesec

momma

srmncm

nmmm

rmnu anwcu mcssnm

2 9 0 3 0 0

am 3 2 0

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Ammmr cccATAm mmmca smoxm

cmcespu nwccscc murmurs

m 3 7 0

: 5 0 2 9 0

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no

4 2 0

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470

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masmm mceav

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comma cmmcss mmaaccc

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rmmmrm rmeemcc MMYCAACG uaacmm

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FOREIGN PATENT

DOCUMENTS

GB

1001949

8 / 1 9 6 5

W0 WO

3 / 1 0 2 4 4

5 / 1 9 9 3

W0 WO

7 / 1 0 3 5 3 3 / 1 9 9 7

W0 WO

9 / 5 1 7 2 4

1 0 / 1 9 9 9

W0 WO 9 / 5 8 6 9 1

1 1 / 1 9 9 9

OTHER PUBLICATIONS

F r a n c h i m o n t , e t a l . , I n d u c t i o n o f

I n ? a m m a t i o n b y Immu

n o l o g i c a l R e a c t i o n s , A g e n t s a n d

c t i o n s , 6 ( 1 3 ) : 2 4 ,

1 9 7 6 .

A n s o n , D . S . , e t a l . ,

C o r r e c t i o n o f

Human M u c o p o l y s a c

c h a r i d o s i s TypeVI F i b r o b l a s t s W i t h R e c o m b i n a n t

N A c e t y l g a l a c t o s a m i n e 4 S u l p h a t a s e , Biochem J 2 8 4 :

7 8 9 7 9 4 ( 1 9 9 2 ) .

B a r t o n , R . W . ,

e t

a l . ,

T h e H u r l e r C o r r e c t i v e F a c t o r , J .

B i o l .

C h e m . 2 4 6 ( 2 4 ) : 7 7 7 3 7 7 7 9 ( 1 9 7 1 ) .

B i e l i c k i , J

,

e t

a l . ,

Recombinant

Human Iduronate2Sul

p h a t a s e :

C o r r e c t i o n

o f M u c o p o l y s a c c h a r i d o s i s T y p e

I I

F i b r o b l a s t s a n d

C h a r a c t e r i z a t i o n

o f t h e

P u r i ? e d Enzyme,

B i o c h e m . J

2 8 9 : 2 4 1 2 4 6

( 1 9 9 3 ) .

( L i s t

c o n t i n u e d

o n n e X t

p a g e . )

P r i m a r y E x a m i n e r M a n j u n a t h

N . R a o

( 7 4 ) A t t o r n e y , A g e n t ,

o r

F i r m M a r s h a l l ,

G e r s t e i n & o r u n

LLP

( 5 7 ) ABST RACT

The p r e s e n t i n v e n t i o n

p r o v i d e s a f o r m u l a t i o n c o m p r i s i n g a

p h a r m a c e u t i c a l c o m p o s i t i o n c o m p r i s i n g a human

r e c o m b i

n a n t

o t - L - i d u r o n i d a s e

o r b i o l o g i c a l l y

a c t i v e

o r muteins

t h e r e o f W i t h a

p u r i t y

o f

g r e a t e r t h a n 99%,

o r i n

c o m b i n a t i o n

W i t h

a

p h a r m a c e u t i c a l l y a c c e p t a b l e

c a r r i e r .

The

p r e s e n t

i n v e n t i o n f u r t h e r p r o v i d e s

m e t h o d s

t o t r e a t c e r t a i n g e n e t i c

d i s o r d e r s i n c l u d i n g o t - L - i d u r o n i d a se

d e ? c i e n c y

a n d

muco

p o l y s a c c h a r i d o s i s

I (MPS

1 )

b y

a d m i n i s t e r i n g s a i d

f o r m u

l a t i o n .

4 6

C l a i m s ,

1 4 Drawing S h e e t s

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L C Y E A C G Y E

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US 6 , 8 5 8 , 2 0 6 B2

P a g e 2

OTHER PUBLICATIONS

C l e m e n t s , e t

a l . , Human

a l p h a L i d u r o n i d a s e 1 . P u r i ? c a

t i o n , m o n o c l o n a l a n t i b o d y p r o d u c t i o n , n a t i v e

a n d

s u b u n i t

m o l e c u l a r m a s s ,

E u r o p e a n

J o u r n a l

o f B i o c h e m i s t r y ,

1 5 2 ( 1 ) : 4 3 4 9 ( 1 9 9 4 ) .

F r i e d m a n ,

T . ,

P r o g r e s s ToWard Human Gene

T h e r a p y ,

S c i e n c e

2 4 4 : 1 2 7 5 1 2 8 1

( 1 9 8 9 ) .

H o o g e r b r u g g e ,

P . M . ,

e t a l . ,

A l l o g e n e i c B o n e MarroW

T r a n s p l a n t a t i o n f o r L y s o s o m a l

S t o r a g e

D i s e a s e s , L a n c e t

3 4 5 : 1 3 9 8 ( 1 9 9 5 ) .

I o a n n o u , Y . A . , e t a l . , O v e r e x p r e s s i o n o f Human a G a l a c

t o s i d a s e A e s u l t s i n

I t s

I n t r a c e l l u l a r A g g r e g a t i o n ,

C r y s t a l

l i Z a t i o n i n

L y s o s o m e s ,

a n d S e l e c t i v e S e c r e t i o n , J . C e l l

B i o l . 1 1 9 ( 5 ) : 1 1 3 7 1 1 5 0

( 1 9 9 2 ) .

K a k k i s , e t a l . ,

E n Z y m e r e p l a c e m e n t

t h e r a p y

i n

muco

p o l y s a c c h a r i d o s i s

I . , New E n g l a n d J o u r n a l o f

M e d i c i n e ,

3 4 4 ( 3 ) : 1 8 2 1 8 8 ( 2 0 0 1 ) .

K a k k i s ,

E . , e t

a l . , S t r o n g T r a n s c r i p t i o n a l

A c t i v a t i o n

o f

T r a n s l o c a t e d CMyc

Genes

O c c u r s

W i t h o u t

a

S t r o n g

Nearby E n h a n c e r o r

P r o m o t e r ,

N u c l e i c A c i d s Res.

1 6 ( 1 ) : 7 7 9 6

( 1 9 8 8 ) .

L e d l e y , F . D . , C l i n i c a l

A p p l i c a t i o n o f

S o m a t i c

G e n e

T h e r a p y i n

I n b o r n

E r r o r s

o f M e t a b o l i s m , I n h e r i t .

M e t a b .

D i s . 1 3 : 5 9 7 6 1 6 ( 1 9 9 0 ) .

L o W r y , RB, t a l . An

U p d a t e

o n

t h e

F r e q u e n c y o f

Muco

p o l y s a c c h a r i d e

S y n d r o m e s i n

B r i t i s h C o l u m b i a , Human

G e n e t i c s 8 5 : 3 8 9 3 9 0 ( 1 9 9 0 ) .

M y e r o W i t Z ,

R . ,

e t

a l . , M a t u r a t i o n o f o t L I d u r o n i d a s e i n

C u l t u r e d

Human F i b r o b l a s t s , J .

B i o l .

Chem.

2 5 6 ( 6 ) : 3 0 4 4 3 0 4 8

( 1 9 8 1 ) .

M o s k o W i t Z ,

S . M . ,

e t a l . , C l o n i n g a n d E x p r e s s i o n

o f

cDNA

E n c o d i n g t h e

Human

L y s o s o m a l E n Z y m e ,

o t L I d u

r o n i d a s e , FASEB J

.6:A77

( 1 9 9 2 ) .

N e l s o n , 1 . , I n c i d e n c e o f t h e M u c o p o l y s a c c h a r i d o s e s i n

N o r t h e r n

I r e l a n d ,

Human

G e n e t i c s 1 0 1 : 3 5 5 3 5 8

( 1 9 9 7 ) .

S c r i v e r ,

C . R . , B e a u d e t , A . L . , S l y ,

WS

n d V a l l e , D .

E d s .

T h e M e t a b o l i c

B a s i s o f

I n h e r i t e d D i s e a s e p p

1 5 6 5 1 5 8 7 ,

McGraW i l l , NeW Y o r k

( 1 9 8 9 ) .

S h u l l , R . M . ,

e t

a l . ,

EnZyme R e p l a c e m e n t i n a

C a n i n e

Model o f H u r l e r S y n d r o m e ,

P r o c .

N a t l .

A c a d . S c i . , USA

9 1 : 1 2 9 3 7 1 2 9 4 1

( 1 9 9 4 ) .

S t o l t Z f u s , L . J . ,

e t

a l . ,

C l o n i n g a n d C h a r a c t e r i Z a t i o n o f

cDNA E n c o d i n g

C a n i n e

o t L I d u r o n i d a s e ,

J .

B i o l .

C h e m . 2 6 7 ( 1 0 ) : 6 5 7 0 6 5 7 5 ( 1 9 9 2 ) .

T a y l o r , J , e t a l . ,

o t L I d u r o n i d a s e

i n Normal and Muco

p o l y s a c c h a r i d o s i s T y p e 1 Human

S k i n

F i b r o b l a s t s , B i o

c h e m J . 2 7 4 : 2 6 3 2 6 8 ( 1 9 9 1 ) .

T o l s t o s h e v , P . ,

e t

a l . ,

G e n e

E x p r e s s i o n U s i n g R e t r o v i r a l

V e c t o r s , C u r r e n t

O p i n i o n s

B i o t e c h . 1 : 5 5 6 1 ( 1 9 9 0 ) .

T u c k e r ,

P . W .

e t a l . , Mouse IgA Heavy

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G e n e

S e q u e n c e :

I m p l i c a t i o n s f o r

E v o l u t i o n

o f

I m m u n o g l o b u l i n

H i n g e

E x o n s ,

P r o c . N a t l .

A c a d .

S c i .

U S A 7 8 ( 1 2 ) : 7 6 8 4 7 6 8 8 ( 1 9 8 1 ) .

U n g e r , E . G . ,

e t

a l . ,

R e c o m b i n a n t

o t L I d u r o n i d a s e :

C h a r

a c t e r i Z a t i o n

o f

t h e P u r i ? e d

EnZyme

a n d C o r r e c t i o n o f Muco

p o l y s a c c h a r i d o s i s

T y p e I

F i b r o b l a s t s ,

B i o c h e m .

J

3 0 4 : 4 3 4 9

( 1 9 9 4 ) .

K a k k i s ,

e t a l . , L o n g T e r m a n d H i g h D o s e T r i a l s

o f

EnZyme

R e p l a c e m e nt T h e r a p y i n t h e

C a n i n e

Model

o f

M u c o p o l y s a c c h a r i d o s i s I , B i o c h e m . M o l .

M e d .

v o l . 5 8 , N o .

2 , p p .

1 5 6 1 6 7 ( 1 9 9 6 ) .

N e u ? e l d ,

e t

a l . ,

T h e

M u c o p o l y s a c c h a r i d o s e s ,

T h e

Meta

b o l i c

B a s i s o f n h e r i t e d D i s e a s e ,

S c r i v e r ,

C . R . , B e a u d e t ,

A .

L . , S l y ,

W . S . , a n d V a l l e , D . E d s . McGraW

i l l , NeW Y o r k ,

p p .

1 5 6 5 1 5 8 7 ( 1 9 8 9 ) .

Rome, e t

a l . , o t L I d u r o n i d a s e f o r Human K i d n e y

Meth

o d s i n

E n z y m o l o g y ,

v o l . 8 3 , p p . 5 7 8 5 8 2 ( 1 9 8 2 ) .

Schuchman,

e t

a l . , Human

o t L I d u r o n i d a s e : P u r i ? c a t i o n

a n d

P r o p e r t i e s

o f

t h e

H i g h

U p t a k e

( H i g h e r M o l e c u l a r

W e i g h t ) a n d t h e

LoW

U p t a k e ( P r o c e s s e d ) F o r m s J . B i o .

C h e m ,

v o l . 2 5 9 ,

N o .

5 ,

p p . 3 1 3 2 3 1 4 0

( 1 9 8 4 ) .

S c o t t , e t a l . , Human o t L I d u r o n i d a s e : cDNA s o l a t i o n

a n d

E x p r e s s io n , P r o c .

N a t l . A c a d . S c i . USA

v o l .

8 8 , p p .

9 6 9 5 9 6 9 9 ,

( 1 9 9 1 ) .

S c o t t , e t a l . , M u l t i p l e P o l y m o r p h i s m s W i t h i n t h e o t L I d u

r o n i d a s e Gene

( I D U A ) : I m p l i c a t i o n s f o r a R o l e

i n Modi?

c a t i o n

o f MPSI

D i s e a s e

P h e n o t y p e , Hum. M o l . G e n e t . v o l .

2 ,

N o . 9 , p p . 1 4 7 1 1 4 7 3 ( 1 9 9 3 ) .

S t o l Z f u s ,

e t a l . ,

M u c o p o l y s a c c h a r i d o s i s

I : c l o n i n g

a n d

c h a r a c t e r i Z a t i o n o f

cDNA

e n c o d i n g c a n i n e

o t L I d u

r o n i d a s e ,

Am. J . Hum.

G e n e t . ,

v o l . 4 7 , N o . 3 ,

p . A 1 6 7 ,

( 1 9 9 0 ) .

C l e m e n t s ,

e t

a l . ,

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a n d c h a r a c t e r i Z a t i o n

o f human o t L i d u r o n i d a s e

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t h e

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a n t i b o d i e s , B i o c h e m . J . v o l .

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p p . 1 9 9 2 0 8 ( 1 9 8 9 ) .

K a k k i s , e t a l . , O v e r e x p r e s s i o n o f

t h e

human l y s o s o m a l

enZyme o t L i d u r o n i d a s e i n C h i n e s e H a m s t e r O v a r y c e l l s ,

P r o t . E x p . P u r i f . v o l .

5 ,

p p . 2 2 5 2 3 2 ( 1 9 9 4 ) .

S c o t t ,

e t a l . , Chromosomal l o c a l i Z a t i o n o f t h e human

o t L i d u r o n i d a s e g e n e (IDUA) t o 4 p 1 6 . 3 Am. J . Hum.

G e n e t .

v o l .

4 7 , p p .

8 0 2 8 0 7

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a l . ,

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o f

r e c o m b i n a n t human

o t L i d u r o n i d a s e s e c r e t e d b y C h i n e s e

H a m s t e r

O v a r y

c e l l s ,

J .

B i o l .

C h e m . v o l . 2 7 3 , N o . 3 6 , p p .

2275822765

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B i o M a r i n

a n d GenZyme R e p o r t p o s i t i v e

One

Y e a r Sum

mary

D a t a F o r M PS 1 P a t i e n t s S e p . 1 4 ,

1 9 9 9 .

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T A C

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C T G G C G C A T G T T T C C

A A G

l G G

A A C

T T C

G A G

A C G T G G A A T G A G C C A G A G C A C

T y r G l y L e u A l a H i s

V a l

S e r L y s T r p A s n P h e G l u T h r T r p A s n G l u P r o A s p H i s

2 1 2 0 2 1 3 0 2 1 4 0 2 1 5 0 2 1 6 0

i v

*

k k

*

1 k

*

~ I r

* i *

C A C G A C T T T

GAC AAC

G T C T C C A T G

ACC

ATG

CAA GGC

T T C

CTG

A A G T A C T A C G A T 6 0 0

H i s A s p P h e A s p A s n V a l S e r M e t T h r M e t G l n G l y P h e L e u A s n T y r T y r A s p A l a

2 1 7 0

2180

2190 2200

2210

2220

T G C TCG

GAG

GGT C T G CGC GCC GCC AGC

CCC

GCC [ Z T G C G G CTG GGA GGC ( I C C GGC GAC

C y s S e r G l u G l y L e u A r g A l a A l a S e r P r o A l a L e u A r g L e u G l y G l y P r o G l y A s p

2 2 3 0 2240 2 2 5 0 2260 2 2 7 0 2280

T C C

T T C

C A C A C C

C C A C C G

C G A T C C C C G C T G A G C T G G GG C C T C

C T G

C G C

C A C

T G C

C A C

S e r

P h e

H i s

T h r

P r o

P r o A r g S e r

P r o L e u

S e r

T r p

G l y

L e u

L e u A r g

H i s

C y s H i s

2 2 9 0 2300 2 3 1 0

2 3 2 0

2 3 3 0 2340

G A C

G G T

ACC AAC T T C

T T C

A C T G G G G A G

G C G

G G C

G T G

C G G

C T G

GAC T A C ATC TCC CTC

A s p

G l y T h r A s n P h e

P h e

T h r

G l y

G l u A l a

G l y V a l

A r g

L e u

A s p T y r

I l e S e r

L e u

2350 2360 2370 2380 2390

CAC

AGG AAG GGT GCG

CGC

AGC

T C C ATC TCC

ATC

CTG GAG CAG

GAG AAG GTC GTC GCG

H i s A r g L y s G l y

A l a

A r g

S e r S e r

I l e S e r I l e

L e u

G l u

G l n

G l u L y s V a l V a l A l a

2400

2410

2420 2430 2440 2450

*

* *

C A G

C A G A T C

C G G C A G

C T C T T C C C C

A A G

T T C GCG

G A C

ACC C C C A T T

T A C

A A C G A C G A G

G l n

G l n I l e A r g

G l n

L e u

P h e

P r o

L y s P h e A l a A s p

T h r P r o I l e T y r A s n A s p G l u

2460 2470 2480 2490 2500 2510

~ k * ' k k

i k *

k

*

* 1 ' *

G C G G A C C C G C T G G T G G G C T G G T C C C T G

CCA C A G C C G

T G G A G G G C G G A E

G T G A C C

T A C

A l a A s p P r o L e u V a l G l y T r p S e r L e u P r o

G l n P r o

T r p A r g

A l a

A s p

V a l

T h r T y r

2520 2530 2540

2 5 5 0 2 5 6 0

* * * 1r

*

w _ - x a 1 : w

G C G G C C A T G

G T G

G T G

A A G G T C

A T C

G C G CAG C A T C A G

A A G

C T G C T A

C T G G C C

A A C A C C

Ala

Ala Met

V a l V a l L y s

V a l

I l e Ala Gln His Gln Asn L e u Leu Leu Ala

Asn Thr

2 5 7 0

2580 2590 2600 2610

2 6 2 0

k

k

*

i

A C C T C C G C C T T C

( I C C T A C

G C G C T C C T G

A G C

A A C

G A C

A A T G C C T T C C T G

A G C

T A C C A C

T h r Ser Ala P h e P r o Tyr Ala L e u L e u Ser A s n Asp A s n A l a P h e L e u S e r Tyr H i s

2 6 3 0

2 6 4 0

2 6 5 0 2 6 6 0 2 6 7 0 2 6 8 0

* * i k ~ I r * i - k * k *

C C G CAC

( I C C

TTC GCG C A G CGC A C G C T C ACC GCG C G C

T T C

C A G GTC AAC AAC ACC CGC

P r o H i s

P r o

P h e A l a Gln A r g Thr

L e u

Thr Ala Arg

P h e

G l n V a l Asn Asn Thr Arg

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U.S. P a t e n t F e b . 2 2 , 2 0 0 5

S h e e t

7 0 f 1 4 U S 6 , 8 5 8 , 2 0 6 B2

F I G . 1 - 7

5 5 1 0 5 5 2 0

5 5 3 0

5 5 4 0

5 5 5 0 5 5 6 0 5 5 7 0

k

*

* ' k

* *

* i * * * * * *

C A A C G T T GT T G C C A T T G C T A C A G G C A T C G T G G T G T C A C G C

T C G T C G T T T G

G T A T G G C T T C A T T C A G C T C C

5 5 8 0

5 5 9 0

5 6 0 0 5 6 1 0

5 6 2 0

5 6 3 0

5 6 4 0

* * * * .

*

f 7 r

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v:

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1

G G TT C C C A A C G A T C A A G G C G A G T T A C A T G A T C C C C C A T G T T G T G C A A A A A A G C G G T T A G C T C C T T C G G T C

5 6 5 0 5 6 6 0 5 6 7 0 5 6 8 0 5 6 9 0 5 7 0 0 5 7 1 0

~ k * * *

i -

* 1 : 1 : * * 1 : r * *

C T C C G A T C G T T G T C A G A A G T A A G T T G G C C G C A G T G T T A T C A C T C A T G G T T A T G G C A G C A C T G C A T A A T T C

5 7 2 0 5 7 3 0

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* * * * a r

1 :

* * ~ x * * 1 : a: *

T C T T A C T G T C A T G C C A T C C G T A A G A T G C T T T T C T G T G A C T G G T G A G T A C T C A A C C A A G T C A T T C T G A G A A

5790

5800

5810

5820

5830 5840 5850

* * * ' * i * * * * a: * * * *

T A G T G T A T G C G G C G A C C G A G

T T G C T C T T G C C C G G C G Y C A A

T A C G G G A T A A

T A C C G C G C C A C A T A G C A G A A

5 8 6 0

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5 8 8 0

5 8 9 0

5 9 0 0 5 9 1 0 5 9 2 0

*

*

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G C T C A T C A T T G G A A A A C G T T C T T C G G G G C G A A A A C T C T C A A G G A T C T T A C C G C T G T T G A G

5 9 3 0 5 9 4 0 5 9 5 0 5 9 6 0 5 9 7 0 5 9 8 0 5 9 9 0

*

1 : 1 1 ' k

a: * = 1 * 1 k * * * * *

A T C C A G T T C G A T G T A A C C C A C T C G T G C A C C C A A C T G A T C T T C A G C A T C T T T T A C T T T C A C C A G C G T T T C T

6 0 0 0 6 0 1 0 6 0 2 0 6 0 3 0 6 0 4 0 6 0 5 0 6 0 6 0

* * * 1 r * * 1r * * w * * * *

G G G T G A G C A A A A A C A G G A A G G C A A A A T G C C G C A A A A A A G G G A A T A A G G G C

G A C A C G G A A A

T G T T G A A T A C

6 0 7 0

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6 1 0 0 6 1 1 0

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a:

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1r

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1 *

1

* * * * *

T C A T A C T C T T C C T T T T T C A A T A T T A T T G A A G C A T T T A T C A G G G T T A T T G T C T C A T G A G C G G A T A C A T A T T

6 1 4 0

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* * 1 1 :

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i * * * * * 1 * * *

T G A A T G T A T T T A GA A A A A T A A A C A A A T A G G G G T T C C G C G C A C A T T T C C C C G A A A A G T G C C A C C T G A C G T C

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yam

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170

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160

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e g r e e s

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K n e e

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F I G . 7

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o

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U.S. P a t e n t F e b . 2 2 , 2 0 0 5 S h e e t

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S l e e p A p n e a

I m p r o v e s

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+ H y p o p n e a r s D u r i n g S l e e p

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P u l m o n a r y

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3

p e r f u s i o n ,

( c ) o x y g e n s a t u r a t i o n

may b e

o p t i m i z e d

t o

a b o u t

4 0 %

b u t may b e a s h i g h

a s

80%, ( d )

m a c r o p o r o u s c e l l u l o s e

m i c r o c a r r i e r s

W i t h a b o u t

5 erum

i n t h e medium i n i t i a l l y ,

may b e u s e d t o p r o d u c e c e l l mass

f o l l o W e d

by a r a p i d

W a s h o u t s h i f t t o p r o t e i n - f r e e medium

f o r p r o d u c t i o n ,

( e )

a

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s c i e n c e s

PF-CHO

p r o d u c t

may

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supplemental

amounts

o f

one

o r

more

i n g r e d i e n t s s e l e c t e d

f r o m t h e g r o u p c o n s i s t i n g o f : g l u t a m a t e , a s p a r t a t e , g l y c i n e ,

r i b o n u c l e o s i d e s , a n d

d e o x y r i b o n u c l e o s i d e s ;

a s t i r r e d

t a n k

s u s p e n s i o n

c u l t u r e

may b e p e r f u s e d

i n a

c o n t i n u o u s p r o c e s s

t o p r o d u c e i d u r o n i d a s e .

I n

a

s e c o n d a s p e c t , t h e p r e s e n t

i n v e nt i o n p r o v i d e s a

t r a n s f e c t e d c e l l l i n e , W h i c h f e a t u r e s

t h e

a b i l i t y t o

p r o d u c e

o t - L - i d u r o n i d a s e

i n a m o u n t s ,

Which e n a b l e u s i n g

t h e

enZyme

t h e r a p e u t i c a l l y . I n p r e f e r r e d

e m b o d i m e n t s , t h e p r e s e n t

i n v e n t i o n

f e a t u r e s

a recombinant Chinese hamster ovary c e l l

l i n e s u c h a s t h e

2 . 1 3 1

c e l l l i n e t h a t s t a b l y a n d r e l i a b l y

p r o d u c e s a m o u n t s o f

o t - L - i d u r o n i d a s e W h i c h

e n a b l e u s i n g

t h e enZyme h e r a p e u t i c a l l y . I n some

p r e f e r r e d

e m b o d i m e n t s ,

t h e c e l l l i n e

may

c o n t a i n

more

t h a n 1 copy o f a n e x p r e s s i o n

c o n s t r u c t .

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t h e

c e l l

l i n e

e x p r e s s e s

recombinant

o t - L - i d u r o n i d a s e

i n

amounts

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a t

l e a s t

2 0 m i c r o g r a m s

p e r 1 c e l l s

p e r d a y .

I n

a

t h i r d

a s p e c t , t h e p r e s e n t i n v e n t i o n

p r o v i d e s

n o v e l

v e c t o r s s u i t a b l e

t o produce

o t - L - i d u r o n i d a s e

i n amounts

W h i c h e n a b l e u s i n g t h e e n Z y m e t h e r a p e u t i c a l l y . I n p r e f e r r e d

e m b o d i m e n t s , t h e

p r e s e n t i n v e n t i o n

f e a t u r e s a n e x p r e s s i o n

v e c t o r

c o m p r i s i n g

a

c y t o m e g al o v i r u s p r o m o t e r / e n h a n c e r

e l e m e n t , a 5 i n t r o n c o n s i s t i n g

o f

a murine

C O .

i n t r o n , a

cDNA encoding a l l o r a fragment o r

mutein

of

an

o t - L

i d u r o n i d a s e ,

a n d a 3 b o v i n e

g r o W t h

h o r m o n e p o l y a d e n y l a

t i o n

s i t e . A l s o ,

p r e f e r a b l y t h e cDNA

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o r

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l e n g t h .

T h i s e x p r e s s i o n v e c t o r may b e t r a n s f e c t e d

a t ,

f o r

e x a m p l e , a 50 t o 1

r a t i o

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g e n e a m p l i ? c a t i o n may b e

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i n d u c e

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copy i n s e r t i o n s .

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t o

t h e p r e s e n t

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gram p r o t e i n .

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a b o u t 8 2 , 0 0 0

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p r e s e n t i n v e n t i o n f e a t u r e s

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e m b o d i m e n t ,

a n a c i d pH

t r e a t m e n t s t e p i s u s e d t o

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i n c r e a s e t h e c a p a c i t y o f t h e l a r g e s c a l e

p u r i ? c a t i o n

p r o c e s s ,

10

1 5

20

25

30

35

40

45

50

55

60

6 5

4

t o

r e d u c e u n d e s i r a b l e l e a c h a b l e s i n a p p r o p r i at e

f o r

l o n g t e r m

p a t i e n t u s e , a n d t o i m p r o v e t h e p u r i t y o f t h e p r o d u c t .

I n a s i x t h a s p e c t , t h e

p r e s e n t i n v e n t i o n f e a t u r e s

n o v e l

methods o f t r e a t i n g

d i s e a s e s

c a u s e d a l l o r i n p a r t by a

d e ? c i e n c y

in o t - L - i d u r o n i d a s e . I n o n e

e m b o d i m e n t ,

t h i s

method f e a t u r e s

a d m i n i s t e r i n g

a recombinant o t - L

i d u r o n i d a s e

o r

a

b i o l o g i c a l l y

a c t i v e

fragment

o r

mutein

t h e r e o f

a l o n e

o r in combination

With

a p h a r m a c e u t i c a l l y

s u i t a b l e

c a r r i e r .

I n o t h e r

e m b o d i m e n t s ,

t h i s

method f e a t u r e s

t r a n s f e r r i n g

a

n u c l e i c a c i d encoding a l l o r

a

p a r t o f a n

o t - L - i d u r o n i d a s e

i n t o one o r more h o s t c e l l s i n

v i v o .

Pre

f e r r e d

e m b o d i m e n t s i n c l u d e o p t i m i Z i n g

t h e d o s a g e

t o

t h e

n e e d s

o f

t h e o r g a n i s m t o b e t r e a t e d , p r e f e r a b l y mammals o r

h u m a n s ,

t o

e f f e c t i v e l y

a m e l i o r a t e

t h e

d i s e a s e

s y m p t o m s . I n

p r e f e r r e d

e m b o d i m e n t s ,

t h e d i s e a s e i s M u c o p o l y s a c c h a r i d o

s i s

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(MPS ) ,

H u r l e r

s y n d r o m e , H u r l e r - S c h e i e s y n d r o m e o r

S c h e i e s y n d r o m e .

I n a s e v e n t h a s p e c t ,

t h e

p r e s e n t i n v e n t i o n f e a t u r e s

n o v e l

p h a r m a c e u t i c a l c o m p o s i t i o n s c o m p r i s i n g

o t - L - i d u r o n i d a s e

u s e f u l

f o r t r e a t i n g a

d i s e a s e

c a u s e d a l l

o r

in p a r t

by

a

d e ? c i e n c y

i n o t - L - i d u r o n i d a s e .

S u c h c o m p o s i t i o n s may b e

s u i t a b l e f o r

a d m i n i s t r a t i o n

i n a number o f

Wa y s

such a s

p a r e n t e r a l ,

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t i o n . Within t h e s c o p e

o f

t h i s a s p e c t a r e

embodiments

f e a t u r i n g n u c l e i c a c i d s e q u e n c e s encoding a l l o r a p a r t

o f a n

o t - L - i d u r o n i d a s e ,

Which may b e a d m i n i s t e r e d

i n

v i v o

i n t o

c e l l s ,

a f f e c t e d

W i t h

a n

o t - L - i d u r o n i d a s e d e ? c i e n c y .

DESCRIPTION

OF THE

FIGURES

F I G .

1 r e p r e s e n t s

t h e n u c l e o t i d e a n d deduced amino a c i d

s e q u e n c e s o f

cDNA

e n c o d i n g

o t - L - i d u r o n i d a s e

(SEQ I D

NOS:1

a n d 2 ) .

N u c l e o t i d e s 1

t h r o u g h

6 2 0 0 a r e

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Amino a c i d s a r e p r o v i d e d s t a r t i n g W i t h t h e ? r s t m e t h i o n i n e

i n

t h e o p e n r e a d i n g

f r a m e .

F I G .

2 r e p r e s e n t s

t h e r e s u l t s from SDS-PAGE r u n s o f

e l u a t e o b t a i n e d

a c c o r d i n g

t o t h e

p r o c e d u r e s a s

d e s c r i b e d

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t h e SDS-PAGE r e s u l t s

o f

p u r i ? e d

o t - L - i d u r o n i d a s e

( 3 m i c r o g r a m s )

a n d

c o n t a m i n a n t s

from t h e

p r o d u c t i o n / p u r i ? c a t i o n

scheme

d i s c l o s e d i n

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x p r ' . P u r i f 5 :

2 2 5 2 3 2

( 1 9 9 4 ) . I n t h e

bottom p a n e l , SDS-PAGE r e s u l t s o f

p u r i ? e d

o t - L

i d u r o n i d a s e W i t h c o n t a m i n a n ts f r o m a n u n p u b l i s h e d

p r i o r

p r o d u c t i o n / p u r i ? c a t i o n p r o c e s s ( U S . p a t e n t a p p l i c a t i o n S e r .

N o s .

0 9 / 0 7 8 , 2 0 9

a n d

0 9 / 1 7 0 , 9 7 7 ) r e f e r r e d t o a s t h e C a r s o n

m e t h o d

i n

L a n e s

2 ( 7 . 5

m i c r o g r a m o t - L - i d u r o n i d a s e )

a n d

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t h a t o f

t h e

p r o d u c t i o n / p u r i ? c a t i o n p r o c e s s o f

t h e

p r e s e n t

i n v e n t i o n r e f e r r e d t o a s t h e G a l l i P r o c e s s

( L a n e 4

5

m i c r o

grams o t - L - i d u r o n i d a s e ) . Lane 1

c o n t a i n s t h e m o l e c u l a r

W e i g h t m a r k e r . F I G .

2

s h o W s

t h a t t h e G a l l i p r o d u c t i o n /

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o f t h e

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p u r i ? e d o t - L - i d u r o n i d a s e

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W i t h f e W e r c o n t a m i n a n t s

i n

c o m p a r i s o n

W i t h

p r i o r

p r o d u c t i o n / p u r i ? c a t i o n

s c h e m e s .

F I G . 3

d e m o n s t r a t e s t h e

o t - i d u r o n i d a s e

p r o d u c t i o n

l e v e l

o v e r a

3 0 - d a y

p e r i o d , d u r i n g Which t i m e c e l l s a r e s W i t c h e d

a t day 5 from a serumcontaining medium t o a s e r u m - f r e e

medium. o t - I d u r o n i d a s e p r o d u c t i o n Was c h a r a c t e r i Z e d b y :

( 1 )

a b s e n c e

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f o r

a d a p t a t i o n When

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from

s e r u m - c o n t a i n i n g t o

s e r u m - f r e e

medium a t

100200

( t o p

a n d

b o t t o m

p a n e l s ) W i t h a n

u n i n t e r r u p t e d

i n c r e a s e

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p r o d u c t i v i t y ( t o p p a n e l ) ; ( 2 )

a

h i g h l e v e l

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i n

e x c e s s o f 4

mg

e r

l i t e r

( 1 0 0 0

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medium ( b o t t o m p a n e l ) ;

a n d

( 3 ) a b o o s t i n o t - i d u r o n i d a s e

p r o d u c t i o n W i t h b u t y r a t e

i n d u c t i o n e v e n t s

( b o t t o m p a n e l ) .

F I G .

4 d e m o n s t r a t e s a d e c r e a s e i n l i v e r volume d u r i n g

enZyme

t h e r a p y

i n M P S p a t i e n t s .

8/10/2019 US 6,858,206-Jul 20

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8/10/2019 US 6,858,206-Jul 20

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7

P a r t i c u l a r l y

p r e f e r r e d e m b o d i m e n t s

o f

t h e m e t h o d f o r

p r o d u c i n g o t - L - i d u r o n i d a s e a c c o r d i n g t o t h e

p r e s e n t

i n v e n

t i o n f e a t u r e o n e , more

t h a n

o n e , o r

a l l

o f t h e o p t i m i Z a t i o n s

d e s c r i b e d

h e r e i n and

may b e

employed a s

d e s c r i b e d

i n

more

d e t a i l b e l o W .

The

p r o d u c t i o n method

o f

t h e

p r e s e n t i n v e n

t i o n m a y , t h e r e f o r e ,

p r o v i d e a p r o d u c t i o n

c u l t u r e p r o c e s s

h a v i n g t h e f o l l o W i n g f e a t u r e s :

1 .

A

i c r o c a r r i e r b a s e d c u l t u r e

u s i n g

m a c r o p o r o u s m i c r o

c a r r i e r

beads

made

o f modi?ed

c e l l u l o s e

o r a n e q u i v a l e n t

t h e r e o f i s p r e f e r a b l y u s e d i n l a r g e s c a l e c u l t u r e ? a s k s W i t h

o v e r h e a d s t i r r i n g

o r

a n e q u i v a l e n t

t h e r e o f .

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o f

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t h e s e

b e a d s may e

a c h i e v e d

by c u l t u r e i n

a

5 e t a l

bovine serum may e added t o DME/F 12

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f r e e medium modi?ed W i t h i n g r e d i e n t s

i n c l u d i n g

r i b o n u c l e o s i d e s , d e o x y r i b o n u c l e o s i d e s , p y r u v a t e ,

n o n

e s s e n t i a l a m i n o a c i d s ,

a n d

HEPES.

A f t e r

a b o u t 3 6 d a y s i n

t h i s medium, a

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i n

W h i c h

p r o t e i n - f r e e

medium

e p l a c e s

t h e s e r u m - c o n t a i n i n g medium

a t

a n

i n c r e a s i n g p e r f u s i o n r a t e

d e p e n d e n t

on t h e

g l u c o s e

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t h e

e n t i r e

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p e r i o d ,

t h e c e l l s

a r e c u l t i v a t e d

i n a

p r o t e i n - f r e e

medium. Th e u s e o f a

p r o t e i n - f r e e medium

i n

enZyme

p r o d u c t i o n

i s

b e n e ? c i a l

i n

r e d u c i n g

t h e

e x p o s u r e

r i s k

o f

b o v i n e s p o n g i f o r m e n c e p h a lo p a t h y

( B S E ) a n d o t h e r

i n f e c t io u s b i o l o g i c a g e n t s s u c h

a s

v i r u s e s t o

p a t i e n t s

b e i n g

t r e a t e d W i t h t h e

enZyme,

Wherein t h e r i s k o f BSE r o t h e r

h a r m f u l a g e n t s i s d e p e n d e n t

on t h e amount

o f

p o t e n t i a l

s e r u m e x p o s u r e . I n

p r i o r

p u b l i s h e d s t u d i e s ,

t h e

c a r r i e r s u s e d

t o groW t h e c e l l s Were b o v i n e g e l a t i n m i c r o c a r r i e r s ,

u s e d a t

1 gram

p e r

l i t e r

o r 100 t i m e s t h e p r o d u c t c o n c e n t r a t i o n .

L e a c h i n g

o f

1 % o f

t h e g e l a t i n

p r o t e i n f r o m

t h e

m i c r o c a r r i e r s

Would r e p r e s e n t a r e l a t i v e

100%

c o n t a m i n a t i o n a n d t h e r e b y

c o n t r i b u t e

t o

t h e r i s k

o f BSE. Thus, n e W

c a r r i e r s a r e e i t h e r

d e x t r a n

o r c e l l u lo s e - b a s e d a n d

c o n s i s t

o f

c a r b o h y d r a t e s ,

and

n o t a n i m a l - d e r i v e d m a t e r i a l s .

F I G . 3 shoWs

t h a t

t h e c e l l s a r e groWn t o a d e n s i t y i n 5

serum

c o n t a i n i n g

medium a n d

t h e n s W i t c h e d W i t h o u t any

a d a p t a t i o n

t o

a

p r o t e i n - f r e e

medium.

F I G .

3

s p e c i ? c a l l y

shoWs

t h a t :

1 )

C e l l s s u r v i v e a n d c o n t i n u e

t o

p r o d u c e i d u

r o n i d a s e When

s h i f t e d

W i t h o u t

a d a p t a t i o n . I n

c o n t r a s t , o t h e r

s t u d i e s

Would s u g g e s t t h a t

a d a p t a t i o n t o a p r o t e i n - f r e e

medium s n e c e s s a r y . I n t h e method

o f

t h e p r e s e n t i n v e n t i o n ,

enZyme p r o d u c t i o n c o n t i n u e s a t l e v e l s comparable t o

serum

c o n t a i n i n g m e d i u m . 2 ) o t - L - I d u r o n i d a s e p r o d u c e d

i n a

p r o t e i n - f r e e medium

r e t a i n s a

l e v e l

o f p r o d u c t i o n i n e x c e s s

o f 4 mg

e r

l i t e r o r 1 , 0 0 0

u n i t s

p e r m l .

3 ) o t - L - I d u r o n i d a s e

p r o d u c e d i n a p r o t e i n - f r e e medium h a s

h i g h u p t a k e

i n d i c a t

i n g t h a t

t h e

s h i f t

i n

medium a n d ,

h e n c e ,

a s h i f t

i n

c a r b o h y

d r a t e s b e i n g f e d t o c e l l s , d o e s n o t a d v e r s e l y a f f e c t t h e h i g h

u p t a k e c h a r a c t e r

o f

t h e e n Z y m e .

E i g h t

l o t s

o f

o t - L

i d u r o n i d a s e h a v e

been

p r o d u c e d a n d r e l e a s e d i n t h i s manner

W i t h a n

u p t a k e h a l f

maximal

v a l u e o f

l e s s

t h a n 2 nM n

a l l

l o t s .

2 . T h e c u l t u r e c o n d i t i o n s

a r e

p r e f e r a b l y maintained a t

a

d i s s o l v e d

oxygen

o f 4 0 % o f

a i r

s a t u r a t i o n

a t a pH

f a b o u t

6 . 8 7 . 0 and a t a t e m p e r a t u r e

o f a b o u t

3537 C .

T h i s

may

b e a c h i e v e d u s i ng a c o n t r o l u n i t , m o n i t o r i n g u n i t a n d a p p r o

p r i a t e p r o b e s

s u c h a s t h o s e p r o d u c e d b y A p p l i k o n o r

M e t t l e r .

H o W e v e r , s k i l l e d

a r t i s a n s

W i l l r e a d i l y a p p r e c i a t e

t h a t t h i s c a n

e a s i l y

b e a c h i e v e d

by e q u i v a l e n t

c o n t r o l s y s

t e m s p r o d u c e d

by

o t h e r

m a n u f a c t u r e r s . An

a i r s a t u r a t i o n o f

a b o u t 4 0 % r e s u l t s i n i m p r o v e d

o t - L - i d u r o n i d a s e

s e c r e t i o n

t h o u g h up t o 80 i r s a t u r a t i o n may b e u s e d . HoWever,

f u r t h e r

i n c r e a s e s

i n

oxygen t o ,

f o r e x a m p l e , 90% a i r

s a t u r a t i o n ,

d o n o t p r o v i d e s i g n i ? c a n t l y e n h a n c e d s e c r e t i o n

o v e r 80

a i r

s a t u r a t i o n . The

d i s s o l v e d

oxygen may be

s u p p l i e d b y i n t e r m i t t e n t o r c o n t i n u o u s o x y g e n s p a r g i n g

10

1 5

20

25

30

35

40

45

50

55

60

6 5

8

u s i n g a 5

micron s t a i n l e s s s t e e l

o r

l a r g e r o p e n i n g s p a r g e r ,

o r

e q u i v a l e n t t h e r e o f . ApH

f

a b o u t 6 . 8 7 . 0 i s o p t i m a l

f o r t h e

a c c u m u l a t i o n

o f t h e o t - L - i d u r o n i d a s e

enZyme.

The enZyme

i s p a r t i c u l a r l y u n s t a b l e

a t

pHs

a b o v e a b o u t 7 . 0 .

BeloW

a

pH

o f a b o u t 6 . 7 ,

t h e s e c r e t i o n

r a t e may d e c r e a s e ,

p a r t i c u l a r l y

beloW a

pH

f

a b o u t

6 . 5 . The

c u l t u r e

i s

t h e r e f o r e m a i n t a i n e d

o p t i m a l l y b e t W e e n a pH

o f

a b o u t

6 . 8 7 . 0 .

3 .

The

p r o d u c t i o n

c u l t u r e

medium

may

b e

a

modi?ed

f o r m o f t h e

c o m m e r c i a l l y

a v a i l a b l e

p r o p r i e t a r y medium

from JR

i o s c i e n c e s

c a l l e d

E x c e l l

PF CHO.

T h i s

medium

s u p p o r t s l e v e l s

o f

s e c r e t i o n

e q u i v a l e n t t o t h a t

o f

serum

u s i n g

a c e l l l i n e

such a s

t h e

2 . 1 3 1

c e l l

l i n e .

I t

may be p r e f e r a b l y

modi?ed

t o i n c l u d e

a n

a c i d i c

pH o f a b o u t 6 . 8 7 . 0

( + 0 . 1 ) ,

and b u f f e r e d

With

HEPES

t 7 . 5 mM

r

15

mM. h e medium

may

c o n t a i n 0 . 0 5 t o 0.1% o f P l u r o n i c s

F - 6 8 (BASF),

a

n o n - i o n i c

s u r f a c t a nt o r

a n

e q u i v a l e n t

t h e r e o f Which

f e a t u r e s

t h e a d v a n t a g e

o f p r o t e c t i n g

c e l l s f r o m s h e a r

f o r c e s

a s s o c i

a t e d W i t h s p a r g i n g .

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medium

may f u r t h e r

c o n t a i n

a

p r o p r i e t a r y

s u p p l e m e n t t h a t

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i n

i n c r e a s i n g t h e

p r o d u c t i v i t y

o f t h e

medium o v e r o t h e r p r o t e i n - f r e e media

t h a t

a r e

p r e s e n t l y a v a i l a b l e . Those

s k i l l e d

i n t h e a r t W i l l

r e a d i l y

u n d e r s t a n d

t h a t

t h e

c h o i c e o f c u l t u r e medium

may e

o p t i m i Z e d

c o n t i n u a l l y

a c c o r d i n g

t o

p a r t i c u l a r

c o m m e r c i a l

e m b o d i m e n t s

a v a i l a b l e a t p a r t i c u l a r

p o i n t s i n

t i m e .

S u c h

c h a n g e s

encompass no more

t h a n

r o u t i n e e x p e r i m e n t a t i o n

and a r e

i n t e n d e d

t o be W i t h i n t h e

s c o p e o f

t h e

p r e s e n t

i n v e n t i o n .

4 .

The p r o d u c t i o n

medium may b e a n a l yZ e d u s i n g a n

a m i n o a c i d a n a l y Z e r

c o m p a r i n g

s p e n t medium

W i t h s t a r t i n g

medium. Such a n a l y s e s h a v e d e m o n s t r a t e d t h a t t h e

2 . 1 3 1

c e l l

l i n e

d e p l e t e s a

s t a n d a r d PF CHO medium o f g l y c i n e ,

g l u t a m a t e a n d a s p a r t a t e t o a

l e v e l o f

a r o u n d 10% o f t h e

s t a r t i n g

c o n c e n t r a t i o n . S u p p l e m e n t a t i o n o f t h e s e a m i n o

a c i d s t o

h i g h e r

l e v e l s may

e s u l t

in e n h a n c e d c u l t u r e d e n s i t y

a n d

p r o d u c t i v i t y t h a t

may

l e a d

t o

a

23 f o l d

h i g h e r p r o d u c

t i o n t h a n a t b a s e l i n e .

S k i l l e d

a r t i s a n s W i l l

a p p r e c i a t e

t h a t

o t h e r

c e l l l i n e s W i t h i n t h e

s c o p e

o f t h e p r e s e n t i n v e n t i o n may

b e

e q u a l l y u s e f u l

f o r p r o d u c i n g

o t - L - i d u r o n i d a s e a c c o r d i n g

t o t h e p r e s e n t

m e t h o d . Hence, more

o r l e s s s u p p l e m e n t a l

n u t r i e n t s

may b e r e q u i r e d t o o p t i m i Z e

t h e

medium. Such

o p t i m i Z a t i o n s

a r e

i n t e n d e d t o

be

W i t h i n t h e s c o p e o f

t h e

p r e s e n t i n v e n t i o n

a n d may b e p r a c t i c e d

W i t h o u t

u n d u e

e x p e r i m e n t a t i o n .

5 . The

medium

may b e s u p p l e m e n t e d W i t h t h e f o u r

r i b o n u c l e o s i d e s

a n d f o u r d e o x y r i b o n u c l e o s i d e s

e a c h

a t

a b o u t 1 0 m g / l i t e r t o s u p p o r t t h e d i h y d r o f o l a t e r e d u c t a s e

d e ? c i e n t

c e l l

l i n e

2 . 1 3 1 . S k i l l e d a r t i s a n s

W i l l

a p p r e c i a t e

t h a t

o t h e r

c e l l

l i n e s W i t h i n

t h e

s c o p e

o f t h e p r e s e n t i n v e n t i o n may

b e

e q u a l l y

u s e f u l f o r

p r o d u c i n g

o t - L - i d u r o n i d a s e a c c o r d i n g

t o t h e p r e s e n t method. Hence , more o r

l e s s

r i b o n u c l e o s i d e s

a n d d e o x y r i b o n u c l e o s i d e s may b e r e q u i r e d t o o p t i m i Z e

t h e

medium,

a n d a l t e r n a t i v e s o u r c e s

o f

p u r i n e s a n d p y r m i d i n e s

f o r n u c l e i c a c i d s y n t h e s i s

may e u s e d s u c h

a s

h y p o x a n t h i n e

a n d

t h y m i d i n e .

Such

o p t i m i Z a t i o n s

a r e

i n t e n d e d W i t h i n

t h e

s c o p e

o f t h e

p r e s e n t i n v e n t i o n

a n d

may

b e

p r a c t i c e d W i t h o u t

u n d u e e x p e r i m e n t a t i o n .

6 . A f t e r r e a c h i n g c o n ? u e n c e a t

a b o u t

3 6 d a y s o f

c u l t u r e ,

a n

i n c r e a s i n g r a t e

o f c o n t i n u o u s

p e r f u s i o n i s i n i t i a t e d . A

c h a n g e o f medium

may e

a c c o m p l i s h e d ,

f o r e x a m p l e ,

u s i n g

a s l a n t f e e d t u b e c o n s t r u c t e d

and

p o s i t i o n e d t o

a l l o W

t h e

u p t a k e

o f medium

i t h o u t removal

o f

t h e m i c r o c a r r i e r s

e v e n

W h i l e t h e c u l t u r e i s s t i r r e d . By

pumping

o u t medium t h r o u g h

t h e s l a n t

f e e d

t u b e ,

m i c r o c a r r i e r s

s e t t l e W i t h i n t h e

body

o f

t h e

t u b e

i n s i d e t h e c u l t u r e and a r e

n o t

removed from

t h e

c u l t u r e

d u r i n g

t h e

c h a n g e

o n medium. I n t h i s m a n n e r , t h e

m i c r o c a r r i e r s

W i t h

t h e c e l l mass

a r e s e p a r a t e d

from

s u p e r

n a t a n t c o n t a i n i n g t h e e n Z y m e .

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