Production of Antistasin Using the Baculovirus Expression System

Chapter 8

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Production of Antistasin Using the Baculovirus Expression System D. Jain, K. Ramasubramanyan, S. Gould, C. Seamans, S. Wang, A. Lenny, and M . Silberklang Merck Sharp and Dohme Research Laboratories, P.O. Box 2000, Rahway, NJ 07065

We have used Spodoptera frugiperda (Sf9) insect cells infected with recombinant baculovirus to produce antistasin, a potent anticoagulant and antimetastatic agent from leech salivary glands. Mature antistasin is a 119-a.a.,15-kDa secreted protein which has an unusually high cysteine content (20/119 residues) and acts by inhibiting coagulation Factor Xa. We have also genetically engineered a truncated (7.5 kDa) form of antistasin, "half antistasin" (Η-ANS) for baculovirus-mediated expression. The efficiency of utilization of baculovirus in this system was improved by the use of a low (0.1) mutiplicity of infection. By monitoring increase in cell diameter during the course of infection and decrease in cell viability, both of which were found to correlate with product accumulation, harvest time could be optimized under various culture conditions. Secreted Η-ANS activity peaked between 72 and 96 hours post-infection. Among the major process variables examined, dissolved oxygen at a low level of 10% and at a high level of 110% air saturation was found to result in a reduced growth rate as well as lower productivity relative to a level of 65%. Data are presented on the kinetics of oxygen uptake during cell growth and the viral infection cycle for the production of H-ANS.

The i n s e c t c e l l - b a c u l o v i r u s e x p r e s s i o n v e c t o r system i s becoming i n c r e a s i n g l y p o p u l a r f o r t h e p r o d u c t i o n o f recombinant p r o t e i n s . B a c u l o v i r u s e s as a c l a s s have been w e l l c h a r a c t e r i z e d because o f

0097-6156/91/0477-0097S06.00/0 © 1991 American Chemical Society In Expression Systems and Processes for rDNA Products; Hatch, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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EXPRESSION SYSTEMS & PROCESSES FOR RDNA PRODUCTS

t h e i r p o t e n t i a l a p p l i c a t i o n s as a g r i c u l t u r a l a g e n t s f o r i n s e c t p e s t c o n t r o l ( 1 / 2 ) . T h i s has p r o v i d e d an e x c e l l e n t b a s i s b o t h f o r t h e development o f e f f i c i e n t e x p r e s s i o n v e c t o r systems u s i n g t h e a b u n d a n t l y e x p r e s s e d v i r a l p o l y h e d r i n gene l o c u s and f o r t h e p r o d u c t i o n o f v i r a l and recombinant p r o t e i n s i n i n s e c t c e l l s u s p e n s i o n c u l t u r e (3-5). A t Merck, we have used t h e b a c u l o v i r u s e x p r e s s i o n v e c t o r system t o produce a number o f recombinant p r o t e i n s , one o f which i s a n t i s t a s i n , a p o t e n t a n t i c o a g u l a n t and a n t i m e t a s t a t i c agent ( 6 ) . A n t i s t a s i n i s a p r o t e i n found i n t h e s a l i v a r y g l a n d s o f t h e M e x i c a n l e e c h Hae/nentaria officinalis (7-9) . I t i s a s t o i c h i o m e t r i c i n h i b i t o r o f F a c t o r Xa (10) i n t h e b l o o d c o a g u l a t i o n pathway and has no homology t o H i r u d i n ( 1 1 - 1 3 ) , a n o t h e r l e e c h (Hirudo medicinalis) d e r i v e d a n t i c o a g u l a n t which i n h i b i t s t h r o m b i n (14). A n t i s t a s i n a l s o i n h i b i t s e x p e r i m e n t a l l y i n d u c e d tumor c e l l m e t a s t a s i s i n a mouse model system ( 1 0 , 1 5 ) . A n t i s t a s i n i s t r a n s l a t e d as a p r e p r o t e i n w i t h a s i g n a l p e p t i d e o f 17 amino a c i d s (6). The mature p r o t e i n has 119 amino a c i d r e s i d u e s , has no N - l i n k e d g l y c o s y l a t i o n s i t e s and has a 17% c y s t e i n e c o n t e n t . There i s a s i g n i f i c a n t t w o - f o l d homology between t h e N- and C- t e r m i n a l h a l v e s , w i t h t h e a c t i v e s i t e residing i n the N-terminal h a l f (16). A n t i s t a s i n p u r i f i e d from n a t u r a l l e e c h p o p u l a t i o n s c o n t a i n s m u l t i p l e p r i m a r y sequence v a r i a n t s (16); two o f t h e v a r i a n t s have been c l o n e d and e x p r e s s e d i n t h e i n s e c t b a c u l o v i r u s e x p r e s s i o n v e c t o r system ( 6 ) , as has a t r u n c a t e d gene e x p r e s s i n g a p o r t i o n o f one v a r i a n t ( " h a l f - a n t i s t a s i n " , J.S. Tung e t . a l . , manuscript i n p r e p a r a t i o n ) . One o f t h e v a r i a n t s was a l s o e x p r e s s e d i n y e a s t and i n a mammalian c e l l l i n e , GH3, a r a t p i t u i t a r y tumor l i n e (17). The i n i t i a l y i e l d s o f a n t i s t a s i n v a r i a n t 1 were h i g h e s t i n t h e i n s e c t - b a c u l o v i r u s system (about 1.7 f o l d b e t t e r t h a n i n t h e y e a s t system and about 3 . 5 f o l d b e t t e r t h a n t h e mammalian s y s t e m ) . The b a c u l o v i r u s system was t h e r e f o r e s e l e c t e d t o s t u d y t h e p r o d u c t i o n o f a n t i s t a s i n v a r i a n t s as w e l l as h a l f - a n t i s t a s i n (H-ANS). I n o n g o i n g s t u d i e s , b o t h in vivo and in vitro, t h e b a c u l o v i r u s - p r o d u c e d homogeneous f u l l a n t i s t a s i n p r o t e i n has been found t o be p h a r m a c o l o g i c a l l y v e r y s i m i l a r t o t h e l e e c h - d e r i v e d p r o t e i n (C. Dunwiddie, P. S i e g l and G.Vlasuk, p e r s o n a l communication). A number o f r e p o r t s have been p u b l i s h e d on t h e l a r g e - s c a l e c u l t i v a t i o n o f i n s e c t c e l l s i n b a t c h and c o n t i n u o u s suspension c u l t u r e (18-20), i n s t i r r e d fermenters (21) and i n a i r l i f t fermenters (22-25). There have been r e p o r t s t h a t i n s e c t c e l l s , l i k e many a n i m a l c e l l l i n e s , a r e s h e a r - s e n s i t i v e ( a g i t a t i o n and/or gas s p a r g i n g ) ( 2 0 , 2 3 , 2 4 , 2 6 , 2 7 ) , a r e a f f e c t e d by t h e p r e s e n c e o f foam and a i r b u b b l e s (22-24,28) and c a n be p r o t e c t e d by t h e p r e s e n c e o f P l u r o n i c F68 (24,28-35) and by c o n t r o l l i n g t h e s i z e o f gas b u b b l e s (25). I t a l s o has been r e p o r t e d t h a t i n s e c t c e l l s have a r e l a t i v e l y h i g h oxygen demand i n comparison t o most mammalian c e l l s (25,36) . We i n i t i a l l y d e v e l o p e d a s c a l a b l e i n s e c t b a c u l o v i r u s s u s p e n s i o n c u l t u r e p r o c e s s f o r the p r o d u c t i o n o f a n t i s t a s i n , which was s u b s e q u e n t l y found t o work e q u a l l y w e l l f o r the g e n e t i c a l l y e n g i n e e r e d Η-ANS. In t h i s p a p e r we p r e s e n t s t u d i e s on t h e

In Expression Systems and Processes for rDNA Products; Hatch, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

8. JAIN ET AL. production centered

of

on

of

oxygen

uptake

have of

Η-ANS

the

growth

Pluronic

99

Antistasin Production Using Baculovirus System

the F68

cells

batch

for

of

as

rate

verified

sparging

in

effect

suspension

dissolved

well

as

the

its

reported

either

cell

(DO)

that

there or

investigation on

the

protein

and

accumulation.

"protective"

growth

Our level

production of

(OUR) d u r i n g p r o d u c t

experimentally

on

culture.

oxygen

properties

is

no

(29)

used

and

deleterious

protein

on

We h a v e

effect

p r o d u c t i o n under

our

conditions.


M a t e r i a l s and Methods Cell

line.

A clonal

isolate,

the

cell

line

the

Fall

A r m y Worm Spodopetra

ATCC

(#CRL

Virus.

1711)

(AcMNPV)

standard

culture

Gibco,

Grand

and

3.3

g/1

for

both

F68

IPL-41 basal NY),

in

liquid

NJ)

to

to

the

insect

NY)

volume

then cell

effect"

of

and l a r g e r

spinner-flasks

volume/volume/min.;

50% a n d

of

100%

air

in

culture

upon

50-60

seeding

flasks 100

of

every

as

the

and oxygen

was

used

to

keep

off-line

4 due

of

cell

head-space

used

gassed

starting the

DO

gas level

measurements

below). Two

fermenters

"Biostat

utilizing

were

E " fermenter

a bubble-free

used

from B.

silicone

for

growth

Braun tube

of

insect

Biotech

gassing

cells,

(Bethlehem,

system

3-

exceed

passage

was

on

density

variation

air

a i r - s a t u r a t i o n based

stir rpm.

to

density

were

ml

culture

started

long-term

For higher

flasks

Glass,

allowed were

at

spinner

8 liters.

experimental

(38,39).

was 20%

M O ) . Thawed tissue

spinner

at

a

not

"aging

ml

8-L

to

cells

from t i s s u e

at

was

suspension

a mixture

all

Iowa)

in

0.1

Sf9

2

(Bellco

cultures

seen

cm

ml to

propagated density

possibility

of

slow-speed magnetic

Dubuque,

250

(see

used

g/1

added

Louis,

75

directly

28 ° C o n

at

between

MI)

was

1.0

was

culture

reported

then

MI)

I P L - 4 1 / 2 % FBS medium

f r o m 100

seeded

at

were

the

in

St,

investigation,

culture, and

(FBS,

Detroit,

I P L - 4 1 medium w i t h in

cells

about

Scientific,

that

NJ)

stock

in

(Sigma,

Fresh suspension

eliminate

seed

suspension

Thermolyne,

Maximal

cells/ml.

to

were

and grown

cultures

cells/ml.

weeks

by

Serum

Detroit,

microcarrier-type

in

cultures

(Barnstead/

6

polyhedrosis

(Difco,

except

culture

Corning,

For this

ranging

flask

2.0xl0

of

the

generated

Bovine

Parsippany,

static

transferred

cultures

5

cultures,

10% DMSO

in

paddle-impeller

Suspension 4xl0

were

same m e d i u m .

flask

from

(from J . R .

Fetal

(Difco,

nitrogen

and then

plates

was

yeastolate

working

in

(Corning Glass,

spinner

g/1

vials

FBS a n d

Vineland,

Η-ANS

medium

A cryopreserved

grown

first

the

3.3

and suspension

cells

the

for

2% h e a t - i n a c t i v a t e d

heat-inactivated

in

gene

(BASF C o r p o r a t i o n ,

growth.

were

nuclear

californica

the

obtained

of

tissue

cultures.

maintained

28°C,

was

ovary

source.

lactalbumin hydrolysate

suspension

flasks

cell

unpublished),

from pupal

(3).

Island,

static

Pluronic

(M. Summers,

derived

frugiperda

Autographa

medium.

CA) w i t h

Sf9

(37) as

containing

procedures

Woodland,

Cell

and used

Recombinant

virus

Cell

IPLB-Sf21-AE

and an


an PA) 18-1

100

EXPRESSION SYSTEMS & PROCESSES FOR

RDNA PRODUCTS

Sulzer-MBR (Woodbury, NY) " S p i n f e r m " u s i n g a 2 Urn (pore s i z e ) s t a i n l e s s s t e e l m i c r o s p a r g e r f o r d i r e c t s p a r g i n g o f oxygen. V i r u s s t o c k p r o d u c t i o n . V i r a l s t o c k s were made by growing Sf9 c e l l s i n s p i n n e r f l a s k s (3; G i b c o , Grand I s l a n d , NY) and i n f e c t i n g them w i t h recombinant b a c u l o v i r u s (AcMNPV) c o n t a i n i n g t h e gene f o r Η-ANS. C e l l s were grown t o a d e n s i t y o f l . O x l O c e l l s / m l u s i n g TNMFH/10% FBS medium, and v i r u s was added a t an MOI o f 0.1 ( e.g., 10 ml o f v i r u s s t o c k h a v i n g a v i r a l t i t e r o f 10 p l a q u e f o r m i n g u n i t s (PFU)/ml added p e r l i t e r o f c u l t u r e ) . C u l t u r e medium c o n t a i n i n g t h e v i r u s was h a r v e s t e d between 72 and 96 hours p o s t i n f e c t i o n (based on 50% l o s s o f v i a b i l i t y ( t r y p a n b l u e e x c l u s i o n method; below) i n t h e c u l t u r e ) . V i r a l t i t e r was o b t a i n e d by endp o i n t d i l u t i o n (3). 6


7

P r o d u c t i o n o f H-ANS. Η-ANS was produced i n b o t h l a r g e (up t o 5 l i t e r ) g a s s e d s p i n n e r f l a s k s and f e r m e n t e r s ( d e s c r i b e d a b o v e ) . C e l l s were grown t o a d e n s i t y o f 1 . 5 x l 0 c e l l s / m l i n IPL-41/2% FBS medium and i n f e c t e d w i t h v i r u s . The c e l l s were m o n i t o r e d f o r change i n modal d i a m e t e r and/or l o s s i n v i a b i l i t y (below). The c u l t u r e was h a r v e s t e d w i t h i n 24 hours o f when t h e modal c e l l d i a m e t e r peaked o r when t h e c e l l v i a b i l i t y dropped t o 50%. 6

C a l c u l a t i o n o f oxygen uptake r a t e (OUR). OUR c a l c u l a t i o n s i n the Braun f e r m e n t e r were made u s i n g t h e f o l l o w i n g p r o c e d u r e . The gas m i x t u r e s u p p l y ( a i r , n i t r o g e n and oxygen) t o t h e f e r m e n t e r was f i r s t t u r n e d o f f . In t h e c a s e o f t h e Braun f e r m e n t e r , t h e s i l i c o n e t u b i n g was purged w i t h n i t r o g e n t o e l i m i n a t e any r e s i d u a l oxygen t r a n s f e r d u r i n g t h e measurement, and t h e exhaust was c l o s e d . F o r t h e S u l z e r f e r m e n t e r , t h e head space was purged w i t h n i t r o g e n and t h e exhaust was c l o s e d . The i n i t i a l DO r e a d i n g was n o t e d . The drop i n DO w i t h time was o b s e r v e d and n o t e d e v e r y 30 seconds f o r 5 m i n u t e s . The s l o p e o f t h e DO vs time c u r v e was used t o c a l c u l a t e the s p e c i f i c OUR. Assay Methods. Η-ANS a c t i v i t y was measured by a c o l o r i m e t r i c F a c t o r Xa i n h i b i t i o n a s s a y u s i n g a commercial (Helena L a b o r a t o r i e s , Beaumont, Texas) F a c t o r Xa a s s a y k i t (G. Cuca, p e r s o n a l communication). The a s s a y was performed i n a 9 6 - w e l l m i c r o - t i t e r p l a t e w i t h a Bio-Rad (Richmond, CA) k i n e t i c m i c r o p l a t e r e a d e r . Η-ANS a c t i v i t y i s r e p o r t e d here as n o r m a l i z e d r e l a t i v e units. G l u c o s e , ammonia and l a c t a t e dehydrogenase were measured w i t h an Ektachem A n a l y s e r Model DT60 e q u i p p e d w i t h a DTSC module ( f o r enzyme a n a l y s i s ) from Kodak (Rochester, NY). O f f - l i n e DO measurements were made w i t h a B l o o d Gas A n a l y s e r from Radiometer, Copenhagen. O n - l i n e measurements were made w i t h a p o l a r o g r a p h i c DO probe from I n g o l d (Wilmington, MA). C e l l c o u n t s were made u s i n g a haemacytometer (AO S c i e n t i f i c I n s t r u m e n t s , B u f f a l o , NY). V i a b i l i t y c o u n t s were made by a t r y p a n - b l u e dye e x c l u s i o n method. A 1:1 d i l u t i o n o f the sample was made w i t h t r y p a n - b l u e (0.4% i n 0.85% s a l i n e , G i b c o , Grand I s l a n d , NY), and c e l l s t h a t p i c k e d up t h e b l u e s t a i n were s c o r e d as n o n - v i a b l e . C e l l s i z e d i s t r i b u t i o n (modal c e l l diameter) was d e t e r m i n e d w i t h a C o u l t e r Counter ( H i a l e a h , FL) Model ZBI


8. JAIN ET AL.

Antistasin Production Using Baculovirus System

101

o u t f i t t e d w i t h a c h a n n e l i s e r , and d a t a were a n a l y s e d on an IBM A T c o m p a t i b l e computer. P a r t i c l e s i z e s between 8 and 21 μια were considered f o r c e l l s i z e determination with t h e C o u l t e r counter.


Results The e f f e c t o f s p a r g i n g on Sf9 c e l l growth was s t u d i e d by growing c e l l s i n two d i f f e r e n t f e r m e n t e r s , one u s i n g b u b b l e - f r e e s i l i c o n e t u b e g a s s i n g (Braun) and t h e o t h e r u s i n g d i r e c t oxygen s p a r g i n g (Sulzer-MBR). A 250 ml s p i n n e r f l a s k e q u i p p e d w i t h head-space g a s s i n g was used as a c o n t r o l . F i g u r e 1 compares growth o f c e l l s i n t h e t h r e e systems. Growth o f c e l l s was s i m i l i a r , and t h e r e was no a p p a r e n t d e l e t e r i o u s e f f e c t o f s p a r g i n g . S p e c i f i c growth r a t e was i n t h e range o f 0.024 t o 0.026 h r ( c e l l d o u b l i n g time o f about 2 7 . 5 h o u r s ) . The l e v e l o f DO i n t h e c u l t u r e medium had a d r a m a t i c e f f e c t on t h e growth r a t e o f c e l l s i n t h e f i r s t 90-100 hours o f growth, as shown i n F i g u r e 2. A t a low DO l e v e l o f 10% a i r s a t u r a t i o n , and a t h i g h DO l e v e l o f 110% a i r s a t u r a t i o n maximum s p e c i f i c growth r a t e was slower (about 25%) t h a n a t a DO l e v e l o f 65% a i r s a t u r a t i o n . We had o b s e r v e d i n i n i t i a l e x p e r i m e n t s i n s p i n n e r f l a s k s t h a t t h e maximum s p e c i f i c growth r a t e i s o b t a i n e d i n t h e f i r s t 90-100 hours o f growth. Growth r a t e s i n f e r m e n t e r s were, t h e r e f o r e , n o t m o n i t o r e d beyond 100 h o u r s . I t i s s i g n i f i c a n t t h a t t h e v i a b i l i t i e s seemed t o be s i m i l i a r (ca. 98%) a t t h e t h r e e DO c o n d i t i o n s (data n o t shown), i m p l y i n g t h a t t h e reduced growth r a t e o f t h e c e l l s i s p r o b a b l y n o t t h e r e s u l t o f an i n c r e a s e i n c e l l d e a t h b u t a d i r e c t e f f e c t o f DO c o n c e n t r a t i o n on growth. In o u r e a r l y e x p e r i m e n t s w i t h a n t i s t a s i n v a r i a n t 1, h i g h e s t p r o d u c t a c c u m u l a t i o n was o b t a i n e d when t h e m u l t i p l i c i t y o f i n f e c t i o n (MOI) was l e s s t h a n 1.0 ( u n p u b l i s h e d o b s e r v a t i o n ) . However, w i t h t h e Η-ANS b a c u l o v i r u s v e c t o r , peak p r o d u c t y i e l d was u n a f f e c t e d by MOI. A t y p i c a l experiment f o r Η-ANS p r o d u c t i o n a t d i f f e r e n t MOI i s shown i n F i g u r e 3; a t an MOI o f 0 . 1 , 0 . 5 , 2.0 o r 5 . 0 , p r o d u c t y i e l d s were s i m i l i a r i n 250 ml s p i n n e r f l a s k c u l t u r e s . Thus, t o c o n s e r v e v i r u s , a l l subsequent p r o d u c t i o n e x p e r i m e n t s used an MOI o f 0 . 1 . S i n c e b a c u l o v i r u s e s a r e l y t i c v i r u s e s , h a r v e s t time s e l e c t i o n c a n be c r i t i c a l f o r p r o d u c t y i e l d . We had p r e v i o u s l y examined m o n i t o r i n g o f a number o f p a r a m e t e r s as i n d i c a t o r s o f h a r v e s t time, i n l i e u o f t h e r e l a t i v e l y t e d i o u s o f f - l i n e p r o d u c t a s s a y , and found a s t r o n g c o r r e l a t i o n ( i n s p i n n e r f l a s k s ) between t h e i n c r e a s e i n modal c e l l d i a m e t e r (as measured by a C o u l t e r Counter) and a n t i s t a s i n p r o d u c t i o n (40, S. G o u l d e t . a l . m a n u s c r i p t i n p r e p a r a t i o n ) . T h i s c o r r e l a t i o n s u b s e q u e n t l y was found t o be c o n s i s t e n t between e x p e r i m e n t s i n s p i n n e r f l a s k s and i n f e r m e n t e r s f o r Η-ANS p r o d u c t i o n . I t was o b s e r v e d t h a t , t y p i c a l l y , modal c e l l d i a m e t e r peaked about 24 hours b e f o r e maximum a n t i s t a s i n o r Η-ANS p r o d u c t i o n . C e l l v i a b i l i t y , though not as a c c u r a t e , i s much more c o n v e n i e n t t o f o l l o w and a l s o c o r r e l a t e s w e l l w i t h p r o d u c t a c c u m u l a t i o n . I t was found i n numerous e x p e r i m e n t s t h a t a 50% drop i n c e l l v i a b i l i t y c o i n c i d e d w i t h t h e peak i n a n t i s t a s i n o r Η-ANS p r o d u c t i o n . F i g u r e 4 i l l u s t r a t e s t h e r e l a t i o n between t h e k i n e t i c s o f Η-ANS a c c u m u l a t i o n and i n c r e a s e _ 1


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