erythropoietin produced using a baculovirus vector ...

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1989 74: 652-657

High-level expression and purification of a recombinant human erythropoietin produced using a baculovirus vector FW Quelle, LF Caslake, RE Burkert and DM Wojchowski

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High-Level

Expression

and

Purification

Produced By Frederick Conditions

presently

have

W. Quelle. been

of a Recombinant

Using

Laurie

established

a Baculovirus

F. Caslake, for

the

Rebecca 80%

high-

level expression and simplified purification of recombinant human erythropoietin produced in Spodoptera frugiperda cells. Expression. as mediated by infection with a recombinant baculovirus. was accomplished in suspension culture using reduced levels of serum and media supplements experimentally determined to provide optimum levels of factor production (500.000 U/I). Purification of this recombinant human erythropoietin to virtual homogeneity (:99%) was accomplished via a simple three-step procedure

involving

isocratic

reverse-phase

high

(HPLC)

on

purified

hormone

890-fold

a C4

elution

performance

medium.

from

purification

and

from

DEAE-Sephacel.

liquid

chromatography

the

single-step

concanavalin was

A agarose.

accomplished

with

in

is

circulating

have select

marrow,2

spleen,3

required

for

erythrocytes.

fetal

liver,4

terminal

of

natural

source.

thropoietin has been human erythropoietin tion

of

active

expression recombinant

erythropoietin

erythropoietins

contain

‘

and

of

this

26,200) apparently oligosaccharides biological

is glycosylated of only limited

activity

relatively

as

assayed

simple

conditions

as

for

well

as

a

In contrast

weight

in vitro.

expression

a simple

we of this

and

[mol

wt

with N-linked possesses high on now

its have

erythropoietin

efficient

wt]

(mol

Based

physicochemistry,

derived 40%

distinct estabat

procedure

a

the

Submitted Supported DK40242. Address vania

high

for

College

Department of Molecular and Cell Biology. State University. University Park, PA. November 22. 1988; accepted April 6, 1989. in part by National Institutes of Health Grant to Don M. Wojchowski. Department ofMolecular

requests

University,

of Science.

102

5 Frear

Laboratory,

its

PhD,

“advertisement”

costs ofthis This article in accordance

indicate this fact. © 1989 by Grune

0006-4971/89/7402-0039$3.00/0

652

cells

(mol

as

erythro-

wt

30.000

oligosaccharide).

to Thus.

uniquely

to

homogeneous

form

homogeneity.

that

should

prove

analyses.

Thus,

this

erythropoietin

is

studies of its cell surface receptor mechanism of action. In addition,

and

homogeneous

and

chemistry

structural

and its

should

prove

analyses.

culture

METHODS of

recombinant

erythropoie-

cells (ATCC) were maintained at 28#{176}C in TMN-FH medium,’6 10% fetal calf serum (FCS). For the expression of recombinant erythropoietin, cells were grown in spinncr flasks to a density of 106 cells/mL, collected by centrifugation, and incubated at l0 cells/mL for one hour with clonal recombinant baculovirus.’0 Following subsequent culture for three days in Grace’s medium, 4% FCS, 20% TMN-FH, spent medium was collected, concentrated 15-fold (tangential flow system, 10,000 mol wt PLGC Spodoptera

and

filter)

frugiperda

dialyzed

against

1 mmol/L

Na2EDTA,

10

mmol/L

PIPES, pH 6.8. Purification of erythropoietin. Concentrated media initially was passed over a column of DEAE-Sephacel (Pharmacia, Piscataway, NJ) (1 mL gel/mL dialyzed media) under isocratic conditions in 0.02% octyl-beta-n-glucopyranoside, 10 mmol/L K2PIPES, pH 6.80. Column eluate and two column volumes of buffer wash were collected,

pooled,

subjected

directly (HPLC)

tography cm

and

concentrated

40-fold.

Concentrate

then

was

to reverse-phase high performance liquid chromaon a C4 medium (Supelcosil LC-304, 25 cm x I Samples were applied in 0.06% trifluoroacetic acid as

column).

mobile

phase,

and

were

chromatographed

using

a linear

to

fractions contain

of acetonitrile fractions were that

were

shown

erythropoietin

MgCI2,

PA

applied

to a column

by bioassay were

1 mmol/L

beta-D-glucopyranoside,

PennsylPark,

AND

expression

No.

article were defrayed in part by page must therefore be hereby marked with 18 U.S.C. section 1 734 solely to Inc.

excep(30%)

glycosylated

complex-type

Cell

MnCl2, 10

and

by Western

resolubilized

mmol/L

ofconcanavalin

in

I mmol/L

CaCl2,

PIPES A agarose

blot

analyses

I mol/L pH

NaC1,

0.02% 6.8,

(Sigma

I

octyl-

and

were

Chemical

Co.

St Louis)

(approximately 1 mL gel/mg protein). The column was washed with 100 mL of the above buffer, and erythropoietin was eluted in buffer containing 250 mmol/L alpha-methyl-D-mannoside. Subsequently, this eluate was dialyzed at 4#{176}C against 0.01% octylbeta-D-glucopyranoside. Biologic

& Stratton,

appears

constitution

The

16802.

The publication charge payment.

26.200)

in

(30% to 50%) in 0.06% trifluoroacetic acid. Eluted collected into vials containing 5 ML of 1% octyl-beta-n-glucopyranoside, and were dried under vacuum. HPLC

and Cell Biology,

University

wt]

mammalian

50%

in direct

mmol/L reprint

State

from

to

simple

gradient

From

[mol

heterogeneously

advantageous

an initial

Pennsylvania

with

erythro-

human erythropoietin as presently produced in an insect cell line comprises not only an abundant source of highly active. readily purified hormone for studies of its mechanism of action and cell surface receptor. but also repro-

tin.

mammalian

erythropoietin

uniformly size, yet

weight

in its oligosaccharide

MATERIALS

as expressed in mammalian amounts of heterogeneous, comhuman

poietins 38.000;

from most

previously have developed that mediates the production

(molecular

contrasted

this purified

possessing a specific activity protein. Chemically. this erythro-

factor

which,

recombinant

uniform

uniquely

of cry-

cells.’0

(molecular

Biologically.

active. U/mg

highly suitable for presently unresolved

to

this

availability

insect

poietin tionally

purification

subse-

urine the

using

in vitro.

M. Wojchowski

sents

through the isolation of the cDNA,5”#{176} and the produc-

in cultured

oligosaccharide

35,000),”

the

hormone We vector

substantial

plex-type

levels,

gene9

recombinant

to human

lished

improved

systems.9” baculovirus

of human

cells,

Recently,

as assayed

poietin is highly vitro of 200.000

Don

advantageous for direct structural ‘a 1989 by Grune & Stratton. Inc.

and

studies

and

of

differentiation

Historically,

E. Burkert,

an

a recovery

been limited by its low availability with patients with aplastic anemia comprising

abundant

and

and

their

Vector

elution

RYTHROPOIETIN is a prime regulator of RBC production in mammals and birds.’ Specifically, this glycoprotein hormone promotes the rapid growth of red cell progenitors

Erythropoietin

Overall.

E

quently

Human

assay

of erythropoietin.

The

biologic

activity

of van-

ous preparations was determined by quantifying the erythropoietindependent incorporation of [3H]thymidine into splenocytes derived

Blood,

Vol

74,

No

2 (August

1),

1989:

pp 652-657


RECOMBINANT

HUMAN

653

ERYTHROPOIETIN

Table

Step

Purification Media

500,000

500,000

C4/Resolubilization

Estimated protein

Processed per Step

Concentration/DEAE

Concanavalin

A

via laser densitometri

and

biological

c analysis

of Recombinant

1 . Purification

Units

Erythropoietin

Recovery

Purification Overall

-

-

>95% >90%’

810

180,000

U/mg

890

200,000

U/mg

before

and after C4 chromatography.

Otherwise,

recoveries

(>95%

protein

ofrecombinant

of

initial

In experiments

designed

amounts Expression

poietin mol

to maximize

below, using

weight

poietin. poietin

these

conditions alternate

wt

24,000

and

in part against

increase

in levels

this

simple

procedure

provides

menting

media of

with

the

Material Sephacel

Evidence conditions

that was

immunoreactive Purification

and

levels

of recombinant

homogeneity steps. First,

was accomplished erythropoietin-containing

1 ) was of

(0.02% pH

6.8).

in

on

concentrated,

cell

recovery

biologically in

any

and

octyl-beta-D-glucopyranoside, eluted

with

of active

other

portion

of the

similar Analysis

of this

material

proteins

erythropoietin,

which

gels

1). No

was detected

profile.

Routinely,

the

U per run resulted of hormone (Table

by SDS-PAGE

contaminating

(Fig

hormone

elution

of 60,000 U to 100,000 elution pattern and recovery

injection

hormone

active

showed

that

several

co-eluted from C4 medium was estimated by densitometry

to

represent

erythropoietin

1 5%

of

represented

total

in a I). with of

protein.

5% of total

By protein

C4

HPLC. Thus, this step provided a threefold A sixfold further purification of erythropoietin accomplished simply by reconstituting this material in

was buffer

containing

Under

these were were

contrast,

as high

NaCl

conditions insoluble

removed

at a high

readily

as 200,000

U/mL

fully

as was

(ie,

l

of contaminating 85% of total

by brief was

erythropoietin

at increasing

molarity

the majority (approximately

proprotein)

microcentrifugation. soluble and

In

at concentrations

established

concentrations,

mol/L).

by its reconstisubsequent

biologi-

at

culture.

40 Percent

The to virtual

passed

over conditions

10 mmol/L directly

of >95%

or immunologically

by an

U/L

three chromatographic media (500,000 U/L, isocratic

chromatography on DEAEand was chromatographed

under these amounts of

erythropoietin

under

Thus, purifica-

by supple-

hydrolysates

routine

dialyzed,

45-fold

cells

in cell lysates. human erythropoietin. via

initial

of the

retained.

C4 medium. As established eluted in 44% acetonitnile, with

a reverse-phase

erythropoietin

estimated

tution

shown

(ie, 500,000

efficiently of detectable

human

98%

was

a significant

achieved

lactalbumin used

DEAE-Sephacel

Erythropoietin

Also,

1) was

secretion proceeded given by the lack

purification

column

FCS.

erythropoietin

yeast

As

lower

erythro-

erythropoietin ofrecombinant

Table

22,000).

an

recovered following was concentrated,

bioassay,

and

of a

of

form of recombinant expressed by Sfrugiperda

Table

nominal

composed forms

media

tion.

teins

erythro-

analyses erythro-

4%

ofsecreted

protein cells in

by Western blot purified human

containing

postinfection;

expression

Also, was

two

In contrast, a singular (mol wt 26,000) was

at 72 hours

by direct

purification.

supplements were FCS at concentra-

substantially.

established prepared

in media

the

with reduced Sfrugiperda

plus

(mol

cultured

20%

diminished

form,

this was antibodies

in media infected

FCS and media in media containing

under

26,000

molecular

of

was

produced wt

were measured

approximately

while

in dialyzed

before

by S frugiperda

activity),

contained

comparison,

erythropoietin

of recombinant erythropoietin content, the effects of culturing

of 95%

assay. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method of Laemmli’9 in 12% gels. In specified instances, laser densitometry of silver-stained gels was used to verify purification factors and recoveries. Western blot analyses were performed essentially as described by Towbin#{176} using antibodies prepared against recombinant human erythropoietin produced in CHO cells. Protein assays were performed using BCA reagent (Pierce Chemical, Rockford, IL) and chromatographically purified BSA (Cappel, Malvern, PA) as a standard.

limiting

230 10.000

45

60.000

mice’8

cells.

Activity

assays.

Electrophoresis

Expression

Specific

300,000

from mice treated with phenylhydrazine.3 Assays were standardized against the World Health Organization Second International Reference Preparation, calibrated recombinant human erythropoietin standards, and purified recombinant human erythropoietin produced in CHO cells (160,000 U/A2, Amgen, Thousand Oaks, CA). The activity of purified hormone also was assayed in vitro using a CFU-e colony forming assay’7 and in vivo in ex-hypoxic, polycythemic

Human

K2PIPES, high

recovery

a

Acetonitrile

Fig 1 . Reverse-phase chromatography of lowing DEAE chromatography. erythropoietin was applied to a reverse-phase (C4) HPLC developed with a linear gradient of acetonitrile mm. 1 mI/mm flow rate). The elution of protein absorbance at 254 nm (-). The concentration various fractions (0.2 ml) was determined (-.-).

erythropoietin. Fol(350 U in 0.1 ml) column. and was (3.9% acetonitrile/ was monitored by of erythropoietin in by biological assay


654

QUELLE

from and

C4 HPLC was adsorbed purified erythropoietin

alpha-methyl-D-mannoside.

The

>99%

(200,000

(Table

1, Fig 2). The

by in vitro assay.

U/mg

However,

little,

if any,

activity

under

above, 20%

product a CFU-e

ex-hypoxic,

Analysis of purified erythropoietin by SDS-PAGE. The of erythropoietin as isolated by chromatography on DEAESephacel. C4 medium. and concanavalin A agarose was assessed by SDS-PAGE (12% gel. reducing conditions) and silver-staining. Scanning laser densitometry (right panel) was used to confirm purity (>99%). Molecular weight standards (mol wt x i0) are indexed in the left margin.

and subjecting Western blot

cal

between

assay.

typically

Final virtual lectin

A

Thus,

the

>90%, purification was

homogeneity affinity

purity

of this

reconstituted

preparation

with a recovery of >90% (Table of this recombinant erythropoietin

of erythropoietin

ofculture.

culture

was

procedure.

accomplished

using

Reconstituted

to

a single-step

material

(ie,

of recombinant

4%

serum,

human

ery-

by S frugiperda molecular weight

form

was shown

gel analysis

slice (Fig

to approximate

eluates to 3). Biological

equivalent and 24,000

these

two

ments in which with N-glycanase.

mol

glycosylation

these purified Treatment

weight

of each

of the

biological assay assay indicated

activities were erythropoietins

higher

to differential

that

This was shown by separating eluting each from gel slices,

associated (Fig 3B).

blotting confirmed the efficient separation plus their approximately equivalent abundance from gel slices (Fig 3A). Evidence that

molecular

derived

pro-

As mentioned

conditions

form

erythropoietin. by SDS-PAGE,

approximately mol wt 26,000

primarily

1).

polypossesses

produced under alternate conditions of culture (see was of some interest. First, the biological activity of

mol wt 26,000 the two forms Fig 2.

determined

erythropoietin

forms

conditions

a single

the mol wt 24,000

purity

was >95%

colony-forming

thropoietin (mol wt 26,000) was produced cells. However, the nature of the two lower forms above)

of

in vivo.

optimized

TMN-FH),

material

was

using

this

of minor

alternate

under

assays

A agarose, mmol/L

a recovery

and

that

250

of this

with

of this

assays

indicate

Characterization duced

activity

preliminary

mice

purity

protein),

proliferation

cythemic

to concanavalin was eluted in

ET AL

form

wt

and that

with the Western

of these forms, and recovery the difference

forms

is

was

given

erythropoietins with N-glycanase to the approximate

attributable by experiwere treated reduced the molecular

262422-

5

4

6

Gel

7

Slice

8

9

10

Number

B

8) U

Fig 3. Isolation of mol wt 26.000 and 24.000 erythropoietins and comparison of specific bioactivities. Erythropoietin expressed under low serum conditions (1 % FCS) and purified via reversephase HPLC (approximately 90% purity) was subjected to SDSPAGE in the absence of 2-mercaptoethanol. Alternate forms were visualized by Western blotting (A. lane A). A parallel gel lane was sectioned into 1 mm slices. and erythropoietins then were eluted into 0.5 ml of phosphate-buffered saline containing 0.02% Tween20 and 0.1 mg/mI BSA. Eluates then were subjected to biological assay (B). and to Western blot analysis (A) in order to confirm

(I) 8)

0

c

Gel

Slice

Number

recoveries

and

separation.


RECOMBINANT

HUMAN

655

ERYTHROPOIETIN

(-)

(+)

DISCUSSION

Systems binant

previously developed erythropoietin

for the production have depended on

human

mammalian

expression

including

COS

hamster

ovary

systems

cells,5’78

baby

cells6”’3

and

and

psi-2

cells.9

erythropoietin, recombinant mammalian cells is heterogeneously plex

and

N-

0-linked

of total

poietin

as presently

tional

in both

and

its

prepared

limited

capacity

26

chanides neutral

to complex oligosaccharides

24

prospect

that

18.9 18A

A

simple

relative of insect

cells

saccharide

at least presently

certain

general,

N-linked

the

oligosac-

in a predominance mannose type.24’25

oligosacchanides

prepared

erythrois excep-

constitution, In

to process

types results of the high

as much

human cells

homogeneity.

with

produced in with com-

comprising

In contrast,

are

of

lines

Chinese

As is the case

in Sfrugiperda

its comparably

associated

erythropoietin

mass.

cells,7’9

hormone glycosylated,

oligosaccharides

molecular

of cell

kidney

urinary

as 50%

a variety

hamster

of recomthe use of

of the this

type

of The

human is sup-

0.900

#{182} 0.800 o

0 700

0

0.600

.

Fig 4.

of N-glycosylation of mol wt 26,000 and 24.000 erythropoietins using N-glycanase. Purified erythropoietins (350 U) expressed under low serum conditions (1 % FCS) were incubated with N-glycanase (3 U) in 0.25% SDS. 0.25 mol/L phosphate pH 8.6. at 37’C for 18 hours. Products (+) were analyzed by

SDS-PAGE ized

in the absence

of 2-mercaptoethanol. and were sample ( - ) was processed in the absence of N-glycanase.

by silver-staining.

same

15

20

25

30

35

40

Analysis

manner

visual-

A control

Stokes

B

?

Radius

()

3.500 3.000

0

in the 2.500 0

2.000 E

weight

of

aglycosyl

form

(Fig

is N-glycosylated,

and

beyond its secreted form. ence in the electrophoretic products

(mol

for this

difference

bind

is not

processed

was

1 8,400) that

as

1.000

proteolytically

The

Fig 4, lane

The third

A)

was

C 0

S 0

basis

form

of

found

to

if at all, to concanavalin A agarose. Due to its low abundance, the specific biological activity of this form has not been estimated directly. However, based on the above this

nm)

onds)

of the

(Fig from

5, Table

based

to

and

(2.56

content

erythropoietin

oligosaccharide

In order weight

0.500

5

1

2

S20.w

3 (sec

4

X 1013)

poorly,

observations, distinct

1.500

.‘

each

residual differendoglycolytic

detectable.

is unresolved.

wt 22,000,

wt

indicating

a minor of these

v 18,400)

presently (mol

4), thus

However, mobilities

wt 18,900

erythropoietin

(mol

apo-erythropoietin

by SDS-PAGE

determined

these

provide

relative and

is believed

independent saccharide

sedimentation

purified

mol

(30%)

estimates content,

a third

wt

is in approximate

26,000 weight estimate

of molecular

the

coefficient

2). A molecular values. Thus, the

on SDS-PAGE.

to possess

constitution. Stoke’s

(2.48 form

were

of 26,200 of relative

accordance

radius

x iO’

with

sec-

determined is predicted sacchanide estimates

Fig 5. Determination of Stoke’s radius and sedimentation coefficient of human erythropoietin produced in S frugiperda cells. The Stoke’s radius (A) of insect erythropoietin was evaluated by HPLC gel filtration chromatography (G2000 SWXL 30 cm x 0.78 cm column, Supelco). Column calibration was performed using DNA (Sigma type Ill). bovine serum albumin. ovalbumin. and cytochrome C standards (O-O).’ The elution of standards was monitored by absorbance at 254 nm. The elution position of erythropoietin (#{149}; 2.56 nm Stokes radius) was confirmed by Western blot analysis. Evaluation of the sedimentation coefficient (2.48 x 10’3sec; B) was estimated by rate sedimentation in a 5% to 20% sucrose density gradient. Purified erythropoietin (#{149}) was sedimented with standards (O-O; bovine serum albumin and cytochrome C) (18 hours at 40.000 rpm in a SWSO.1 rotor). Fractions (0.16 ml) were collected and analyzed by SDS-PAGE.


QUELLE

656

Table

2.

Physicochemical

Properties

of Human

in S fugiperda

Produced

Sedimentation

coefficient

Calculated

molecular

Molecular

weight

Saccharide

based procedures

2.56

nm’

2.48

x iO’

see’

tional

by SDS-PAGE

26,000

by the development ever, this approach

on measured

calculated

molecular

stokes

radius

and

sedimentation

on

weight

less the predicted

weight

of

dure

by its comparably 30%) and reactivity

The apparent high uniformity wt 26,000 erythropoietin (Fig indication

that

22,000)

two

produced

serum

alternate

glycanase molecular

S frugiperda

to

alternatively

is based first mol wt 26,000

yields weight

erythropoietin three

forms

derive with

from the

mol

sites

with

the

of glycopeptide.

Thus,

the

utilization

differential

wt 26,000

site. Importantly, possessed by this relatively

well

and in

low

products.

of nearly equivalent Second, the failure

Third,

consensus

correlates

24,000

of lower molecular against any possible

products.

of three

lation5’3”5

wt

cultured

glycosylated

peptides v 18,400).

peptides argues

of secreted

(mol cells

on the observation that treatand 24,000 forms with N-

erythropoietin (ie, I 8,900

detect erythropoietin Western blot analysis proteolysis

forms

by

correspond

This conclusion ment of the

among molecules of this mol 2) is evidenced further by the

form

the

here

weight by nonspecific

for N-linked

these

glycosy-

different

glycosylated

in

occurrence

may sites,

uniformly

the

saccharide

structures267

is a likely

at each

lack of in vivo activity.’3 Previously reported procedures

human

recombinant

explanation

erythropoietins

the generally

have

on

C I /C8 160-fold

fully

conventhe

isola-

somewhat

CM

purity

was

blue,

lower

is

a procedure

which

involves

Q

three chroma-

(FPLC),

and

a

This procedure resulted in a a product with a specific

reported (ie,

simple

erythropoietin

ie, sequential Mono

medium.3’ yielding

the overall

three

erythropoie-

human

reported,

SDS

additional the proce-

purification

in preparation,

procedures, Affi-Gel

via

of bioactive

recombinant

cells

Howmonowith

1). An 890-fold was

of

reverse-phase purification

substantially

separate

at least two In contrast,

>99%

recovery

the these

simplified

of 84,000 U/mg. In contrast gradient elution of products was

activity

dune,

to the required

recovery

35%),

and

present proceat each step.

of this

its

final

product

purity

was

was

not

assessed.

The baculovirus-mediated developed for the production provides

highly cal

growth

properties

of

unique

factor. this

source

of milligram the simple

erythropoietin

aimed

analyses

structural

features

system human

Whereas

have

to virtual homogeneity, human erythropoietin also

structural

and

expression of recombinant

a convenient

active

at

amounts

of

glycochemifacilitated

it is anticipated should facilitate

identifying

essential

presently erythro-

its

that

this direct

physicochemical

to its bioactivity.

ACKNOWLEDGMENT The

for

purification

80%

chromatographic

tography

for

of erythropoietin

(Table

manuscript

in BHK

distinct

their

Fisher, for

this

expressed

normal interaction with the cell surface receptor of mammahan erythroid cells. The absence of sialic acid in insectderived

with purification

purification

the high specific in vitro biological activity purified erythropoietin confirms that this level of glycosylation is sufficient for its

limited

the

poietin of

forms

of glycosylation

While

for

been

and steps.

achieves

steps

In addition, to

occurrence

observed

tin.

al

of an efficient immunosorbant.”#{176} requires the use of a proprietary

chromatographic

low saccharide content (approxiwith concanavalin A (Table 1).

has

et

Commonly, Alternatively,

3,29

52.

the denaturation

accomplished ported mately

of five or more

use

epitope recognition, chromatographic

described

Miyake

the

hormone

antibody,

for efficient conventional

(ie. 18,400).

erythropoietin

by

erythropoietin.

of recombinant

clonal based

aglycosyl

involve

tion

30%1

developed urinary

chromatographic

26,200t

coefficients.23 Based

methods of human

weight

content

‘See Fig 5. fCalculated

mature.

on

isolation

radius

Stokes

Erythropoietin

Cells

ET AL

of been

assays

authors generous Tulane

gratefully support

acknowledge and technical

Medical

School,

and providing

for

antiserum

the Supelco assistance, assisting

in

to recombinant

Corporation for and Dr James CFU-e and in vivo human erythropoie-

tin.

REFERENCES 1 . Nigon V, Godet J: Genetic and morphogenetic factors in hemoglobin synthesis during higher vertebrate development: An approach to cell differentiation mechanisms, in Bourne GH, Danielli iF, Jean KW (eds): International Review of Cytology, vol 46. San Diego, Academic, 1976, p79 2.

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SB,

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cryArch