Large scale preparation of hirudin

THROMBOSIS Printed

RESEARCH in the

United

LARGE

SCALE

D. Bagdy, Department

~01.2, pp. Pergamon

States

PREPAFTION

E.

OF

Barabds

HIRUDIN

and L. Graf

of Biochemistry,

for Pharmaceutical

229-238, 1973 Press, Inc.

Research

Chemistry,

Institute

Budapest,

Hungary

(Received 8.1.1973; in revised form 6.2.1977. Accepted by Editor S. Magnusson)

ABSTRACT

A process for large scale production of hirudin from whole leeches is described. Crude hirudin is prepared by aqueous extraction, adsorption and elution on/from bentonite and subsequently some other fractionation steps. Purification of crude hirudin is carried out by chromatography on Ecteola-cellulose followed by rechromatography on CM-Sephadex C-25 and Sephadex G 75. Homogeneity of the end product was tested by end group analysis and ultracentrifugation. The process presented here can be regarded as the first large scale preparation of hirudin.

INTRODUCTION After hirudin,

more

than half a centry

the anticoagulant

by Markwardt remarkable

et al.

papers

of its recognition

substance

(3, 4). During

were published

of leeches

(1, 21,

was rediscovered

the intervening

period

in this field except

no

that of

Waldschmidt-Leitz related

et al. (51, who described some observations -to the extraction and partial purification of hirudin

as well

as to its behaviour

Markwardt's

group was the first, pure

ing chemically with

thrombin

lished

against

hirudin

several

however,

proteolytic

to succeed

and characterizing

(6, 7). In addition,

Markwardt

et al. --

enzymes.

in preparits reaction (8, 9) pub-

effects of hirudin, on the -in vivo antithrombin the resorption, elimination and toxicity of the mole-

experiments

including cule.

All methods

for the isolation

(10, 11, 12) have a common

feature, 229

of hirudin

so far published

i.e., they start

from the

PREPARATTON OF 1lTRTJDTN

330

Vol..Z,No.Y

head-part containing the antithrombin substance of the leeches. The main disadvantage of this method is that it is extremely laborious and time-wasting to decapitate, one by one, the many thousands of leeches required to obtain a minimum quantity of pure material. This paper presents the first process for large scale preparation of hirudin. MATERIAL AND METHODS Leeches were collected and transported by MAVAD (Hungarian Cooperative Enterprise for Game Trading).

Each consignment con-

tained leeches of 20 - 60 mm length without any selection or classification. Starvation at 4 - 10°C lasted about 2 - 14 days, after which the leeches were frozen and kept at -1O'C until processing. All chemicals used in the isolation process were technical or pure grade materials: sodium chloride; hydrochloric acid; trichloracetic acid (TCA); ammonium hydroxide; ethanol; acetone; etc. Celite 535 and Hyflo Supercel (Manson Co.) served as filter acids. Bentonite

(Hungarian standard) activated with hydro-

chloric acid was used for adsorbing hirudin. Carboxymethyl (CM 11) and Ecteola

(ET 30) celluloses were Whatman's products;

Sephadex G-50, G-75 and CM Sephadex C-25 were obtained from Pharmacia Fine Chemicals. Molecular weight determination was performed by the low speed method in a MOM 3121 ultracentrifuge equipped with Rayleigh interferometer optics. The measurements were made at 20°C with a rotor speed of 18000 rpm. For these experiments hirudin was dissolved in 0.1 M TRIS buffer, pH 7,4, to give 2,2 mg protein/ml. The value used for the partial specific volume (v) was 0.741, as determined experimentally by Triebel and Walsmann

(13).

N-terminal end-group analysis was performed by the dansylmethod of Gray (14). To identify the dansyl amino acid residue, two-dimensional polyamide thin-layer chromatography was applied (15)* Amino acid analysis was carried out in a JEOL (JLC-5AH) amino acid analyzer after hydrolysis of the peptide in 6 M HCl for 40 hrs at llO°C in sealed, evacuated tubes. Half-cystine was determined as cysteic acid in 20 hr HCl hydrolysates of

PREPARATION

Vo1.2,No.3

performic

acid oxidized

Determination samples

(Hoffman-LaRoche;

Bovine

out according

per mg)

to Markwardt

of high purity

of hirudin

from whole

leeches

Two kilograms

was prepared

of starved

added

leeches

at 0 - 5OC for 10 minutes.

carried

out after

to the ground

addition

solvents

thawed

per cent cold aqueous

material

and the mixture

Extraction

of sodium

constant

pH 2,s - 3,5. The insoluble and the extraction

of hirudin

chloride

acetone

was precipitated

-1O'C.

The precipitate

washed

with

residue

was was

to a final

buffer

solution

under

containing

suitable

at -5'C, washed

to 100,000

hirudin

of 80 volumesper

of cold acetone

hirudin

conditions

at

and

was dissolved

in a

(~0.1, pH 5 - 6,

and discarding

was precipitated The yield

of which

at

by decantation

was centrifuged

antithrombin

activity

minutes

and TCA. The combined

2 volumes

and dried.

- 140,000

the specific

NaCl

separating

hirudin

separated

2 liters

Thereafter,

0 - 5OC). After crude

with

by adding

for 30 - 60

was

containing

cold acetone.

residue,

stirring

was repeated

cent cold aqueous

leeches,

organic

of 0.2 - 0.5 M and TCA to a final concentration

of 0.1 - 0.4 M with

amounts

fibrinogen

kept at -1O'C were

of 80 volumes

stirred

or acetone

of

(20)

were

soluble

by the method bovine

with water-miscible

at O'C. 6 liters

temperature

substances

AND RESULTS

acetone

extract

(Hungary;

AG) was used.

1. Extraction

concentration

(16). Thrombin

(17, 18) were used as reference

EXPERIMENTS

and minced

(ATU) of hirudin

(19), but in some experiments

et al.

(Behringwerke

activity

53 NIH units per mg) and Phylaxia

fibrinogen

Barab&

231

peptide.

of antithrombin

was carried

250 NIH units

OF HIRUDIN

with

the incold ethanol

of crude hirudin

units

(ATU) per kg of

is 200 - 400 ATU/mg

protein. 2. Extraction

of hirudin

in aqueous

medium

from whole

leeches

(21) By studying

the conditions

Waldschmidt-Leitz acidic

or alkaline

of the extraction

et al. (5) established -aqueous medium yields

of hirudin

that the extraction a very poor quantity

in

PREPARATION

272

of hirudin. Subsequently,

OF HIRUDIN

Vo1.2,No.3

all methods published for the extract-

ion of hirudin used water-miscible

organic solvents. By reinvest-

igating the problem, we have found that hirudin can be extracted from whole leeches even in aqueous medium under appropriate conditions. The yield of hirudin in the aqueous extracts equals that obtained with water-miscible organic solvents. llowever, the aqueous extracts contained much less pigments. 50 kilogrammes of ground whole leeches were mixed and stirred at room temperature with 150 liters of 0.5 M NaCl solution for 30 - 60 minutes. After acidifying with 3 M HCl to pH 2.0 2.5, the temperature was raised and kept at 70°C for 15 minutes with constant stirring. The insoluble residue was separated by centrifugation. The extract was adjusted to pH 6.5 - 7.0 and the precipitate formed was separated and discarded. 3. Preparation of crude hirudin from the aqueous extract (21) By addition of 1.5 volume of ethanol to 1 volume 06 the extract, a considerable quantity of impurities was precipitated and removed. The supernatant was then concentrated to 15 - 30 liters in vacua at 30 - 40°C. A further purification was carried out by fractionation with acetone. To one volume of the concentrate one volume of acetone was added and the precipitate formed was separated and discarded; thereafter hirudin was precipitated from the supernatant by addition of 4 volumes of cold acetone at 0 to -5'C. The precipitate was dissolved in 5 - 8 liters of cold 0.1 M TCA and after removing the insoluble residue by centrifugation, the solution was diluted with 4 - 5 volumes

of

water. Then 20 g of activated bentonite was added

cold distilled

per 1000 ml of solution with constant stirring. Adsorption of hirudin on bentonite became somplete within one hour. Elution was performed in two steps with 45 volumes per cent aqueous acetone at pH 8.0 - 8.5 and room temperature. concentrated pH was

in vacua

adjusted

discarded,

at 20 - 30°C to about

The eluate 5 liters

to 4.5 - 5.0; the precipitate

the supernatant

containjng

hirudin

was

and its

was separated was

filtered

and and

The average yield was 0.2 g of crude hirudin per kg of leeches, and the specific activity varies from 400 - 700 ATU/mg protein. A flow chart of the large scale process for lyophilized.

isolation of crude hirudin is shown in Fig. 1.

PREPARATION

Vol.Z,No.3

OF HIRUDIN

FLOW CHART LARGE

i ii iii

SCALE

PREPARATION

OF CRUDE HIRUDIN

Refrigeration

and storage

and grinding

in aqueous

Precipitation

with

residue

ethanol

3

Supernatant Concentration

in vacua --

4

-1 Fractionation

with

r

6

Concentration

7

Isoeledtric

discarded

of impurities

acetone

and elution

I

the extraction

discarded

Precipitate

Adsorption

starting

medium

Precipitate

5

LEECHES

(at -10 to -2OOC)

before

Insoluble 2

FROM WHOLE

(2-14 days at 4-6'C)

Extraction

1

of the

Starvation

Thawing

233

formed

on/from

at 50 volumes per cent acetone discarded

Bentonite

in vacua -~

I

precipitation

of impurities

Precipitate Drying

8

from the frozen - 140,000

YIELD:

80,000

SPECIFIC

ACTIVITY:

discarded

state ATU/kg

It was

of leeches

400 - 700 ATU/mg

FIGURE

4. Chromatography

of crude hirudin

adsorbed

Ecteola

in a mild

Cellulose

on Ecteola

strength.

Elution

strength

stepwise

increase

in specific

starting

material.

may be carried or gradually. activity

Gradient

obtained

on anionic acidic

protein

1

found that crude hirudin

can be completely

at pH 4.50

Cellulose form whole

celluloses,

medium

of about

elution

leeches

especially

on

and at low ionic

out by increasing Stepwise

(22)

elution

the ionic resulted

3 - 5 times

proved,

related

however,

in an to the

much more

vo1.2,No.3

PREPARATION OF HIRUDIN

234

FLOW CHART of the PURIFICATION OF CRUDE HIRUDIN PREPARED FROM WHOLE LEECHES 1

Chromatography on Ecteola Cellulose

2

1 Chromatography on CM-Sephadex C-25

3

Gelfiltration on Sephadex G 75

4

Lyophilization

Average yield:

70% ATU activity related to the crude hirudin

Specific activity:

6000 - 8000 ATU/mg protein FIGURE 2

effective resulting in a purity increase of about 8 - 10 times. Fractions containing the antithrombin activity were separated very sharply between ionic strength 0.19 - 0.22. Hirudin was precipitated with 4 volumes of cold acetone at -5'C from the eluate after concentrating in vacua at 30°C and desalted by dialysis. The yield of antithrombin activity amounts to 85 - 90%. Despite

the definite increase in specific activity, the samples

contained a significant quantity of yellow-brownish pigments, too, A typical experiment can be seen in Fig. 3.

*nu

ANhl

W

(0

Fmcrw

Muam

FIGURE 3 Chromatography of crude hirudin (400 mg, 260 ATU/mg) on a 1.5 x 23 cm column of Ecteola Cellulose equilibrated with 0.02 M ace-

PREPARATION

Vol.p,No.?

tate buffer M NaCl

of pH 5.0. Gradient

at room temperature.

fraction

OF HIRUDIN

volumes

23 - 31. Yield:

Mere

was carried

The flow rate was

5 ml. Hirudin

activity

of which

ditions.

It

was

on Sephadex

found that an optimal

hirudin

fractions

remained

containing

by increasing

after

concentrating

This

4 volumes

with

approximately

is demonstrated

Sephadex

was submitcon-

can be realized contaminating

the

colourless

could be eluted

was precipitated

of cold acetone

and

at -5'C

of pH 5.0. Gradient

increase

Equilibration

elution

in specific

4

(260 mg, 2000 ATU/mg)

of hirudin

CM C-25 column.

a 3-fold

in Fig. 4.

FIGURE Chromatography

to

in vacua at 30°C and desalted by dialysis. -an 80 percent yield related to the starting acti-

vity was reached activity.

with

activity

amounts

different

and practically

Hirudin

the ionic strength.

from the eluate

On an average,

on the column

the antithrombin

dried

separation

0.02. Pigments

at pH 5.0 and an ionic strength crude

CM C-25 under

and

activity).

5. Chromatography of hirudin on Sephadex CM C-25 Hirudin partially purified on Ecteola Cellulose ted to chromatography

0.5

in fractions

..o... antithrombin

absorbance,

out with

30 ml/hr

was contained

44 mg, the specific

(

2000 ATU/mg

elution

235

with

with

0.5 M NaCl

on a 0.8 x 25 cm

0.02 M acetate

buffer

at room temperature.

PREPARATION OF HIRUDIN

236

Vo1.2,No.Y

Flow rate 20 ml/hr; fraction volume, 5 ml. Hirudin was found in fractions 19 - 23. The yield was 66 mg of hirudin. The specific activity was 5800 ATU/mg protein (

absorbance; 0. -a - - ATU

activity). 6. Gel filtration and characterization of hirudin Hirudin partially purified on Ecteola Cellulose and subsequently on Sephadex CM C-25 was chromatographed on Sephadex G-75. Separation of three components could be observed. Only the second contained antithrombin activity

(Fig. 5).

.” :

20 -

m

:

: : .

:

PO0

.

. .

AJ?#

Amh

:

do-

wlxl

FIGURE 5 Gel filtration of hirudin

(42 mg, 5800 ATU/mg) on a 1.2 x 52 cm

Sephadex G-75 column. Equilibration with 0.1 M NaCl. Flow rate 30 ml/hr; fraction volume, 5 ml. Hirudin was contained in fractions 11 - 15 and dried from the frozen state. The yield was 29 mg with a specific activity 6800 ATU/mg protein sorbance; -*----

ab-

ATU activity).

This active component was subjected to physico-chemical and chemical analysis. The molecular weight of hirudin was determined by equilibrium sedimentation and found to be 12,200. This value is in the range of the values (9,000 - 16,000) obtained for hirudin previously by different techniques (13, 23). The homogeneity of our preparation was also proved by Nterminal end-group analysis. The N-terminal amino acid residue was identified as valine instead of isoleucine, which was pre-

PREPARATION

Vo1.2,No.3

viously

found

to be the N-terminal

Experimental

details

the apparent

contradiction

described

AsP14,

Ile3, Leu5, This isolated

that the molecular

Ser6, Tyr3,

Glu16, Phe2,

composition

data

(11, 24). reason

for

in the literature

(25). weight

of hirudin

is 12,200,

Gly13,

Ala3,

l/2 Cys6,

Va17,

Lys5, His2. appears

to be close

to that of hirudin

by de la Llosa -et al. (111, save for two One difference pertains to methionine, which is

and Walsmann

and the preparations

(24). Secondly,

et al. we found alanine --

to be present

reported

in contrast in higher

by

to de la l,losa,

concentrations.

ACKNOWLEDGEMENTS Thanks performing

are due to Mrs.

Zs. Lakatos

the ultracentrifugal

and Mr. A. Patthy

and amino

acid analyses,

for resp.

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(1957). 7.

is

is as follows: Pro6,

in our preparation

Markwardt

of hirudin

and analyzed

differences. absent

communication

237

and the possible

of previous

acid composition

Thr6,

residue

of our analysis

in another

Assuming the amino

OF IITRUDIN

F. MARKWARDT Chem. 312,

and P. WALSMANN, 85

8.

F. MARKWARDT,

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