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Biochemical, Molecular and Preclinical Characterization of a Double Virus-Reduced Human Butyrylcholinesterase Preparation Designed for Clinical Use Plasma Product Biotechnology Meeting, Cyprus May 12, 2011 Harald Arno Butterweck, Alfred Weber, Ursula Mais-Paul, Wolfgang Teschner, Laura Lei, Eva-Maria Muchitsch and Hans Peter Schwarz Baxter Innovations GmbH

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Page 1: Biochemical, Molecular and Preclinical Characterization of a … · 2018-02-10 · Biochemical, Molecular and Preclinical Characterization of a Double Virus-Reduced Human Butyrylcholinesterase

Biochemical, Molecular and Preclinical

Characterization of a Double Virus-Reduced

Human Butyrylcholinesterase Preparation

Designed for Clinical Use

Plasma Product Biotechnology Meeting, Cyprus

May 12, 2011

Harald Arno Butterweck, Alfred Weber, Ursula Mais-Paul, Wolfgang

Teschner, Laura Lei, Eva-Maria Muchitsch and Hans Peter Schwarz

Baxter Innovations GmbH

Page 2: Biochemical, Molecular and Preclinical Characterization of a … · 2018-02-10 · Biochemical, Molecular and Preclinical Characterization of a Double Virus-Reduced Human Butyrylcholinesterase

Background

History:

Purification and partial characterization of serum cholinesterase published in the

second half of last century by Michael, H.O. 1949; Haupt et al.,1966 and 1971;

Muensch et al., 1976

Lyophilized serum cholinesterase from Behringwerke AG on the market - indicated

for the treatment of prolonged apnoe caused by succinylcholine chloride sensitivity

or detoxification – product discontinued

Attempts to purify butyrylcholinesterase (BChE) from human plasma in large scale

(eg. Lockridge and La Du, 1978; Ralston et al. 1983; Lockridge, 1990; Lynch,

1993; Grunwald et al., 1997; Lynch et al., 1997; Lockridge et al., 2005)

Large scale production of recombinant BChE (Huang et al., 2007) – no product on

the market

Situation:

Organophosphates are the basis of many insecticides, herbicides and nerve

agents (Soman, VX, Sarin and Tabun)

Conventional approaches to treat organophosphate poisoning are still not satisfactory

No prophylactic treatment available

No prevention of performance deficits, loss of conciousness, permanent brain

damage

May 12, 2011 PPB, Cyprus 2

Page 3: Biochemical, Molecular and Preclinical Characterization of a … · 2018-02-10 · Biochemical, Molecular and Preclinical Characterization of a Double Virus-Reduced Human Butyrylcholinesterase

Human BChE as Bioscavenger

May 12, 2011 PPB, Cyprus 3

OP: Organophosphate

ACh: Acetylcholine

AChE: Acetylcholinesterase

Mechanism of nerve

gas toxification:

1. Acetylcholinesterase

is inactivated by nerve

agents.

Neurotransmitter

acetylcholine cannot

be cleaved.

2. High concentration of

acetylcholine leads to

uncontrolled

stimulation of

receptors.

3. Convulsions,

permanent brain

damage, ...

Page 4: Biochemical, Molecular and Preclinical Characterization of a … · 2018-02-10 · Biochemical, Molecular and Preclinical Characterization of a Double Virus-Reduced Human Butyrylcholinesterase

Tetrameric enzyme

4 identical subunits (574 amino acids) arranged as a dimer of dimers

Subunit structure:

Highly glycosylated (9 asparagine-linked carbohydrate chains)

Essential for maintaining the tetrameric structure of BChE

Essential for the long half-life (~6 days) of BChE (recombinant products

with different glycosylation pattern are cleared within hours)

Human BChE Structure

May 12, 2011 PPB, Cyprus 4

Active site

Catalytic triade

Modified from

„Butyrylcholinesterase, its

function and Inhibitors“,

edited by Ezio Giacobini,

F. Nachon p39ff

Page 5: Biochemical, Molecular and Preclinical Characterization of a … · 2018-02-10 · Biochemical, Molecular and Preclinical Characterization of a Double Virus-Reduced Human Butyrylcholinesterase

Human BChE – Literature Search

Characteristics of the Tetramer:

• Activity: modified assay to Ellman et al., Biochem. Pharmacol. 7: 88-95 (1961)

• Molecular size: 342 kDa

• UV extinction factor: 1.8

• Isoelectric point : 4.0

• Plasma concentration: 5 µg/mL plasma

Purification methods starting from Cohn IV-4 precipitate:

• PEG precipitation

• Ion exchange chromatography

• Size exclusion chromatography

• Affinity chromatography

Stabilization:

• EDTA, pasteurization, lyophilization, glycerol

May 12, 2011 PPB, Cyprus 5

Page 6: Biochemical, Molecular and Preclinical Characterization of a … · 2018-02-10 · Biochemical, Molecular and Preclinical Characterization of a Double Virus-Reduced Human Butyrylcholinesterase

New Human BChE Process Flow Chart

SD Treatment

IV-4 suspension

depth filtration

Fraction IV-4

Nanofiltration

Diafiltration and

concentration

Anion exchange

chromatography

Clarification

Solvent detergent

inactivation of

lipid enveloped

viruses

Virus removal

by size

exclusion

Procainamide affinity

chromatography

Capture step

Polishing step

Formulation

May 12, 2011 PPB, Cyprus 6

Lot Size:

10,000 L of plasma

Page 7: Biochemical, Molecular and Preclinical Characterization of a … · 2018-02-10 · Biochemical, Molecular and Preclinical Characterization of a Double Virus-Reduced Human Butyrylcholinesterase

Human BChE Final Container Properties

Sterile and endotoxin-free liquid formulation in sodium

phosphate and sodium chloride at neutral pH

Protein concentration: 25 mg/mL

Dose: 200 mg

Specific activity: ~ 700 U/mg

Purity: >95%

SD reagents and procainamide below specification limits

May 12, 2011 PPB, Cyprus 7

Page 8: Biochemical, Molecular and Preclinical Characterization of a … · 2018-02-10 · Biochemical, Molecular and Preclinical Characterization of a Double Virus-Reduced Human Butyrylcholinesterase

HBChE Final Container Impurity Profile

May 12, 2011 PPB, Cyprus 8

Protein

Relative concentrations as a percent of total protein

Intermediates

(mean, n=3) Final products

IV-4 Eluate #03 #04 #05 #06

hBChE 0.08 9.68 99.75 99.94 99.80 99.81

a1-acid glycoprotein 0.06 9.46 < 0.01 < 0.01 < 0.01 0.01

a1-antichymotrypsin 0.85 61.30 < 0.01 < 0.01 < 0.01 < 0.01

a1-antitrypsin 0.14 0.41 < 0.01 < 0.01 < 0.01 < 0.01

a2-HS glycoprotein 2.78 2.87 < 0.01 < 0.01 0.01 < 0.01

a2-macroglobulin 0.23 0.50 0.19 0.02 0.08 0.10

Albumin 38.06 0.29 0.01 < 0.01 0.02 0.01

C1-Inhibitor 0.03 4.94 < 0.01 < 0.01 0.01 0.01

C4b-binding protein < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01

Ceruloplasmin 0.01 3.72 < 0.01 < 0.01 < 0.01 < 0.01

Complement C4 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01

Fibrinogen < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01

Factor XII 0.01 0.02 < 0.01 < 0.01 < 0.01 < 0.01

Haptoglobin 0.35 0.68 0.02 0.02 0.05 0.03

Histidin-rich glycoprotein < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01

HMW kininogen 0.25 2.34 0.01 < 0.01 0.01 < 0.01

IgA 0.87 1.75 0.01 < 0.01 0.01 0.01

IgG 1.58 0.06 < 0.01 < 0.01 < 0.01 < 0.01

IgM < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01

Inter-a-trypsin inhibitor < 0.01 0.08 < 0.01 < 0.01 < 0.01 < 0.01

Plasminogen < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01

Prealbumin 0.04 0.34 < 0.01 < 0.01 < 0.01 < 0.01

Prekallikrein 0.27 0.03 < 0.01 < 0.01 < 0.01 < 0.01

ProteinC < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01

Protein S < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01

Factor II < 0.01 0.02 < 0.01 < 0.01 < 0.01 < 0.01

Transferrin 45.45 0.16 < 0.01 < 0.01 < 0.01 < 0.01

Vitamin-D-binding protein 8.69 0.06 < 0.01 < 0.01 < 0.01 < 0.01

Vitronectin 0.24 1.30 < 0.01 < 0.01 < 0.01 < 0.01

Factor XI < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01

Four exemplary hBChE final container show a purity of >99% after the determination of

30 plasma proteins with ELISA

Page 9: Biochemical, Molecular and Preclinical Characterization of a … · 2018-02-10 · Biochemical, Molecular and Preclinical Characterization of a Double Virus-Reduced Human Butyrylcholinesterase

HBChE Final Container Impurity Profile

May 12, 2011 PPB, Cyprus 9

Protein

Relative concentrations as a percent of total protein

Starting material

IV-4

(mean, n=3)

IEX

Eluate

(mean, n=3)

Final products

#03 #04 #05 #06

hBChE 0.08 9.68 99.75 99.94 99.80 99.81

a1-acid glycoprotein 0.06 9.46 < 0.01 < 0.01 < 0.01 0.01

a1-antichymotrypsin 0.85 61.30 < 0.01 < 0.01 < 0.01 < 0.01

a1-antitrypsin 0.14 0.41 < 0.01 < 0.01 < 0.01 < 0.01

a2-HS glycoprotein 2.78 2.87 < 0.01 < 0.01 0.01 < 0.01

a2-macroglobulin 0.23 0.50 0.19 0.02 0.08 0.10

Albumin 38.06 0.29 0.01 < 0.01 0.02 0.01

C1-Inhibitor 0.03 4.94 < 0.01 < 0.01 0.01 0.01

Ceruloplasmin 0.01 3.72 < 0.01 < 0.01 < 0.01 < 0.01

Haptoglobin 0.35 0.68 0.02 0.02 0.05 0.03

HMW kininogen 0.25 2.34 0.01 < 0.01 0.01 < 0.01

IgA 0.87 1.75 0.01 < 0.01 0.01 0.01

IgG 1.58 0.06 < 0.01 < 0.01 < 0.01 < 0.01

Inter-a-trypsin inhibitor < 0.01 0.08 < 0.01 < 0.01 < 0.01 < 0.01

Prealbumin 0.04 0.34 < 0.01 < 0.01 < 0.01 < 0.01

Prekallikrein 0.27 0.03 < 0.01 < 0.01 < 0.01 < 0.01

Transferrin 45.45 0.16 < 0.01 < 0.01 < 0.01 < 0.01

Vitamin-D-binding protein 8.69 0.06 < 0.01 < 0.01 < 0.01 < 0.01

Vitronectin 0.24 1.30 < 0.01 < 0.01 < 0.01 < 0.01

a2 macroglobulin and haptoglobulin are the main contaminants in the hBChE final container

Page 10: Biochemical, Molecular and Preclinical Characterization of a … · 2018-02-10 · Biochemical, Molecular and Preclinical Characterization of a Double Virus-Reduced Human Butyrylcholinesterase

Molecular Size Distribution

Size exclusion chromatography confirms molecular integrity of the tetramer

No aggregates and no procainamide in the final container

May 12, 2011 PPB, Cyprus 10

0.100

0.200

0.300

10 15 20 25 30 35

Retention time (min)

OD

(2

80

nm

)

hBChE

hBChE, heat treated

660 k

Da

15

8 k

Da

44

kD

a

17

kD

a

Protein Standard Biorad

HP-SEC profile on TSK G4000SW of the hBChE preparation

in comparison to a heat-aggregated hBChE

Page 11: Biochemical, Molecular and Preclinical Characterization of a … · 2018-02-10 · Biochemical, Molecular and Preclinical Characterization of a Double Virus-Reduced Human Butyrylcholinesterase

Electrophoretic Analyses

PI in the range of pH 4.0 confirmed

Single band indicates homogeneity

May 12, 2011 PPB, Cyprus 11

pI

+

pI

3.5

pI

9.5

St A1 A2 A3 B1 B2 B3 St 1 2 3 4 St 1 2 3 4

kDa

250

Isoelectric focusing Native PAGE of final container Coomassie Activity staining

150

50

75 100

37

3.50

5.20

5.85

6.55

4.55

Single band in Comassie staining

Enzymatic activity of the tetramer shown

A1, B1: Crude BChE after filtration of dissolved fraction

IV-4 (lots A and B)

A2, B2: Corresponding eluate from the IEX column

A3, B3: Corresponding final product

Lane 1 to 4: Four lots of final product

Page 12: Biochemical, Molecular and Preclinical Characterization of a … · 2018-02-10 · Biochemical, Molecular and Preclinical Characterization of a Double Virus-Reduced Human Butyrylcholinesterase

Mass Spectrometry Data

Tandem mass spectrometry (MS) spectra of N- and C-terminal tryptic

peptides confirmed the presence of the unaltered protein

Peptide mapping resulted in 97% sequence coverage (data not shown)

May 12, 2011 PPB, Cyprus 12

N-terminal peptide C-terminal peptide

Page 13: Biochemical, Molecular and Preclinical Characterization of a … · 2018-02-10 · Biochemical, Molecular and Preclinical Characterization of a Double Virus-Reduced Human Butyrylcholinesterase

N-Glycan Profile of HBChE

May 12, 2011 PPB, Cyprus 13

N-glycan profiling identified a diantennary complex structure as the main

oligosaccharide, which was about 60% fully sialylated

The presence of at least 8 of the predicted N-glycans confirmed (Kolarich et al.,

Proteomics 2008)

The symbols used are as

proposed by the

Consortium of

Functional Glycomics

Page 14: Biochemical, Molecular and Preclinical Characterization of a … · 2018-02-10 · Biochemical, Molecular and Preclinical Characterization of a Double Virus-Reduced Human Butyrylcholinesterase

In Vivo Pre-Clinical Results

May 12, 2011 PPB, Cyprus 14

Safety studies

Studies on acute toxicity in rats (7.5 to 500 mg/kg i.v.; 3 to 100 mg/kg i.m.),

bronchospastic activity in guinea pigs (250 mg/kg), blood pressure lowering activity in

rats (7.5 to 500 mg/kg) and thrombogenic potential in rabbits using a modified Wessler

test (250 mg/kg) revealed a similar safety profile as intravenous immunoglobulin

preparations

Pharmacokinetics in rats

Efficacy studies in guinea pigs as a post exposure therapy published recently:

Mumford H, Price ME, Cerasoli DM, et al.: Efficacy and physiological effects of human butyryl- cholinesterase as a post-exposure therapy against percutaneous poisoning by VX in the guinea-pig. Chem Biol Interact 2010; 187:304–308

Animal Dosis

mg/kg

In vivo recovery

after 120 h (%)

Mean residence

time (h)

Terminal half-life

(h)

1 500 4.8 36.0 28.1

2 500 5.9 38.5 29.6

3 250 6.9 40.5 30.4

4 250 4.2 37.2 26.3

5 7.5 5.8 41.1 42.7

6 7.5 5.8 43.4 34.0

Page 15: Biochemical, Molecular and Preclinical Characterization of a … · 2018-02-10 · Biochemical, Molecular and Preclinical Characterization of a Double Virus-Reduced Human Butyrylcholinesterase

Summary

May 12, 2011 PPB, Cyprus 15

Scalable hBChE purification process with two dedicated virus

reduction steps

High purity stable liquid hBChE preparation suitable for i.v.

administration

Tetrameric enzyme with confirmed molecular integrity and

high specific activity

Excellent pre-clinical safety and efficacy profile

GMP material already used in clinical phase I study

Ideal candidate for prophylactic or immediate treatment after

toxin exposure

Page 16: Biochemical, Molecular and Preclinical Characterization of a … · 2018-02-10 · Biochemical, Molecular and Preclinical Characterization of a Double Virus-Reduced Human Butyrylcholinesterase

Acknowledgements

For more detailed information please see also Vox Sanguinis, 2010:

Biochemical, molecular and preclinical characterization of a double-virus-reduced

human butyrylcholinesterase preparation designed for clinical use

Development and scale-up of the purification process:

Patrick Gavit, Alex Zaydenberg, Po-Shing Wah, Thomas Neubauer, Anna Nürnberger,

Michaela Chytil, Martin Rohrer, Bernhard Kölbl, Theresa Bauer, Leopold

Bruckschwaiger and Katayoun Maljic

Analytics:

Daniel Kolarich, Friedrich Altmann,

Andrea Engelmaier, Christina Leb

and Sabine Riedler

Preclinical animal studies:

Wilfried Auer

Project team lead:

Irmtraud Bernwieser

This project was funded in part by United States Federal funds from the United States Department of the Army under

Contract Number W9113M-05-C0131 awarded to DynPort Vaccine Company, Prime Contractor, and Baxter

International Inc, Subcontractor.

May 12, 2011 PPB, Cyprus 16