Post on 26-Apr-2020
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
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
Human BChE as Bioscavenger
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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, ...
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
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
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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
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Lot Size:
10,000 L of plasma
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
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HBChE Final Container Impurity Profile
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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
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
Molecular Size Distribution
Size exclusion chromatography confirms molecular integrity of the tetramer
No aggregates and no procainamide in the final container
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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
Electrophoretic Analyses
PI in the range of pH 4.0 confirmed
Single band indicates homogeneity
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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
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)
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N-terminal peptide C-terminal peptide
N-Glycan Profile of HBChE
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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
In Vivo Pre-Clinical Results
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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
Summary
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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
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.
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