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Chemo-enzymatic peptide synthesis (CEPS): a generally
applicable, traceless ligation technology for the synthesis
of longer peptides and peptide-to-protein conjugates.
Belgian peptide group meeting
February 18th, 2016
Timo Nuijens
[email protected]
Enzypep: The Company
Founded:
July 2012 as spin-off from DSM
Location:
Brightlands Campus in Geleen (NL)
Team:
Dr. Timo Nuijens
Dr. Ana Toplak, B.Sc.
B. Sc. Mathijs B.A.C. van de Meulenreek
M.Sc. Michel Goldbach
M.Sc. Marcel Schmidt
Dr. Peter J.L.M. Quaedflieg (hire in basis)
Funding:
2 Private investors and 3 investment funds
Goals:
Development of processes for “long” (pharmaceutical) peptides, cyclic
peptides and peptide-to-protein conjugates with significantly lower costs
(CEPS).
2
Enzypep: Initial CEPS Concept
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During SPPS, yield loss increases exponentially with chain length.
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Many close eluting impurities, difficult prepartive HPLC purification
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Better use short fragments and segment condensation
•
Less and better separated impurities
Full SPPS
Yield as Function of Chain Length and
Coupling Efficiency
100.0%
Crude Yield
80.0%
99.5%
60.0%
99.0%
40.0%
98.0%
Fragment approach
97.0%
20.0%
95.0%
90.0%
0.0%
0
5
10 15 20 25 30 35 40
Number of Amino Acids
3
Fragment condensation: SPPS-LPPS and NCL
Chemical Hybrid (SPPS-LPPS) Fragment Condensation
• racemization except sequences with C-terminal Gly or Pro
• fully protected peptides have poor solubility and are difficult to purify
Other chemical ligation techniques (such as NCL or KAHA)
• sequence dependent
• NCL, thioester instability
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Fragment condensation: CEPS
Chemo-enzymatic Peptide Synthesis (CEPS) in Aqueous Solution
• no racemization
• no side-chain protection = better solubility, easier purification
• selective, does not recognize D-amino acids
• no side-reactions as encountered using chemical fragment condensation
Potential drawbacks:
• C-terminal ester needed (can be expensive/unstable)
• hydrolytic side-reactions: synthesis to hydrolysis ratio (S/H ratio)
• tight sequence specificity (not generally applicable)
• enzyme instability
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Chemo-enzymatic Peptide Synthesis (CEPS)
Most often used: proteases
How can hydrolytic side-reactions (intrinsic hydrolase activity) be minimized?
• Reaction optimization
• Omit water
• Special esters, substrate mimetics (Bordusa et al.)
• Natural occurring ligases:
Sortases
Inteins
Butelase
Aqueous media
Require specific amino acid motifs
May not be traceless
Peptides tuned to requirements of enzymes
• Designed ligases (mutagenesis):
Peptiligases
Enzymes tuned to requirements of peptides
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Ligases derived from nature
Sortase Reaction
Not traceless
sortase footprint
Butelase Reaction
butelase footprint
Butelase 1
• Sortases, butelase and inteins for a thioester intermediate
• Transpeptidation  able to cleave a peptide amide bond
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Subtiligase, designed ligases
Ligases by mutagenesis, protein design
- Streptoligase (Elloitt et al.)
- Subtiligase (Wells et al.)
Subtilisin (serine protease)
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•
•
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S221C mutation at active site
thioester intermediate
P225A mutation to prevent steric crowding
“double mutant” called subtiligase
can be used to
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•
•
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ligate peptide fragments
cyclize peptides >12 amino acids
couple peptides to proteins
synthesize thioacids and thioesters
Limitations:
•
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average yield of 50% using 10 equivalents of one fragment
Subtiligase is labile, is Ca2+ dependent, difficult to produce and purify
Distinct need for more stable and efficient enzymes with higher S/H ratios
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Peptiligase
Starting from a hyperstable and cation (calcium) independent variant of subtilisin
(Bryan et al.) that was
- stable to additives (urea and guanidiniumchloride)
- stable to organic co-solvents (DMF and DMSO, up to 50 vol%)
- thermostable (unfolding temperature = 70⁰C)
- very hydrolytically active
we incorporated the S221C and P225A mutations into this enzyme scaffold.
Surprisingly, this new enzyme “peptiligase” exhibited
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2-fold higher S/H ratio
very fast ligation reaction
retention of stability towards detergents, co-solvents and temperature
Furthermore
• peptiligase was easy to produce and purify
• little enzyme was needed (~1 mg enzyme/gram of 20-mer peptide)
• perfect scaffold for further protein engineering
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Peptiligase condensation
• Peptide Cam-esters synthesized via SPPS (Nuijens et al.)
Leu-Wang Resin, coupling of iodoacetic acid, Fmoc-amino acid and DiPEA
• Usually side-chain unprotected peptides in buffer (pH 8) at 20⁰C.
• Ligation yield usually > 90% synthesis and < 10% hydrolysis for a 10-mer + 10-mer
coupling using only 1.1 equivalents of the amine or Cam-ester fragment
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Peptiligase Specificity
medium
medium
Limitations: very tight amine fragment substrate profile, preference for Gly, Ala and Ser
Action:
broaden peptiligase specificity, S/H ratio, activity via site-directed mutagenesis
Started more than 4 years ago
Peptiligase Specificity :P’1 pocket
HPLC product yield*
Development of 1st, 2nd and 3rd generation mutants: P'1 pocket
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Improvement of the
P'1 pocket specificity
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All amino acids in P’1
pocket except Pro are
now well accepted
*Suboptimal conditions to demonstrate differences
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Peptiligase Specificity: P’2 Pocket
Development of 1st, 2nd and 3rd generation mutants: P‘2 pocket
90%
80%
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Improvement of the
P‘2 pocket specificity
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>80% of the amino
acids in P’2 pocket
are now well
accepted
•
In development:
“Omniligase”, a
peptiligase that can
couple almost all
sequences.
Commercially
available (Iris)
HPLC product yield*
70%
60%
Ptl
50%
1st
40%
2nd
30%
3rd
20%
10%
0%
*Suboptimal conditions to demonstrate differences
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Peptiligase Specificity: Selectivity
Development of 1st, 2nd and 3rd generation mutants: P'1 pocket
90.0%
•
Also highly specific
enzymes
-Large/small
-Polar/hydrophobic
-Charge
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For both P’1 and P’2
HPLC product yield*
80.0%
70.0%
60.0%
50.0%
Sel-1
40.0%
Sel-2 •
30.0%
20.0%
10.0%
•
0.0%
Often no N- or Cterminal protecting
groups are needed
>10,000 unique
(specific) ligases now
at our disposal
*Suboptimal conditions to demonstrate differences
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S/H Ratio in 1st, 2nd & 3rd Generation Mutants
S:H Ratio
S/H Ratio for 1st, 2nd and 3rd generation Peptiligase mutants
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8
7
6
5
4
3
2
1
0
S/H Ratio
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Besides improving the
substrate scope, the S/H
ratio is also much higher
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An S/H of 9 means 90%
synthesis and 10% Camester hydrolysis
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We performed 10 + 10
coupling reactions with S/H
ratio’s of >50, corresponding
to 98% synthesis and only
2% hydrolysis
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No other side reactions as
encountered using chemical
fragment condensation
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Example 1: Exenatide (2 Fragment Approach)
Exenatide has no Gly or Pro residues at strategic positions and is therefore most often
synthesized via full SPPS. Typical yield of commercial processes = 20 – 25%.
1-21
22-39
Cam-ester Fragment (unprotected):
H-His1-Gly2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Leu10-Ser11-Lys12-Gln13-Met14Glu15-Glu16-Glu17-Ala18-Val19-Arg20-Leu21-OCam-Leu-OH
Amine Fragment (unprotected):
H-Phe22-Ile23-Glu24-Trp25-Leu26-Lys27-Asn28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34Ala35-Pro36-Pro37-Pro38-Ser39-NH2
Coupling:
2nd generation peptiligase
Amine Fragment, using Rink resin:
Crude fragment:
70.6% purity, 65% yield;
Single purification:
96.0% purity, 52% yield
Cam-ester Fragment, using CTC resin
Crude fragment:
72.5% purity, 69% yield,
Single purification:
97.1% purity, 56% yield
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CEPS of Exenatide (2-Fragment Strategy)
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2nd generation peptiligase
At gram scale
No protection at all, API in one step
1.1 equivalent amine fragment
Conversion to product 87%
30 g/L Exenatide product
Crude reaction mixture for prep HPLC
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CEPS of Exenatide (2-Fragment Strategy)
Exenatide
(Pure
Fragments)
Scale:
Crude peptide:
Single purification:
Overall yield:
1.0 g, 3.0 wt%, amine excess = 1.1 equiv.
78.9% purity, 87% yield
99.6% purity, 78% yield (based on Cam-ester)
43.5% (based on resin loading of Cam-ester)
NaClO4-ACN
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TFA-ACN
Overall yield 2-fold to better than straight-through SPPS
Purity exceeds innovator’s specifications
Chiral integrity confirmed by C.A.T.
Cost-price reduction estimated around 40% - 50%
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CEPS
What more can we do more with our ligases ??
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Head-to-tail cyclisation
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N- to C- polymerisation
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Maximum ligation length ?
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Peptide to protein conjugation
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Peptide to polymer
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Etc, etc
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Macrocylic Peptides: Exenatide, 39-mer
H-Phe22-Ile23-Glu24-Trp25-Leu26-Lys27-Asn28-Gly29-Gly30-Pro31-Ser32-Ser33-Gly34Ala35-Pro36-Pro37-Pro38-Ser39-His1-Gly2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Leu10Ser11-Lys12-Gln13-Met14-Glu15-Glu16-Glu17-Ala18-Val19-Arg20-Leu21-OCam-Leu-OH
Parameter
Conditions
Enzyme
Buffer
Temperature
Time
Conversion
Broad specificity Ptl
Phosphate buffer, pH 8.3
20°C
5 min
98%
1
39
N to C
coupling point
• Efficiently cyclised many more peptides ranging fom 12-50 amino acids
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N-Terminal Conjugation: Exenatide to Serum Albumin
+
1 + 1 Condensation
selective Ptl
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N-Terminal Conjugation: Peptide to XTEN(144) and XTEN(864)
model peptide
+
1 + 1 Condensation
selective Ptl
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Conclusions
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Peptiligase (extremely stable ligase)
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Efficient condensation of unprotected peptide segments in water
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1st, 2nd and 3rd generation variants:
-”Omniligase”, can couple almost all peptide sequences
-”Sequence-specific ligases”, very selective
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Synthesis of Exenatide
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Head-to-tail cyclisation
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Peptide-to-protein or peptide to polymer conjugation
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Acknowledgements
University of Groningen:
Prof Dick B. Janssen
Dr. Ana Toplak
Dr. Bian Wu
(now at Enzypep)
Enzypep:
Dr. Peter J.L.M. Quaedflieg
B.Sc. Mathijs B.A.C. van de Meulenreek
M.Sc. Marcel Schmidt
M.Sc. Michel Goldbach
M.Sc. Francesco Ventura
Scientific advisory board:
Prof Dick B. Janssen
Dr. Paul ten Kortenaar
Dr. Rodney Lax
Dr. Roland Callans
Dr. Rinus Broxterman
(University of Groningen)
(MSD, Diosynth)
(PolyPeptide Group)
(Corden)
(DSM)
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