Design ester hydrolysis

Report
Insulin Ester Hydrolysis
Megan Palmer
Chee 450
Conversion of Insulin Ester
Following enzymatic cleavage, must de-protect ThrB30 ester into ThrB30
carboxylic acid to convert to final functional human insulin product
Thr30)-OR  Thr30)-OH
Deesterification Reaction
Reaction rates slow at neutral pH: direct water attack
II) Base Catalysis:
Saponification
Water is a weak nucleophile!
Two mechanisms increase rates of hydrolysis (water addition):
I) Acid Catalysis
Equilibrium
Conversion<100%
Ester Breaks Up
Completely
Formation of desamidoinsulins
Amide groups also undergo hydrolysis!
Most prominent nonenzymatic degradation
reaction of insulin is deamidation
Six Resides are potential deamidation sites:
Deamidation Rates:
Asn >> Gln
GlnA5, GlnA15, AsnA18, AsnA21, AsnB3, and
GlnB4
C-terminal residue AsnA21 is very labile in
acidic pH  forms desamido (A21 isulin)
Changes charge and hydrophilic/hydrophobic
properties -- forces controlling protein structure!
Asp/IsoAsp deamidation products
In neutral solutions, deamidation
primarily occurs at residue AsnB3
Formation of both IsoAsp and Asp
derivatives
IsoAsp introduces another carbon
Rate Limiting step for neutral pH
is cyclic succinimide intermediate
Intramolecular
rearrangement
Rate is highly temperature
dependent: increases possibility
of main and side chains to
assume conformation for ring
formation
More alkaline pH increases
deprotenation of peptide bond
hydrogen and rate of succinimide
formation
Maximum stability against deamidation
at around pH 6, where reaction is 5 to
10-fold slower than at pH 7.4
Peptide Bond Cleavage
Peptide bonds can also be hydrolyzed!
Peptide bond between ThrA8 and SerA9
residues is most susceptible
Exposes hydrophobic core of protein, so
easily separated by HPLC due to relatively
long retention time
B3 transformations and A8-A9 split are
highly temperature dependent, therefore
minimized using low reaction temperatures
Half life of model solutions at
pH 2.5 and 400C has been
estimated to be as short as
50 hours (acid hydrolysis). At
pH 5-7, the degradation rate
is expected to be slower
Other side reactions
Disulphide Exchange of cysteine residues can occur at neutral
pH but requires close proximity of disulphide bonds
Higher temperatures cause conformational changes in
protein structure to increase bond proximities
Transamidation (inter- or intra-molecular) reactions
Peptide Bond Formation:
Iinsulin covalent dimerization primarily (B30-A1)
Cyclic B30-A21 single-chain insulin
Rates much smaller than for other deamidation reactions
Most susceptible
transamidation and
disulphide exchange sites:
Selectivity in Reaction Rates
Amide functionality and peptide bonds
less reactive to hydrolysis than the ester
functionality
VS
Multiple Parallel reactions!
Mission is therefore to maximize rate of
ester hydrolysis while minimizing
degradation products – need kinetic data!
Selectivity 
Ratedeesterification
Ratepeptidehydrolysis  Ratepolyerization  Ratedeamidatioon  RateDi sup lphiderearrangement
Modelling Rates of Reaction
Not an easy task!
Rates of deesterification and degradation are a function of:
- Primary sequence
- 2o and 3o structure
-Temperature
- pH
- Ion strength
- Other intermolecular interactions
Kinetics of Hydrolysis!
Rate Law for Hydrolysis typically
modeled as pseudo-first order at a
constant pH: [RX] independence as
[H+], [OH-], [H2O] constant
Exponential decrease in reactant
concentration
Conversion concentration independent
Observed rate is combination of three
hydrolysis mechanisms
Plot ln Ester conc vs t to determine
observed kh from slope
Determine dependence of rate of
hydrolysis on pH for each reaction and
individual ka, kb, kN by plotting kh vs pH
d Ester
 k h ,obs Ester
dt
k h ,obs  k a H   kb OH   k N H 2O 

   
k
 k H  k OH  k
kK
k  k H 
k
H 
Ex acid : logk  log k  logH   log k

h ,obs

a

h
'
b
b
a
N
'
W

N

h
a
a
 pH
Temperature Dependence
Arrhenius Plots: Rate constants also
examined as a function of
temperature at a specified pH
Larger activation energy = larger
temperature dependence
Need to balance increased rate of
hydrolysis with increased rates of
disulphide exchange etc. due to
thermal rearrangement in protein
structure
Choosing Reaction Conditions
Maximum selectivity at pH which maximizes differences in kh
O
R1
C
N
R2
log kh
R3
O
R1
O
pH
At mildly acidic/neutral
conditions, expect kN, kB to
dominate for ester hydrolysis
Ideal pH range 6-8
C
R2
Reaction Rate Monitoring
Analytical HPLC and gel electrophoresis used to
monitor kinetics of degradation vs. hydrolysis
Separated by physical properties:
Deamidation products differ in charge compared to
insulin as a negative functionality is introduced (Ovs OR)
Split products differ in hydrohobicity/hydrophilicity
Dimerization products have increased MW
Conditions selected to optimize degradation vs.
batch times for reaction vs. recycle ratio
Basic Process Flow Sheet
Alkaline pH (~14)
306 kg Insulin Ester
426 kg eluted NaOH
8293 kg WFI
Up to 82 kg Recycled Insulin Ester
?
1) 388 kg HCl
600 kg Water
(or buffer)
Cooling
Water
2) HCl
pH
control
pH (6-8)
Low T
(ambient)
~12.3 m3
~16 m3 (with recycle)
HPLC
etc.
213 kg Insulin
83 kg Ester
11.2 kg Denatured
By-products
pH 3-4
213kg Insulin
By-products
Some Process Details
Initial pH is very alkaline, rates of base catalysed hydrolysis high
Parallel acid feed used to attain pH 6-8 of incoming mixed solution
For unbuffered system specified, hydrolysis rate constants will change over
course of reaction as OH-, H+ consumed (2nd order kinetics)
Can monitor reaction progress by pH change – need tight control !
Alternative pH control uses buffer solution to attain desired pH
Reaction scheme specifies 73% desired conversion, 3.8% degradation
At constant pH kh (ester hydrolysis) 33x kh (peptide/amide hydrolysis)
Temperature controlled at around ambient temperature by cooling jacket/coils
Final step is acidification to bring to optimal pH for subsequent HPLC
pH 3-4, below pI of Insulin of 5.4, so protein remains soluble
Recovery of unconverted insulin ester from purification can be recycled back to
increase total process yield
Nuts and Bolts
Well mixed reactor run in batch mode increases ease of monitoring pH and
temperature changes as well as controlling batch to batch specifications
Kinetic data is proprietary! Difficult to estimate reaction times….
1994 Capital cost estimation using computer assisted design for similar scale
porcine purification scheme yields a unit operation cost of $109, 000 for 12.3
m3 agitated reactor manufactured by Novo Nordisk (leader in recombinant
insulin production)
Integrated Process Design and
Economics
Questions?
I’m sure you have many….

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