Insulin Ester Hydrolysis

Megan Palmer

Chee 450

Conversion of Insulin EsterFollowing enzymatic cleavage, must de-protect ThrB30 ester into ThrB30 carboxylic acid to convert to final functional human insulin product

Thr30)-OR Thr30)-OH

Deesterification ReactionReaction rates slow at neutral pH: direct water attack

I) Acid Catalysis

II) Base Catalysis: Saponification

Two mechanisms increase rates of hydrolysis (water addition):

Water is a weak nucleophile!

Equilibrium

Conversion<100% Ester Breaks Up Completely

Formation of desamidoinsulinsAmide groups also undergo hydrolysis!

Most prominent nonenzymatic degradation reaction of insulin is deamidation

Six Resides are potential deamidation sites:

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!

Deamidation Rates: Asn >> Gln

Asp/IsoAsp deamidation productsIn 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

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

Intramolecular rearrangement

Peptide Bond CleavagePeptide 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 reactionsDisulphide 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 RatesAmide functionality and peptide bonds less reactive to hydrolysis than the ester functionality

  

VS

Mission is therefore to maximize rate of ester hydrolysis while minimizing degradation products – need kinetic data!

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Multiple Parallel reactions!

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

Modelling Rates of Reaction

Kinetics of Hydrolysis!

pHkHkk

kH

KkHkk

kOHkHkk

OHkOHkHkk

Esterkdt

Esterd

aah

NWb

ah

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obsh

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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

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

R1 CO

N R2

R3

R1 CO

O R2

log k

h

pH

Maximum selectivity at pH which maximizes differences in kh

At mildly acidic/neutral conditions, expect kN, kB to dominate for ester hydrolysis

Ideal pH range 6-8

Reaction Rate MonitoringAnalytical 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 (O- vs 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

1) 388 kg HCl 600 kg Water(or buffer)

Alkaline pH (~14)306 kg Insulin Ester426 kg eluted NaOH8293 kg WFI

HPLC etc.

Up to 82 kg Recycled Insulin Ester

213 kg Insulin83 kg Ester11.2 kg DenaturedBy-productspH 3-4

By-products

213kg InsulinpH (6-8)

Low T (ambient)

Cooling Water

Basic Process Flow Sheet

2) HCl

~12.3 m3

pH control

?

~16 m3 (with recycle)

Some Process DetailsInitial 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 BoltsWell 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….