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Insulin Ester Hydrolysis Megan Palmer Chee 450. Conversion of Insulin Ester Following enzymatic cleavage, must de-protect Thr B30 ester into Thr B30 carboxylic.

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Presentation on theme: "Insulin Ester Hydrolysis Megan Palmer Chee 450. Conversion of Insulin Ester Following enzymatic cleavage, must de-protect Thr B30 ester into Thr B30 carboxylic."— Presentation transcript:

1 Insulin Ester Hydrolysis Megan Palmer Chee 450

2 Conversion of Insulin Ester Following enzymatic cleavage, must de-protect Thr B30 ester into Thr B30 carboxylic acid to convert to final functional human insulin product Thr 30 )-OR  Thr 30 )-OH

3 Deesterification Reaction Reaction 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

4 Formation of desamidoinsulins Amide groups also undergo hydrolysis! Most prominent nonenzymatic degradation reaction of insulin is deamidation Six Resides are potential deamidation sites: Gln A5, Gln A15, Asn A18, Asn A21, Asn B3, and Gln B4 C-terminal residue Asn A21 is very labile in acidic pH  forms desamido (A21 isulin) Changes charge and hydrophilic/hydrophobic properties -- forces controlling protein structure! Deamidation Rates: Asn >> Gln

5 Asp/IsoAsp deamidation products In neutral solutions, deamidation primarily occurs at residue Asn B3 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

6 Peptide Bond Cleavage Peptide bonds can also be hydrolyzed! Peptide bond between Thr A8 and Ser A9 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 40 0 C has been estimated to be as short as 50 hours (acid hydrolysis). At pH 5-7, the degradation rate is expected to be slower

7 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:

8 Selectivity in Reaction Rates Amide 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! Multiple Parallel reactions!

9 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

10 Kinetics of Hydrolysis! Rate Law for Hydrolysis typically modeled as pseudo-first order at a constant pH: [RX] independence as [H + ], [OH - ], [H 2 O] 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 k h from slope Determine dependence of rate of hydrolysis on pH for each reaction and individual k a, k b, k N by plotting k h vs pH

11 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

12 Choosing Reaction Conditions log k h pH Maximum selectivity at pH which maximizes differences in k h At mildly acidic/neutral conditions, expect k N, k B to dominate for ester hydrolysis Ideal pH range 6-8

13 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 (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

14 1) 388 kg HCl 600 kg Water (or buffer) Alkaline pH (~14) 306 kg Insulin Ester 426 kg eluted NaOH 8293 kg WFI HPLC etc. Up to 82 kg Recycled Insulin Ester 213 kg Insulin 83 kg Ester 11.2 kg Denatured By-products pH 3-4 By-products 213kg Insulin pH (6-8) Low T (ambient) Cooling Water Basic Process Flow Sheet 2) HCl ~12.3 m 3 pH control ? ~16 m 3 (with recycle)

15 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 (2 nd 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 k h (ester hydrolysis) 33x k h (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

16 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 m 3 agitated reactor manufactured by Novo Nordisk (leader in recombinant insulin production)

17 Integrated Process Design and Economics

18 Questions? I’m sure you have many….

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