Insulin Ester Hydrolysis

1. Insulin Ester Hydrolysis Megan Palmer Chee 450

2. 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

3. 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

4. Formation of desamidoinsulins Amide 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) Deamidation Rates: Asn >> Gln Changes charge and hydrophilic/hydrophobic properties -- forces controlling protein structure!

5. 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 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 Intramolecular rearrangement Maximum stability against deamidation at around pH 6, where reaction is 5 to 10-fold slower than at pH 7.4

6. 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

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

9. 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

10. 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

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. log kh pH Choosing Reaction Conditions 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

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. 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) 2) HCl pH control Cooling Water pH (6-8) Low T (ambient) 213kg Insulin HPLC etc. 213 kg Insulin 83 kg Ester 11.2 kg Denatured By-products pH 3-4 By-products ~12.3 m3 ~16 m3 (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 (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

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 m3 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….