Dosanjh, J.K.S.; (2011) Protein damage during purification: understanding the effects of size exclusion chromatography on the structure of biosynthetic human insulin (BHI). Doctoral thesis, UCL (University College London).
Full text not available from this repository.
Purification of Biosynthetic Human Insulin (BHI or Humulin), involves multimodal chromatographies as well as intermediate crystallizations. Size exclusion chromatography (SEC) is the final chromatographic step in BHI purification, which is conducted in acetic acid at high protein concentrations. The purpose of the SEC step is to remove the higher-molecular weight species proinsulin (molecular weight (MW) ≈ 10 kDa) and covalently linked insulin dimer (MW ≈ 11.6 kDa) from native insulin monomer (MW 5808 Da). Because covalent insulin dimers are recognised as a significant factor in immunological responses seen in diabetic patients (Darrington & Anderson 1995), limiting the amount of covalent in the final active pharmaceutical ingredient (API) is a key requirement for the development of the purification process. The presence of covalent dimers in insulin process streams results from chemical degradation of BHI either through intermolecular disulphide bond formation or from transamidation. Complete characterisation of the covalent dimeric species present in commercial SEC feed streams has not been performed. However, because specific purification technology is employed to remove disulphide bonded polymeric impurities upstream of the SEC step, dimeric impurities are thought to be predominantly transamidated species. Further, recent laboratory-scale SEC studies have indicated that there is a covalent dimer form present in the SEC feed streams that is difficult to separate from the native BHI molecule by SEC. The objectives of this study were to determine the types of insulin covalent dimer entering the BHI SEC step, understand the impact of the covalent insulin dimer type on SEC separation of dimer from the native insulin and to learn more about the SEC step itself i.e., investigations into the effect the SEC resin (G-50 Sephadex), may have had on the BHI structure. In order to achieve this, various techniques were explored to analyse BHI conformation, purity and degradation trends. Such techniques included Sodium dodecyl sulphate polyacrylamide gel electrophoresis SDS-PAGE, High Performance Liquid Chromatography (Reverse Phase and Size Exclusion Chromatography), Circular Dichorism and Surface Enhanced Laser Desorption Ionisation (SELDI). Breakdown studies on BHI were carried out using reducing agents DTT and GdnHCl and the effects of environmental conditions (pH, temperature and protein concentration), on native BHI as well as the reduced protein were investigated. Further to this, studies carried out at Eli Lilly and Company explored the occurrence of the fronting observed after SEC purification. Here the human proinsulin (HPI), high molecular weight polymer (HMWP) and BHI were thought to exist together and were separated from the main BHI peak, and then recycled in order to separate and recapture BHI in order to increase yield. Studies were conducted to understand the occurrence of the fronting better, as well as to see if factors such as presence of Zinc could alter the fronting and whether this could ultimately lead to steps being taken to modify the manufacturing process to make it more efficient and economically sound. Finally, the folding reaction where human proinsulin s-sulfonate (HPSS) is converted to HPI during BHI manufacture was investigated. During this step, the HPI purity and behaviour was assessed in order to determine whether changes could be made here in order to make the manufacturing process more efficient. Overall it was found that the species present during BHI analysis were BHI monomer and transamidated dimer. Upon reduction by denaturing agents such as DTT with GdnHCl, the BHI monomer was found to become reduced to form its constituent A and B chains. More of a dimer peak was observed also, which was a combination of transamidated dimer, but also BHI monomer being eluted sooner during HPLC due to its reversible formation of self-associated dimer as it flowed through the column. An additional small peak was also observed which was suspected to be an AA-AA dimer, but further experimentation is required to confirm its identity. Addition of Sephadex caused BHI monomer to be more stable, thus more difficult to reduce using denaturing agents such as DTT and GdnHCl to its constituent A and B Chains. The sephadex induced a shift in equilibrium between BHI and Chains A and B such that the BHI protein remained in a more folded state, thus making it harder for the DTT to access and reduce the disulphide bonds. With regards to environmental conditions, it was found that an increase of temperature lead to an increase of degradation/reduction of BHI monomer, resulting in an increase of all the other peaks. The general trend observed with regards to pH was that BHI monomer was most stable at pH 3 at all the temperatures tested, and pH 4 was the most unstable except at 40°C where an increase of pH appeared to encourage misfolding and avoid aggregation. Complete unfolding of BHI was only found to occur at pH 9 (where all the pH’s examined were 1.5, 2, 3, 3.5, 4, and 9). Concentration changes resulted in an expected increase in peak areas in SEC HPLC, and it did show that more BHI dimer was likely to form as a result of increased concentration. A number of theories were tested to understand the occurrence of fronting in the BHI elution peak from SEC. It was found to result from the BHI forming a self-associated dimer on the column, thus eluting earlier but then returning to its original monomeric state. This explained why BHI monomer was found in the fronting area along with HMWP and HPI, and therefore had to be recycled and repurified. It was also found that chelating zinc ions from the BHI sample did not have an effect on the equilibrium between monomer and dimer, or on the fronting observed on the large scale SEC HPLC. It was seen however that when the G-50 SEC column was saturated with ZnCl2 the BHI monomer peak shifted towards being eluted at a higher MW (towards the left), thus promotion of zinc induced BHI dimerisation. Although this resulted in increased fronting, this is more likely now to be due to the harmless self-associated dimers that are reversibly formed as opposed to non-reversible covalently formed dimers, so the need for the cut and recycle step to remove the covalent dimer, would no longer be needed. However it was also found that the HPI content was significantly increased in the Main Stream (MS) step (as illustrated in Figure 7.1) which decreased the final BHI purity, and so overall the saturation with zinc did not allow the recycle step to be avoided during manufacturing. Thus it was better to keep the initial fronting and avoid Zn induced dimerisation. Finally with regards to HPI purity during the manufacturing step where HPSS was converted to HPI, it was found that disulphide shuffling occurred with a corresponding increase of HMWP, but only for the first 40-50 hours, after which the polymer fraction decreased and HPI fraction increased until a plateau was reached. A variation in the time to reach the plateau is likely to be due to slight variations in added cysteine concentration. It is recommended that the cysteine concentration should be investigated in further experimentation in order to speed the reaction up so that the plateau could be reached faster. This decreased manufacturing time would result in a more time and cost efficient process.
|Title:||Protein damage during purification: understanding the effects of size exclusion chromatography on the structure of biosynthetic human insulin (BHI)|
|Additional information:||Permission for digitisation not received|
|UCL classification:||UCL > School of BEAMS > Faculty of Engineering Science > Biochemical Engineering|
Archive Staff Only: edit this record