Combined optimization of N‐terminal site‐specific PEGylation of recombinant hirudin using response surface methodology and kinetic analysis

Abstract

In this study, a combined optimization method was developed to optimize the N‐terminal site‐specific PEGylation of recombinant hirudin variant‐2 (HV2) with different molecular weight mPEG‐propionaldehyde (mPEG‐ALD), which is a multifactor‐influencing process. The HV2‐PEGylation with 5 kDa mPEG‐ALD was first chosen to screen significant factors and determine the locally optimized conditions for maximizing the yield of mono‐PEGylated product using combined statistical methods, including the Plackett–Burman design, steepest ascent path analysis, and central composition design for the response surface methodology (RSM). Under the locally optimized conditions, PEGylation kinetics of HV2 with 5, 10, and 20 kDa mPEG‐ALD were further investigated. The molar ratio of polyethylene glycol to HV2 and reaction time (the two most significant factors influencing the PEGylation efficiency) were globally optimized in a wide range using kinetic analysis. The data predicted by the combined optimization method using RSM and kinetic analysis were in good agreement with the corresponding experiment data. PEGylation site analysis revealed that almost 100% of the obtained mono‐PEGylated‐HV2 was modified at the N‐terminus of HV2. This study demonstrated that the developed method is a useful tool for the optimization of the N‐terminal site‐specific PEGylation process to obtain a homogeneous mono‐PEGylated protein with desirable yield.

Kinetic Optimization and Scale-Up of Site-Specific Thiol-PEGylation of Loxenatide from Laboratory to Pilot Scale

“…This model considered three reactions concerning the generation of mono-, di-, and tri-PEGylated proteins, and one reaction concerning the deactivation of PEG reagent itself, which might be used as a general PEGylation reaction kinetics model to describe most of the PEGylation reactions. The parameter identifiability of this model and its simplified form has been demonstrated to be simple and valid in our previous studies. − In this study, the constructed reaction kinetics model for site-specific thiol-PEGylation of loxenatide can be regarded as its further simplified form, and the results (Figure , Tables S1–S7, and Table ) also showed that the parameter identifiability of the constructed model is simple and valid.…”

“…However, the optimization and scale-up of the PEGylation reaction process to a pilot or industrial scale requires the understanding of the underlying mechanism of the PEGylation reaction. In this context, the reaction kinetic model can provide detailed information about the mechanism of PEGylation, and has been proved to be a powerful tool to optimize the amino-PEGylation of proteins and peptides on a laboratory scale. − However, a reaction kinetic model for the thiol-PEGylation of proteins and peptides has not yet been reported to date. The aim of this study is to develop a reaction kinetic model based method for the optimization of site-specific thiol-PEGylation of loxenatide and for scaling up the PEG-loxenatide production process to a pilot scale, thereby providing basis for the production of PEG-loxenatide on an industrial scale.…”

Site-selective fatty acid chain conjugation of the N-terminus of the recombinant human granulocyte colony-stimulating factor

“…Two unknown elution peaks at retention times of 22.8 and 23.8 min, respectively, were collected and detected by MS ( Supplementary Figures S2, S3 ), which indicated that both the peaks corresponded to single-fatty acid chain-modified rhG-CSF. Previous studies demonstrated that aldehyde chemistry was preferential and high-selective modification at α -amine of N-terminus, and concurrently minor competitive modification at ε -amine of lysine residues under acidic condition ( Wang et al, 2011 ; Wang et al, 2018 ). Therefore, the peak at retention time of 22.8 min was identified as single-fatty acid chain-modified rhG-CSF at α -amine of N-terminus, which was named as PAL-PEG 3 -Ph-rhG-CSF.…”

“…Commercially available rhG-CSF injections are typically stored in acetic acid buffer at a pH of around 4.2, and the isoelectric point of rhG-CSF is 6.1 ( Liu et al, 2020 ). Besides, the ratio of competitive modification at ε-amine of lysine residues increased with the increasing pH ( Wang et al, 2011 ; Wang et al, 2018 ). Hence, pH 5.0 was selected as the optimized condition for subsequent experiments.…”

“…When the molar ratio reached 6:1, further increasing the molar ratio did not significantly increase the reaction yield. Besides, multi-fatty acid chain-modified rhG-CSF products might generate with the increasing molar ratio ( Wang et al, 2011 ; Wang et al, 2018 ). Therefore, we selected 6:1 as the optimized molar ratio of PAL-PEG 3 -Ph-CHO to rhG-CSF.…”