微生物来源的谷氨酰胺转氨酶(mTG)介导的单一定点修饰的PEG IFN-α2a(英文)

PEG修饰被认为是改善重组蛋白药物特性的最有效手段,包括增加蛋白质药物在体内的血浆半衰期,降低免疫原性和抗原性。目前典型的PEG修饰手段为将PEG连接至蛋白质的游离氨基,包括赖氨酸和N-末端,但这种连接缺乏选择性,产物为混合物,活性及工艺稳定性差,难以控制。酶法PEG化修饰能有效克服上述缺点,其中谷氨酰胺转氨酶(TGase)可以作为PEG化定点修饰用酶。文中选择重组人干扰素α2a(IFNα2a)进行酶法修饰反应,通过计算机模拟预测IFNα2a可以在第101位Gln特异性定点修饰。将IFNα2a与40 kDa的Y型PEG在微生物来源的谷氨酰胺转氨酶(mTG)催化下进行定点PEG化修饰。结果显示,mTG可以介导IFNα2a特异性位点Gln的单一定点PEG修饰,产生分子量为58 495.6 Da的PEG-Gln101-IFNα2a分子。圆二色谱结果显示,PEG-Gln101-IFNα2a与未修饰的IFNα2a具有相同的二级结构。SD大鼠药代结果显示,与IFNα2a相比,PEG-Gln101-IFNα2a能有效提高药代动力学参数,强于已上市PEGIFNα2a-PEGASYS?。     

Chromatographic methods for characterization of poly(ethylene glycol)-modified polyamidoamine dendrimers

The objective of this study was to develop chromatographic methods for the determination of the modification degree and the characterization of poly(ethylene glycol)-modified polyamidoamine dendrimers (PEG–PAMAMs). The PEG–PAMAMs were prepared by reacting PAMAM generation 4 with monomethoxy PEG–nitrophenyl carbonate (mPEG–NPC). The modification degrees of PEG–PAMAMs were determined by quantifying 4-nitrophenol released from mPEG–NPC after PEGylation reaction using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The PEG–PAMAMs, which have poor UV absorbances, were characterized by HPLC with charged aerosol detection. This study demonstrates that the combination of these two detectors is a powerful tool for the preparation and characterization of PEG–PAMAMs.     

Shielding effect of a PEG molecule of a mono-PEGylated peptide varies with PEG chain length

Abstract

‘Shielding’ effect of a conjugated PEG molecule could cause a change in the electrostatic interaction characteristics of a PEGylate. We investigated how PEG chain length (or molecular weight) alters the electrostatic interaction potential of exenatide variants using their mono-PEGylates in a branched and linear form as model PEGylates. First, we performed the experiments to demonstrate the elution time changes of the mono-PEGylates conjugated with various MW PEGs (5, 10, 20, and 40 kD) using cation exchange chromatography (HiTrap^® SP) at various pHs (2.5, 3.0, 3.5, and 4.0). Then, we calculated the net surface charge of each mono-PEGylate to propose the PEG molecule’s shielding range in terms of the number of amino acids adjacent to the conjugation residue, assuming that a PEG molecule in solution sweeps out a spherical space and an exenatide molecule have a secondary structure. The net charge calculation result was well-correlated with the experimental elution time data, where 5, 10, 20, and 40 kD PEG hindered the electrostatic potential of 5, 8, 12, and 17 amino acid residues in maximum, respectively, on each side of the conjugation point.     

Purification,immobilization, and biochemical characterization of l‐arginine deiminase from thermophilic Aspergillus fumigatus KJ434941: Anticancer activity in vitro

l ‐Arginine deiminase (ADI) has a powerful anticancer activity against various tumors, via arginine depletion, arresting the cell cycle at G₁ phase. However, the current clinically tried bacterial ADI displayed a higher antigenicity and lower thermal stability. Thus, our objective was to purify and characterize this enzyme from thermophilic fungi, to explore its catalytic and antigenic properties for therapeutic uses. ADI was purified from thermophilic Aspergillus fumigatus KJ434941 to its electrophoretic homogeneity by 5.1‐fold, with molecular subunit 50 kDa. The purified ADI was PEGylated and covalently immobilized on dextran to explore its catalytic properties. The specific activity of free ADI, PEG‐ADI, and Dex‐ADI was 26.7, 21.5, and 18.0 U/mg, respectively. At 50°C, PEG‐ADI displays twofold resistance to thermal denaturation (t_1/2 13.9 h), than free ADI (t_1/2 6.9 h), while at 70°C, the thermal stability of PEG‐ADI was increased by 1.7‐fold, with similar stability to Dex‐ADI with the free one. Kinetically, free ADI had the higher catalytic affinity to arginine, followed by PEG‐ADI and Dex‐ADI. Upon proteolysis for 30 min, the residual activity of native ADI, PEG‐ADI, and Dex‐AD was 8.0, 32.0, and 20.0% for proteinase K and 10.0, 52.0, and 90.0% for acid protease, respectively. The anticancer activity of the ADIs was assessed against HCT, HEP‐G2, and MCF7, in vitro. The free and PEG‐ADI exhibits a similar cytotoxic efficacy for the tested cells, lower than Dex‐ADI. The free ADI had IC₅₀ value 22.0, 16.6, and 13.9 U/mL, while Dex‐ADI had 3.98, 5.18, and 4.43 U/mL for HCT, MCF7, and HEPG‐2, respectively. The in vitro anticancer activity of ADI against HCT, MCF7, and HEPG‐2 was increased by five‐, three‐, and threefold upon covalent modification by dextran. The biochemical and hematological parameters of the experimented animals were not affected by ADIs dosing, with no signs of anti‐ADI immunoglobulins in vivo. The in vivo half‐life time of free ADI, PEG‐ADI, and Dex‐ADI was 29.7, 91.1, 59.6 h, respectively. The present findings explored a novel thermostable, less antigenic ADI from thermophilic A. fumigatus, with further molecular and crystallographic analyses, this enzyme will be a powerful candidate for clinical trials. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:396–405, 2015     

Enzymatic activity and thermal stability of PEG-α-chymotrypsin conjugates

α-Chymotrypsin was chemically modified with methoxypoly(ethylene glycol) (PEG) of different molecular weights (700, 2,000, and 5,000 Da) and the amount of polymer attached to the enzyme was varied systematically from 1 to 9 PEG molecules per enzyme molecule. Upon PEG conjugation, enzyme catalytic turnover (k _cat) decreased by 50% and substrate affinity was lowered as evidenced by an increase in the K _M from 0.05 to 0.19 mM. These effects were dependent on the amount of PEG bound to the enzyme but were independent of the PEG size. In contrast, stabilization toward thermal inactivation depended on the PEG molecular weight with conjugates with the larger PEGs being more stable.     

PEG化重组人白细胞介素-6制品游离PEG测定方法的研究

为了建立PEG化学修饰的重组人白细胞介素-6(PEG-rhIL-6)的部分质量控制方法,参照2005年版《中华人民共和国药典》三部附录III B高效液相色谱法,以RP-HPLC方法分离PEG-rhIL-6原液中的不同成分,用蒸发光散射监测器检测游离PEG,用外标法测定计算样品中残留PEG的含量。PEG修饰rhIL-6结构稳定;制品中的游离PEG含量符合要求。RP-HPLC法检测游离PEG结果可靠。     

An oriented adsorption strategy for efficient solid phase PEGylation of recombinant staphylokinase by immobilized metal-ion affinity chromatography

Conjugation of truncated recombinant staphylokinase (trSak) with polyethylene glycol (PEG) is an effective way to overcome its short plasma half-life and enhance its therapeutic potential. However, conventional amine directed PEGylation chemistry inevitably led to modification at its functionally important N terminus, which resulted in a significantly reduced bioactivity of trSak. In this study, a novel solid phase PEGylation process was developed to shield the N-terminal region of the protein from PEGylation. The process was achieved by oriented adsorption of an N-terminally His-tagged trSak (His-trSak) onto an immobilized metal-ion affinity chromatography (IMAC). His-trSak was efficiently separated and retained on IMAC media before reaction with succinimidyl carbonate mPEG (SC-mPEG, 5, 10 or 20 kDa). The IMAC derived mono-PEGylated His-trSak showed structural and stability properties similar to the liquid phase derived conjugate. However, isoelectric focusing electrophoresis analysis revealed that mono-PEGylated His-trSaks via solid phase PEGylation were more homogeneous than those from liquid phase PEGylation. Moreover, tryptic peptide mapping analysis suggested that a complete N-terminal blockage of IMAC bound His-trSak from PEGylation with 10 kDa- and 20 kDa-SC-mPEG. In contrast, only partial protection of the N-terminal region was obtained for 5 kDa-SC-mPEG. Bioactivities of 10 kDa- and 20 kDa-PEG-His-trSak conjugates without N-terminal PEGylation were significantly higher than those of randomly PEGylated products. This further demonstrated the advantage of our new on-column PEGylation strategy.     

Propylbenzmethylation at Val-1(α) markedly increases the tetramer stability of the PEGylated hemoglobin: A comparison with propylation at Val-1(α)

Background

Hemoglobin (Hb)-based oxygen carriers (HBOCs) are potential pharmaceutical agents that can be used in surgery or emergency medicine. PEGylation can modulate the vasoactivity of Hb and is a widely used approach to develop HBOCs. However, PEGylation can significantly enhance the tetramer–dimer dissociation of Hb, which may perturb the structure of Hb and increase its observed adverse effect. Thus, it is necessary to increase the tetramer stability of the PEGylated Hb.

Methods

Propylbenzmethylation at Val-1(α) of HbA was carried out to stabilize the Hb tetramer. The propylbenzmethylated Hb at Val-1(α) (PrB-Hb) was used as the starting material for site-specific PEGylation at Cys-93(β) of Hb using maleimide PEG. Structural and functional properties, autoxidation rate and thermal stability of the resultant product (PEG-PrB-Hb) were measured.

Results

Propylbenzmethylation at Val-1(α) led to 25-fold and 24-fold decreases in the tetramer–dimer dissociation constant of HbA and PEG-Hb, respectively. The increased tetramer stability is due to the enhanced hydrophobicity of the area around Val-1(α) and the increased polar interaction of Hb upon propylbenzmethylation. Thus, the structural and functional properties of PEG-Hb were improved, and its autoxidation rate and thermal denaturation were decreased.

Conclusion

Propylbenzmethylation at Val-1(α) showed higher ability than propylation at Val-1(α) to improve the structural and functional properties and decrease the side effect of PEG-Hb.

General significance

Our study can facilitate the biotechnological development of stable PEGylated Hb as more advanced HBOC. Our study is also expected to improve the stability of the tetrameric or dimeric proteins (e.g., uric oxidase) by propylbenzmethylation at their N-terminus.     

聚乙二醇对PEG化重组人促红细胞生成素热稳定性和生物学活性的影响

为了研究聚乙二醇对PEG化rhEPO热稳定性和生物学活性的影响,用酶学方法切除rhEPO和PEGrhEPO分子中的N连接糖基,研究酶切产物在37℃时287nm和350nm吸光度随时间的变化;用MTT比色法,网织红细胞法和HCT法比较体内和体外生物学活性的改变。结果显示聚乙二醇修饰能显著减少无N连接糖基的rhEPO分子间的聚集,降低rhEPO体外生物学活性,但增高体内生物学活性;无N连接糖基的PEG化rhEPO具有与rhEPO相当的体内生物学活性。因此聚乙二醇能提高rhEPO热稳定性;聚乙二醇可替代糖基,维持rhEPO的体内生物学活性。用PEG修饰原核细胞表达的rhEPO是开发rhEPO制品的新思路。     

A zeolite as a tool for successful refolding of PEGylated proteins and their reassembly with tertiary structures

In the present study, we demonstrated zeolites' potential contribution to establish a method for preparing successfully refolded and reassembled PEGylated protein nanoparticles without the use of protein denaturants through the proteins' reassembly process. At first, the PEGylated nanoparticles are disassembled into identical PEGylated protein subunits by means of protein denaturants, and then the denatured subunits are adsorbed to zeolites. After the complete removal of denaturants, high-molecular-weight poly(ethylene glycol) (PEG) molecules are added to a solution where the zeolites suspend. Consequently, the PEGylated proteins are gradually reassembled into nanoparticles because the subunits are desorbed from the zeolites by the steric hindrance of the added PEG molecules. The present study reveals that PEGylated encapsulin was reassembled and hollow encapsulin nanoparticles were obtained. The results clearly demonstrate the usefulness of zeolites as a tool for the successful refolding of PEGylated proteins and their reassembly with tertiary structures.