重组人粒细胞集落刺激因子的N端氨基定点PEG化修饰

尽管重组粒细胞集落刺激因子(rhG-CSF)具有重大的治疗价值,然而在实际应用却受到体内半衰期过短因而需要频繁重复注射的限制．为了解决这一问题,我们利用两种不同分子量（5 kD和 20 kD）的单甲氧基聚乙二醇丙醛（mPEG-PAL）对rhG-CSF的N端氨基进行了定点PEG化修饰．通过正交实验的统计学方法得到了最适修饰条件．研究发现,PEG化后的rhG-CSF具有了更高的体外稳定性,其体内活性也得到了很大提高,体内作用时间得到很大延长．因此,对于rhG-CSF的N端氨基定点PEG化修饰,可以显著提高rhG-CSF的临床应用价值．     

Preparation and characterization of PEGyated Concanavalin A for affinity chromatography with improved stability

In order to improve its stability, immobilized Concanavalin A (Con A) on Toyopearl adsorbents was conjugated with monomethoxy poly(ethylene glycol) succinimidyl propionate (mPEG-SPA) with different molecular weight. A colorimetric method using ninhydrin is proposed to determine the degree of PEGylation; this method has proved to be easy applicable and reproducible. The PEGylation reaction was studied in detail to elucidate how parameters such as molar ratio of mPEG-SPA to Con A and molecular weight of mPEG-SPA affect the degree of PEGylation. The adsorption isotherms of glucose oxidase (GOD) onto native and PEGylated Con A adsorbents showed that the modification did not alter substantially the specificity of the carbohydrate binding ability of Con A. However, the binding capacity for GOD was slightly reduced probably due to the steric hindrance caused by mPEG chains. Adsorption kinetic studies revealed a lower adsorption rate after PEGylation which was attributed to the steric effect. The dynamic adsorption capacity for modified Con A depended very much on the degree of PEGylation and the molecular weight of mPEG derivatives. The adsorption capacity could be highly preserved for Toyopearl Con A modified by mPEG2k (90% of the original adsorption capacity) even with a degree of PEGylation up to 20% (the ratio of primary amino groups of PEGylated immobilized Con A to that of native immobilized Con A). Studies show that the binding capacity of PEGylated Con A was highly preserved under mild process conditions. PEGylated Con A also exhibited obviously higher stability against more stressful conditions such as the exposure to organic solvents and high temperatures. Conjugation of Con A with mPEG2k provided better adsorption performance thus has greater potential for application in affinity separation processes compared with mPEG5k. The fact that PEGylation stabilizes the properties of Con A may greatly expand the range of applications of unstable proteins to bioprocessing (e.g. biocatalysis and downstream separation) as well as other protein applications (e.g. medication, industrial use, etc.).     

重组人睫状神经营养因子的聚乙二醇化修饰简

目的：研究重组人睫状神经营养因子（rhCNTF）突变体的聚乙二醇（PEG）化修饰,对rhCNTF的PEG化产物进行初步分离纯化及相关生物活性检测。方法：采用分子生物学技术经点突变得到rhCNTF的突变体cNm通过实验设计研究CN10的最佳PEG化条件;采用分子筛层析方式对偶联产物进行初步纯化,最后用ELISA和小鼠体重增长抑制法检测PEG化后的CN。。蛋白的生物活性。结果：能运用mPEG—MAL对CN,。进行定点修饰,PEG化后用Superdex200能够分离CN10;PEG化后的CN10每2d腹腔注射1次,对小鼠体重的增长抑制率可达50％,与rhCNTF每天注射2次的体重增长抑制作用相当。结论：CN10蛋白在PEG化修饰后,其减重效应持续时间明显延长。     

Disulfide–bridging PEGylation during refolding for the more efficient production of modified proteins

Proteins that are modified by chemical conjugation require at least two separate purification processes. First the bulk protein is purified, and then after chemical conjugation, a second purification process is required to obtain the modified protein. In an effort to develop new enabling technologies to integrate bioprocessing and protein modification, we describe the use of disulfide‐bridging conjugation to conduct PEGylation during protein refolding. Preliminary experiments using a PEG‐mono‐sulfone reagent with partially unfolded leptin and unfolded RNAse T1 indicated that the cysteine thiols underwent disulfide‐bridging conjugation to give the PEGylated proteins. Interferon‐β1b (IFN‐β1b) was then expressed in E.coli as inclusion bodies and found to undergo disulfide bridging‐conjugation during refolding. The PEG‐IFN‐β1b was isolated by ion‐exchange chromatography and displayed in vitro biological activity. In the absence of the PEGylation reagent, IFN‐β1b refolding was less efficient and yielded protein aggregates. No PEGylation was observed if the cysteines on IFN‐β1b were first modified with iodoacetamide prior to refolding. Our results demonstrate that the simultaneous refolding and disulfide bridging PEGylation of proteins could be a useful strategy in the development of affordable modified protein therapeutics.     

Site-specific PEGylation at histidine tags

The efficacy of protein-based medicines can be compromised by their rapid clearance from the blood circulatory system. Achieving optimal pharmacokinetics is a key requirement for the successful development of safe protein-based medicines. Protein PEGylation is a clinically proven strategy to increase the circulation half-life of protein-based medicines. One limitation of PEGylation is that there are few strategies that achieve site-specific conjugation of PEG to the protein. Here, we describe the covalent conjugation of PEG site-specifically to a polyhistidine tag (His-tag) on a protein. His-tag site-specific PEGylation was achieved with a domain antibody (dAb) that had a 6-histidine His-tag on the C-terminus (dAb-His(6)) and interferon α-2a (IFN) that had an 8-histidine His-tag on the N-terminus (His(8)-IFN). The site of PEGylation at the His-tag for both dAb-His(6)-PEG and PEG-His(8)-IFN was confirmed by digestion, chromatographic, and mass-spectral studies. A methionine was also inserted directly after the N-terminal His-tag in IFN to give His(8)Met-IFN. Cyanogen bromide digestion studies of PEG-His(8)Met-IFN were also consistent with PEGylation at the His-tag. By using increased stoichiometries of the PEGylation reagent, it was possible to conjugate two separate PEG molecules to the His-tag of both the dAb and IFN proteins. Stability studies followed by in vitro evaluation confirmed that these PEGylated proteins retained their biological activity. In vivo PK studies showed that all of the His-tag PEGylated samples displayed extended circulation half-lives. Together, our results indicate that site-specific, covalent PEG conjugation at a His-tag can be achieved and biological activity maintained with therapeutically relevant proteins.     

Solid-phase N-terminus PEGylation of recombinant human fibroblast growth factor 2 on heparin-sepharose column

Recombinant fibroblast growth factor-2 (FGF-2) has been extensively studied and used in several clinical applications including wound healing, bone regeneration, and neuroprotection. Poly(ethylene glycol) (PEG) modification of recombinant human FGF-2 (rhFGF-2) in solution phase has been studied to increase the in vivo biostabilities and therapeutic potency. However, the solution-phase strategy is not site-controlled and the products are often not homogeneous due to the generation of multi-PEGylated proteins. In order to increase mono-PEGylated rhFGF-2 level, a novel solid-phase strategy for rhFGF-2 PEGylation is developed. RhFGF-2 proteins were loaded onto a heparin-sepharose column and the PEGylaton reaction was carried out at the N-terminus by PEG20 kDa butyraldehyde through reductive alkylation. The PEGylated rhFGF-2 was purified to near homogeneity by SP sepharose anion-exchange chromatography and the purity was more than 95% with a yield of mono-PEGylated rhFGF-2 of 58.3%, as confirmed by N-terminal sequencing and MALDI-TOF mass spectrometry. In vitro biophysical and biochemical measurements demonstrated that PEGylated rhFGF-2 has an unchanged secondary structure, receptor binding activity, cell proliferation, and MAP kinase stimulating activity, and an improved bio- and thermal stability. Animal assay showed that PEGylated rhFGF-2 has an increased half-life and reduced immunogenicity. Compared to conventional solution-phase PEGylation, the solid-phase PEGylation is advantageous in reaction time, production of mono-PEGylated protein, and improvement of biochemical and biological activity.     

Chemically defined polyethylene glycol siRNA conjugates with enhanced gene silencing effect

The therapeutic application of siRNA suffers from poor bioavailability caused by rapid degradation and elimination. The covalent attachment of PEG is a universal concept to increase molecular size and enhance the pharmacokinetic properties of biomacromolecules. We devised a facile approach for attachment of PEG molecules with a defined molecular weight, and successful purification of the resulting conjugates. We directly conjugated structurally defined PEG chains with twelve ethylene glycol units to the 3′-terminal hydroxyl group of both sense and antisense strands via an aminoalkyl linker. The conjugates were easily purified by HPLC and successful PEGylation and molecule integrity were confirmed by ESI-MS. The evaluation of in vitro gene knockdown of two different targets in MCF-7 breast cancer cells showed stable pharmacologic activity when combined with a standard transfection reagent. Sense strand PEGylation even increased the silencing potency of a CRCX4-siRNA which had modest activity in its wild-type form. The results indicate that PEG chains at the 3′-terminus of both strands of siRNA are well tolerated by the RNAi effector. The attachment of short, chemically defined PEG chains is a feasible approach to improve the pharmacokinetic properties of siRNA, and can be combined with other targeted and untargeted delivery vehicles.     

Optimal site-specific PEGylation of mutant TNF-alpha improves its antitumor potency

Recently, we created a lysine-deficient mutant tumor necrosis factor-alpha [mTNF-alpha-Lys(-)] with full bioactivity in vitro compared with wild-type TNF-alpha (wTNF-alpha), and site-specific PEGylation of mTNF-alpha-Lys(-) was found to selectively enhance its in vivo antitumor activity. In this study, we attempted to optimize this PEGylation of mTNF-alpha-Lys(-) to further improve its therapeutic potency. mTNF-alpha-Lys(-) was site-specifically modified at its N-terminus with linear polyethylene glycol (LPEG) or branched PEG (BPEG). While randomly mono-PEGylated wTNF-alpha (ran-LPEG5K-wTNF-alpha) with 5 kDa of LPEG (LPEG5K) had about only 4% in vitro bioactivity of wTNF-alpha, mono-PEGylated mTNF-alpha-Lys(-) [sp-PEG-mTNF-alpha-Lys(-)] with LPEG5K, LPEG20K, BPEG10K, and BPEG40K had 82%, 58%, 93%, and 65% bioactivities of mTNF-alpha-Lys(-), respectively. sp-LPEG-mTNF-alpha-Lys(-) and sp-BPEG10K-mTNF-alpha-Lys(-) had much superior antitumor activity to those of both unmodified TNF-alphas and ran-LPEG5K-wTNF-alpha, though sp-BPEG40K-mTNF-alpha-Lys(-) did not show in vivo antitumor activity. Thus, the molecular shape and weight of PEG may strongly influence the in vivo antitumor activity of sp-PEG-mTNF-alpha-Lys(-).     

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?。     

Proteolytically stabilizing fibronectin without compromising cell and gelatin binding activity

Excessive proteolytic degradation of fibronectin (FN) has been implicated in impaired tissue repair in chronic wounds. We previously reported two strategies for stabilizing FN against proteolytic degradation; the first conjugated polyethylene glycol (PEG) through cysteine residues and the second conjugated PEG chains of varying molecular weight on lysine residues. PEGylation of FN via lysine residues resulted in increased resistance to proteolysis with increasing PEG size, but an overall decrease in biological activity, as characterized by cell and gelatin binding. Our latest method to stabilize FN against proteolysis masks functional regions in the protein during lysine PEGylation. FN is PEGylated while it is bound to gelatin Sepharose beads with 2, 5, and 10 kDa PEG precursors. This results in partially PEGylated FN that is more stable than native FN and whose proteolytic stability increases with PEG molecular weight. Unlike completely PEGylated FN, partially PEGylated FN has cell adhesion, gelatin binding, and matrix assembly responses that are comparable to native FN. This is new evidence of how PEGylation variables can be used to stabilize FN while retaining its activity. The conjugates developed herein can be used to dissect molecular mechanisms mediated by FN stability and functionality, and address the problem of FN degradation in chronic wounds. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:277–288, 2015     

PEGylation of native disulfide bonds in proteins

PEGylation has turned proteins into important new biopharmaceuticals. The fundamental problems with the existing approaches to PEGylation are inefficient conjugation and the formation of heterogeneous mixtures. This is because poly(ethylene glycol) (PEG) is usually conjugated to nucleophilic amine residues. Our PEGylation protocol solves these problems by exploiting the chemical reactivity of both of the sulfur atoms in the disulfide bond of many biologically relevant proteins. An accessible disulfide bond is mildly reduced to liberate the two cysteine sulfur atoms without disturbing the protein's tertiary structure. Site-specific PEGylation is achieved with a bis-thiol alkylating PEG reagent that sequentially undergoes conjugation to form a three-carbon bridge. The two sulfur atoms are re-linked with PEG selectively conjugated to the bridge. PEGylation of a protein can be completed in 24 h and purification of the PEG-protein conjugate in another 3 h. We have successfully applied this approach to PEGylation of cytokines, enzymes, antibody fragments and peptides, without destroying their tertiary structure or abolishing their biological activity.     

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

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.     

Characterization of site-specific ScFv PEGylation for tumor-targeting pharmaceuticals

New radiopharmaceuticals are possible using site-specific conjugation of small tumor binding proteins and poly(ethylene glycol) (PEG) scaffolds to provide modular multivalent, homo- or heterofunctional cancer-targeting molecules having preferred molecular size, valence, and functionality. Residence time in plasma can be optimized by modification of the size, number, and charge of the protein units. However, random PEG conjugation (PEGylation) of these small molecules via amine groups has led to variations of structural conformation and binding affinity. To optimize PEGylation, scFvs have been recombinantly produced in a vector that adds an unpaired cysteine (c) near the scFv carboxy terminus (scFv-c), thus providing a specific site for thiol conjugation. To evaluate the general applicability of this unpaired cysteine for PEGylation of scFv-c, conjugation efficiency was determined for four different scFvs and several PEG molecules having thiol reactive groups. The effect of the PEG molecular format on scFv-c PEG malignant cell binding was also addressed. ScFvs produced as scFv-c and purified by anti E-TAG affinity chromatography were conjugated using PEG molecules with maleimide (Mal) or o-pyridyl disulfide (OPSS). Conjugations were performed at pH 7.0, with 2 molar excess TCEP/scFv and PEG-(Mal) or PEG-OPSS, using 5:1 (PEG/scFv). PEG-Mal conjugation efficiency was also evaluated with 1:5 (PEG/scFv). PEGylation efficiency was determined for each reaction by quantitation of the products on SDS-PAGE. ScFv-c conjugation with unifunctional maleimide PEGs resulted in PEG conjugates incorporating 30-80% of the scFv-c, but usually above 50%. Efficiency of scFv-c conjugation to both functional groups of the bifunctional PEG-(Mal)2 varied between the PEG and scFv-c molecules studied. A maximum of 45% of scFv-c protein was conjugated as PEG- (scFv-c)2 using the smallest PEG-(Mal)2 (2 kDa). No significant increase in scFv-c conjugation was observed by the use of greater than a 5 molar excess of PEG/scFv-c. Under the same conjugation conditions, PEG as OPSS yielded less than 10% PEG-scFv-c. PEG-(scFv)2 conjugates had increased binding in ELISA using malignant cell membranes, when compared with unmodified scFv-c. PEGylated-scFv binding was comparable with unmodified scFv-c. In summary, scFv-c can be PEGylated in a site-specific manner using uni- or bivalent PEG-Mal, either linear or branched. ScFv-c was most efficiently conjugated to smaller PEG-Mal molecules, with the smallest, 2 kDa PEG-Mal, usually PEGylating 60-90% of the scFv-c. ScFv-c conjugation to form PEG-(scFv-c)2 reached greatest efficiency at 45%, and its purified form demonstrated greater binding than the corresponding scFv-c.     

Preparation and stability of N-terminal mono-PEGylated recombinant human endostatin

Endostatin can specifically inhibit endothelial proliferation and potently inhibit angiogenesis and tumor growth. N-Terminal site-specific mono-PEGylation of recombinant human endostatin (mPEG-rhES) was accomplished by using methoxy poly-ethylene glycol (mPEG) propionaldehyde with an average molecular weight of 5000 Da through a reactive terminal aldehyde group. The site-specific mPEG conjugation was conducted under optimal conditions, which were identified through a statistical L(9)(3(4)) orthogonal test. In this study, we have investigated the stability and antitumor activity of mPEG-rhES. SDS-PAGE, RP-HPLC, and UV spectrophotometric analysis were used to identify the purity and stability of mPEG-rhES. When incubated with protease or placed in an extreme environment, mPEG-rhES was more stable than rhES. The unmodified and PEGylated rhES were tested for their ability to inhibit the tumor growth of mouse H22 liver cancer in male mice. In a multiple versus single doses comparison study, daily administration of 0.25, 0.50, and 1.00 micromol/kg of unmodified rhES for 7 days resulted in 26.9%, 43.0%, and 64.9% reductions in tumor weight, respectively, while single doses of 0.13, 0.25, and 0.50 micromol/kg of the PEGylated protein per day resulted in 24.8%, 38.0%, and 64.5% reductions, respectively. Both treatments resulted in statistically significant reductions in mean tumor weight as compared to the physiological saline solution (control)-treated mice, with the dose of mPEG-rhES being a half of rhES, respectively, while the tumor inhibition rates were similar. Therefore, it is suggested that PEGylation enhances the stability of rhES and improves its antitumor activity.     

Site-specific PEGylation of protein disulfide bonds using a three-carbon bridge

The covalent conjugation of a functionalized poly(ethylene glycol) (PEG) to multiple nucleophilic amine residues results in a heterogeneous mixture of PEG positional isomers. Their physicochemical, biological, and pharmaceutical properties vary with the site of conjugation of PEG. Yields are low because of inefficient conjugation chemistry and production costs high because of complex purification procedures. Our solution to these fundamental problems in PEGylating proteins has been to exploit the latent conjugation selectivity of the two sulfur atoms that are derived from the ubiquitous disulfide bonds of proteins. This approach to PEGylation involves two steps: (1) disulfide reduction to release the two cysteine thiols and (2) re-forming the disulfide by bis-alkylation via a three-carbon bridge to which PEG was covalently attached. During this process, irreversible denaturation of the protein did not occur. Mechanistically, the conjugation is conducted by a sequential, interactive bis-alkylation using alpha,beta-unsaturated beta'-monosulfone functionalized PEG reagents. The combination of (a) maintaining the protein's tertiary structure after disulfide reduction, (b) the mechanism for bis-thiol selectivity of the PEG reagent, and (c) the steric shielding of PEG ensure that only one PEG molecule is conjugated at each disulfide bond. PEG was site-specifically conjugated via a three-carbon bridge to 2 equiv of the tripeptide glutathione, the cyclic peptide hormone somatostatin, the tetrameric protein l-asparaginase, and to the disulfides in interferon alpha-2b (IFN). SDS-PAGE, mass spectral, and NMR analyses were used to confirm conjugation, thiol selectivity, and connectivity. The biological activity of the l-asparaginase did not change after the attachment of four PEG molecules. In the case of IFN, a small reduction in biological activity was seen with the single-bridged IFN (without PEG attached). A significantly larger reduction in biological activity was seen with the three-carbon disulfide single-bridged PEG-IFNs and with the double-bridged IFN (without PEG attached). The reduction of the PEG-IFN's in vitro biological activity was a consequence of the steric shielding caused by PEG, and it was comparable to that seen with all other forms of PEG-IFNs reported. However, when a three-carbon bridge was used to attach PEG, our PEG-IFN's biological activity was found to be independent of the length of the PEG. This property has not previously been described for PEG-IFNs. Our studies therefore suggest that peptides, proteins, enzymes, and antibody fragments can be site-specifically PEGylated across a native disulfide bond using three-carbon bridges without destroying their tertiary structure or abolishing their biological activity. The stoichiometric efficiency of this approach also enables recycling of any unreacted protein. It therefore offers the potential to make PEGylated biopharmaceuticals as cost-effective medicines for global use.     

Novel poly(ethylene glycol) derivatives for preparation of ribosome-inactivating protein conjugates

This study describes the synthesis, characterization, and reactivity of new methoxypoly(ethylene glycol) (mPEG) derivatives containing a thioimidoester reactive group. These activated polymers are able to react with the lysyl epsilon-amino groups of suitable proteins, generating an amidinated linkage and thereby preserving the protein's positive charge. mPEG derivatives of molecular weight 2000 and 5000 Da were used, and two spacer arms were prepared, introducing chains of different lengths between the hydroxyl group of the polymer and the thioimidate group. These mPEG derivatives were used to modify gelonin, a cytotoxic single-chain glycoprotein widely used in preparation of antitumoral conjugates, whose biological activity is strongly influenced by charge modification. The reactivity of mPEG thioimidates toward lysil epsilon-amino groups of gelonin was evaluated, and the results showed an increased degree of derivatization in proportion to the molar excesses of the polymer used and to the length of the alkyl spacer. Further studies showed that the thioimidate reactive is able to maintain gelonin's significant biological activity and immunogenicity. On the contrary, modification of the protein with N-hydroxysuccinimide derivative of mPEG strongly reduces the protein's cytotoxic activity. Evaluation of the pharmacokinetic behavior of native and PEG-grafted gelonin showed a marked increase in plasma half-life after protein PEGylation; in particular, the circulating life of the conjugates increased with increased molecular weight of the polymer used. The biodistribution test showed lower organ uptake after PEGylation, in particular by the liver and spleen.     

Effects of linear and branched polyethylene glycol on PEGylation of recombinant hirudin: Reaction kinetics and in vitro and in vivo bioactivities

To improve the therapy efficacy of recombinant hirudin variant-2 (HV2), its PEGylation was investigated using linear mPEG-succinimidyl carbonate (mPEG-SC) and branched mPEG₂-N-hydroxysuccinimide (mPEG₂-NHS). The reaction mixtures of PEGylation were analyzed by RP-HPLC and the mono-PEG-HV2 products were purified by anion exchange chromatography (IEC). Effects of linear and branched PEG on the hydrolysis kinetics of the PEG reagent, the PEGylation kinetics of HV2 and the in vitro and in vivo bioactivity of mono-PEG-HV2 were investigated. The RP-HPLC and IEC analyses showed that linear and branched PEG-HV2 with identical molecular weight had different chromatographic behaviors. The reaction kinetics showed that branched mPEG₂-NHS displayed higher hydrolysis rate but lower PEGylation rates than linear mPEG-SC. Consequently, HV2 conjugated with mPEG₂-NHS required a greater molar ratio of PEG to HV2 than that of mPEG-SC to achieve the identically desired yield of mono-PEG-HV2. The in vitro and in vivo bioactivities of mono-PEG-HV2 showed that branched PEG-HV2 had higher therapeutic efficacy than linear PEG-HV2 with identical molecular weight. The in vivo bioactivity of mono-B-PEG40k-HV2 (mono-PEG-HV2 derived from 40 kDa branched mPEG₂-NHS) had a markedly longer duration in rabbits than did unmodified HV2, which showed its potential to be developed as a candidate antithrombotic drug.     

PEGylation of lysine residues improves the proteolytic stability of fibronectin while retaining biological activity

Excessive proteolysis of fibronectin (FN) impairs tissue repair in chronic wounds. Since FN is essential in wound healing, our goal is to improve its proteolytic stability and at the same time preserve its biological activity. We have previously shown that reduced FN conjugated with polyethylene glycol (PEG) at cysteine residues is more proteolytically stable than native FN. Cysteine‐PEGylated FN supported cell adhesion and migration to the same extent as native FN. However, unlike native FN, cysteine‐PEGylated FN was not assembled into an extracellular matrix (ECM) when immobilized. Here, we present an alternative approach in which FN is preferentially PEGylated at lysine residues using different molecular weight PEGs. We show that lysine PEGylation does not perturb FN secondary structure. PEG molecular weight, from 2 to 10 kDa, positively correlates with FN–PEG proteolytic stability. Cell adhesion, cell spreading, and gelatin binding decrease with increasing molecular weight of PEG. The 2‐kDa FN–PEG conjugate shows comparable cell adhesion to native FN and binds gelatin. Moreover, immobilized FN–PEG is assembled into ECM fibrils. In summary, lysine PEGylation of FN can be used to stabilize FN against proteolytic degradation with minimal perturbation to FN structure and retained biological activity.     

Molecular Insight into the Steric Shielding Effect of PEG on the Conjugated Staphylokinase: Biochemical Characterization and Molecular Dynamics Simulation

PEGylation is a successful approach to improve potency of a therapeutic protein. The improved therapeutic potency is mainly due to the steric shielding effect of PEG. However, the underlying mechanism of this effect on the protein is not well understood, especially on the protein interaction with its high molecular weight substrate or receptor. Here, experimental study and molecular dynamics simulation were used to provide molecular insight into the interaction between the PEGylated protein and its receptor. Staphylokinase (Sak), a therapeutic protein for coronary thrombolysis, was used as a model protein. Four PEGylated Saks were prepared by site-specific conjugation of 5 kDa/20 kDa PEG to N-terminus and C-terminus of Sak, respectively. Experimental study suggests that the native conformation of Sak is essentially not altered by PEGylation. In contrast, the bioactivity, the hydrodynamic volume and the molecular symmetric shape of the PEGylated Sak are altered and dependent on the PEG chain length and the PEGylation site. Molecular modeling of the PEGylated Saks suggests that the PEG chain remains highly flexible and can form a distinctive hydrated layer, thereby resulting in the steric shielding effect of PEG. Docking analyses indicate that the binding affinity of Sak to its receptor is dependent on the PEG chain length and the PEGylation site. Computational simulation results explain experimental data well. Our present study clarifies molecular details of PEG chain on protein surface and may be essential to the rational design, fabrication and clinical application of PEGylated proteins.     

The inhibitory potencies of monoclonal antibodies to the macrophage adhesion molecule sialoadhesin are greatly increased following PEGylation

PEGylation of antibodies is known to increase their half-life in systemic circulation, but nothing is known regarding whether PEGylation can improve the inhibitory potency of antibodies against target receptors. In this paper, we have examined this question using antibodies directed to Sialoadhesin (Sn), a macrophage-restricted adhesion molecule that mediates sialic acid dependent binding to different cells. Anti-Sn monoclonal antibodies (mAbs), SER-4 and 3D6, were conjugated to PEG 5 kDa or and PEG 20 kDa, resulting in the incorporation of up to 3 molecules of PEG per mAb molecule. Following purification of PEGylated mAbs by anion exchange chromatography, it was shown that PEGylation had little or no effect on antigen binding activity but led to a dramatic increase in inhibitory potency that was proportional to both the size of the PEG and the degree of derivatization. Thus, PEGylation of antibodies directed to cell surface receptors could be a powerful approach to improve the therapeutic efficacy of antibodies, not only by increasing their half-life in vivo, but also by increasing their inhibitory potency for blocking receptor-ligand interactions.