PEG chain length impacts yield of solid-phase protein PEGylation and efficiency of PEGylated protein separation by ion-exchange chromatography: Insights of mechanistic models

The mechanisms behind protein PEGylation are complex and dictated by the structure of the protein reactant. Hence, it is difficult to design a reaction process which can produce the desired PEGylated form at high yield. Likewise, efficient purification processes following protein PEGylation must be constructed on an ad hoc basis for each product. The retention and binding mechanisms driving electrostatic interaction-based chromatography (ion-exchange chromatography) of PEGylated proteins (randomly PEGylated lysozyme and mono-PEGylated bovine serum albumin) were investigated, based on our previously developed model Chem. Eng. Technol. 2005, 28, 1387–1393. PEGylation of each protein resulted in a shift to a smaller elution volume compared to the unmodified molecule, but did not affect the number of binding sites appreciably. The shift of the retention volume of PEGylated proteins correlated with the calculated thickness of PEG layer around the protein molecule. Random PEGylation was carried out on a column (solid-phase PEGylation) and the PEGylated proteins were separated on the same column. Solid-phase PEGylation inhibited the production of multi-PEGylated forms and resulted in a relatively low yield of selective mono-PEGylated form. Pore diffusion may play an important role in solid-phase PEGylation. These results suggest the possibility of a reaction and purification process development based on the mechanistic model for PEGylated proteins on ion exchange chromatography.     

PEGylated protein separation using different hydrophobic interaction supports: Conventional and monolithic supports

Protein hydrophobicity can be modified after a PEGylation process. However, hydrophobic interaction chromatography (HIC) has been used to separate PEGylation reaction products less frequently than other techniques. In this context, chromatographic monoliths represent a good alternative to continue exploring the separation of PEGylated proteins with HIC. In this work, the separation of PEGylated proteins using C4 A monolith as well as Toyopearl Butyl 650C and Butyl Sepharose was analyzed. Three proteins were used as models: RNase A, β‐lactoglobulin, and lysozyme. All proteins were PEGylated in the N‐terminal amino groups with 20 kDa methoxy poly(ethylene glycol) propionaldehyde. The concentration of ammonium sulfate (1 M) used was the same for all stationary phases. The results obtained demonstrated that the C4 A monolith could better resolve all protein PEGylation reaction mixtures, since the peaks of mono‐ and di‐PEGylated proteins can be clearly distinguished in the chromatographic profiles. On the contrary, while using Butyl Sepharose media only the PEGylation reaction mixtures of RNase A could be partially separated at 35 and 45 CVs. PEGylated proteins of β‐lactoglobulin and lysozyme could not be resolved when Toyopearl Butyl 650C and Butyl Sepharose were used. It is then clear that monoliths are an excellent choice to explore the purification process of PEGylated proteins exploiting the advantages of HIC. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:702–707, 2016     

A novel solid-phase site-specific PEGylation enhances the in vitro and in vivo biostabilty of recombinant human keratinocyte growth factor 1

Keratinocyte growth factor 1 (KGF-1) has proven useful in the treatment of pathologies associated with dermal adnexae, liver, lung, and the gastrointestinal tract diseases. However, poor stability and short plasma half-life of the protein have restricted its therapeutic applications. While it is possible to improve the stability and extend the circulating half-life of recombinant human KGF-1 (rhKGF-1) using solution-phase PEGylation, such preparations have heterogeneous structures and often low specific activities due to multiple and/or uncontrolled PEGylation. In the present study, a novel solid-phase PEGylation strategy was employed to produce homogenous mono-PEGylated rhKGF-1. RhKGF-1 protein was immobilized on a Heparin-Sepharose column and then a site-selective PEGylation reaction was carried out by a reductive alkylation at the N-terminal amino acid of the protein. The mono-PEGylated rhKGF-1, which accounted for over 40% of the total rhKGF-1 used in the PEGylation reaction, was purified to homogeneity by SP Sepharose ion-exchange chromatography. Our biophysical and biochemical studies demonstrated that the solid-phase PEGylation significantly enhanced the in vitro and in vivo biostability without affecting the over all structure of the protein. Furthermore, pharmacokinetic analysis showed that modified rhKGF-1 had considerably longer plasma half-life than its intact counterpart. Our cell-based analysis showed that, similar to rhKGF-1, PEGylated rhKGF-1 induced proliferation in NIH 3T3 cells through the activation of MAPK/Erk pathway. Notably, PEGylated rhKGF-1 exhibited a greater hepatoprotection against CCl(4)-induced injury in rats compared to rhKGF-1.     

Mono-PEGylation of ribonuclease A: High PEGylation efficiency by thiolation with small molecular weight reagent

PEGylation can improve the therapeutic potential of ribonuclease A (RNase A), a cancer chemotherapeutic agent. However, the common PEGylation that targets at the ?-amino groups of proteins can lead to imprecise control of the stoichiometry of the protein-PEG conjugate (i.e., mono-, di- and multi-PEGylated protein). To prepare a PEGylated therapeutic protein, it is desirable that the protein is mono-PEGylated for industrial production, convenient purification and analytical characterization. Here, N-hydroxysuccinimide esters of S-acetylthioacetic acid (SATA) and 2-iminothiolane (IT) were used to introduce thiol groups on RNase A, followed by maleimide chemistry based PEGylation of the thiolated RNase A. Interestingly, the yield of mono-PEGylated RNase A was higher than 60%, and di- or multi-PEGylated RNase A were absent in the PEGylated product. Presumably, the limited number and low solvent accessibility of the introduced thiol group favored mono-PEGylation of RNase A. As compared to the unmodified RNase A, the mono-PEGylated RNase A showed slightly decreased enzymatic activity, increased anti-proliferative ability and unchanged structural properties. Our study is expected to control the PEGylation process and optimize the industrial pharmaceutical production of PEGylated 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.     

Capillary electrophoretic characterization of PEGylated human parathyroid hormone with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A capillary electrophoretic method (CE) for characterizing PEGylated human parathyroid hormone 1-34 (PTH) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is described. CE was used to optimize the PEGylation of PTH through control of the reaction pH and the molar ratio of reactants with the advantages of minimal sample consumption and high separation capacity. The mono-PEGylated PTH (mono-PEG-PTH) was isolated and then digested with endoproteinase Lys-C. Resistance to Lys-C digestion on the PEGylation sites in the mono-PEG-PTH resulted in patterns of CE electropherograms different from that of the native PTH, and the PEGylation sites were assigned accordingly. The extent of positional isomers present in the mono-PEG-PTH was also determined by quantifying PEGylated fragments in the same CE electropherogram. In conclusion, the CE analysis of the Lys-C-digested sample allowed for simultaneous analysis of the PEGylation site and the extent of positional isomers in the mono-PEG-PTH. The results were confirmed by MALDI-TOF MS. This method will be applicable for characterizing PEGylation of other therapeutic peptides.     

In situ PEGylation of recombinant hirudin on an anion exchange chromatography column

In this study, an integrated process was developed for successive solid-phase PEGylation of recombinant hirudin variant-2 (HV2) and separation of PEGylated HV2 species on an anion exchange chromatography column (so-called in situ PEGylation). The effects of different PEG sizes, ion exchange resins and reaction conditions on in situ PEGylation were investigated. The results showed that in situ PEGylation efficiently integrates the reaction, separation and purification into a single-unit operation using the same column. In situ PEGylation could improve the selectivity of PEGylation reactions by significantly reducing the formation of multi-PEG-HV2. The pore sizes and internal surface structures of different resins had a significant impact on the yield of mono-PEG-HV2. In contrast to liquid-phase PEGylation, the yield of mono-PEG-HV2 decreased as PEG size increased during the in situ PEGylation process, indicating that in situ PEGylation is a pore diffusion-controlled process. The in vitro and in vivo anticoagulant activities of mono-PEG-HV2 derived from in situ PEGylation were higher than those from liquid-phase PEGylation, indicating that in situ PEGylation could enhance the bioactivity retention of mono-PEG-HV2. The results of this study demonstrated that in situ PEGylation can be used as an effective approach for the development of PEGylated protein drugs.     

Site-specific PEGylation of a lysine-deficient TNF-alpha with full bioactivity

Addition of polyethylene glycol to protein (PEGylation) to improve stability and other characteristics is mostly nonspecific and may occur at all lysine residues, some of which may be within or near an active site. Resultant PEGylated proteins are heterogeneous and can show markedly lower bioactivity. We attempted to develop a strategy for site-specific mono-PEGylation using tumor necrosis factor-alpha (TNF-alpha). We prepared phage libraries expressing TNF-alpha mutants in which all the lysine residues were replaced with other amino acids. A fully bioactive lysine-deficient mutant TNF-alpha (mTNF-alpha-Lys(-)) was isolated by panning against TNF-alpha-neutralizing antibody despite reports that some lysine residues were essential for its bioactivity. mTNF-alpha-Lys(-) was site-specifically mono-PEGylated at its N terminus. This mono-PEGylated mTNF-alpha-Lys(-), with superior molecular uniformity, showed higher bioactivity in vitro and greater antitumor therapeutic potency than randomly mono-PEGylated wild-type TNF-alpha. These results suggest the usefulness of the phage display system for creating functional mutant proteins and of our site-specific PEGylation approach.     

Purification of singly PEGylated α-lactalbumin using charged ultrafiltration membranes

One of the challenges in producing a PEGylated therapeutic protein is that the PEGylation reaction typically generates a mixture of both singly and multiply PEGylated species. The objective of this study was to examine the feasibility of using ultrafiltration for the purification of a singly PEGylated protein from the multiply PEGylated conjugates. Data were obtained with α‐lactalbumin that was PEGylated with a 20 kDa activated PEG, with the ultrafiltration performed over a range of pH and ionic strength using both unmodified and negatively charged composite regenerated cellulose membranes. Purification of the singly PEGylated α‐lactalbumin from the multiply PEGylated species was accomplished using a diafiltration process with a negatively charged membrane at pH 5 and an ionic strength of 0.4 mM, conditions that maximized the electrostatic exclusion of the multiply PEGylated species from the charged membrane. The diafiltration process provided more than 97% yield with greater than 20‐fold purification between the singly and doubly PEGylated proteins and nearly complete removal of the more heavily PEGylated species. The singly PEGylated α‐lactalbumin was recovered as a dilute filtrate solution, although this dilution could be eliminated using a cascade filtration or the final product could be re‐concentrated in a second ultrafiltration as part of the final formulation. These results demonstrate the feasibility of using ultrafiltration for the purification of singly PEGylated protein therapeutics. Biotechnol. Bioeng. 2011; 108:822–829. © 2010 Wiley Periodicals, Inc.     

聚乙二醇定点修饰集成干扰素突变体Ⅱ

目的：用聚乙二醇（PEG）修饰集成干扰素突变体Ⅱ（IFN-Con-m2,IIFNm2）,通过纯化获得新型修饰分子并对该分子进行抗胰蛋白酶水解稳定性及初步药代动力学研究。 方法：将mPEG20000定点偶联到IIFNm2的第86位Cys残基上,修饰后的产物经CM层析后,以SDS-PAGE考察其纯度,用WISH-VSV系统进行生物活性测定;在0.1%胰蛋白酶条件下考察体外抗酶解稳定性;并以SD大鼠进行初步药代动力学研究,绘制血药浓度-时间曲线。采用3P87软件进行数据拟合,分析药物动力学参数。 结果：干扰素修饰率约为50%,且绝大多数以单修饰体（mono-PEG- IIFNm2）形式存在;提纯后mono-PEG-IIFNm2 的纯度大于98%,比活性约为修饰前IIFNm2的1%。抗胰蛋白酶水解试验表明：30min后,IIFNm2抗病毒活性残留为8%,mono-PEG-IIFNm2为41%。初步药代动力学研究显示：IIFNm的消除半衰期为(1.57±0.34)h,mono-PEG-IIFNm2为(18.0±4.0)h。 结论：成功地偶联了PEG和IIFNm2,建立了mono-PEG-IIFNm2的纯化工艺,PEG修饰能增加IIFNm2的体外抗胰蛋白酶水解稳定性,并显著延长体内半衰期。     

溶葡球菌酶的定点突变与PEG巯基定点修饰

为了建立聚乙二醇 (PEG) 巯基定点修饰溶葡球菌酶的方法,并检验假定连接区的突变与修饰对酶活的影响,对溶葡球菌酶的假定连接区进行了巯基聚乙二醇定点修饰研究。通过分析溶葡球菌酶的结构特征,选择两个结构域之间的氨基酸 (133-154aa) 进行定点突变引入半胱氨酸残基。使用单甲氧基聚乙二醇马来酰亚胺 (mPEG-MAL) 进行定点修饰,对修饰后的酶进行纯化并测定酶活性。结果表明定点突变的半胱氨酸残基PEG修饰效率高、产物单一,运用简便的Ni2+-NTA柱亲和层析法实现了一步分离,获得了高纯度的目标蛋白,但在连接区进行定点突变及PEG定点修饰后的酶活有不同程度的降低,表明假定连接区部分位点的PEG修饰会对溶葡球菌酶的催化活性产生一定影响。     

PEGylated protein separations: challenges and opportunities

PEGylation, the covalent attachment of polyethylene glycol (PEG) chains to protein, isa promising method for making an efficient protein drug. Several PEGylated protein drugs, such as PEGylated interferons, are already on the market and others are presently in their clinical trials. However, the PEGylation reaction is very product specific so that generalized or platform processes for both reaction and purification have not yet been established. In the current issue of Biotechnology Journal, Günter Allmaier and colleagues report a modified microchip capillary gel electrophoresis (MCGE), which allows for a rapid separation (one minute) of PEGylated proteins of different degrees of PEGylation.     

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.     

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.     

Coupling purification and on-column PEGylation of tumor necrosis factor alpha analogue

Trends in preparation of PEGylated protein drugs strive for simple, fast, and cheap processes, resulting in well-defined homogeneous products. We investigated the on-column PEGylation of tumor necrosis factor alpha (TNF-α), where purification and conjugation were performed in one step by using immobilized metal affinity chromatography (IMAC). The same quality of the PEGylated product was obtained by the on-column approach starting from either the crude Escherichia coli protein extract or the purified protein. In comparison with the PEGylation in solution, the on-column approach resulted in more homogeneous PEGylated product. The on-column PEGylation reduces the number of production steps, costs, and preparation time.     

PEGylation of proteins in organic solution: a case study for interferon beta-1b

Conventional protein PEGylation is carried out in aqueous solution. However, some hydrophobic proteins seem to be stable in organic solution. In this study, a novel approach of PEGylating IFN-β-1b in an organic solution of 2-butanol (2-BuOH) was investigated. Compared with protein PEGylation in aqueous solution, the overall modification yields increased more than 37%, while the yield of mono-PEGylated products could be increased by 36%. Furthermore, the PEGylated IFN-β-1b, which was obtained in organic solution, demonstrated 18% more antiviral potency than those derived from aqueous solution. The PEGylation step could be directly connected to the previous protein separation step for process integration. Dynamic light scattering (DLS) and atomic force microscope (AFM) analysis revealed that IFN-β-1b formed aggregates both in water and in 2-BuOH solutions. However, the aggregates were much smaller and more homogeneous in 2-BuOH than those in aqueous solution, thereby providing larger solvent accessible protein surfaces, which resulted in a more productive PEGylation process. In addition, the results of circular dichroism (CD), fluorescence spectra, and peptide mapping suggested that the increased bioactivity came from the difference in PEGylation site distribution due to solution environment that induced conformational discrepancy. The results of this study show that PEGylation of IFN-β-1b in organic solution is a facile and efficient process, which might find applications for other hydrophobic proteins.     

Optimization of octreotide PEGylation by monitoring with fast reversed-phase high-performance liquid chromatography

The purpose of this study was to develop a fast reversed-phase high-performance liquid chromatography (HPLC) method for monitoring the octreotide PEGylation reaction in order to find optimal conditions for the production of the desired mono-PEGylated octreotide. The fast HPLC method could separate the positional isomers of two mono-PEGylated octreotides, di-PEGylated octreotide, and unmodified octreotide within 4.5 min. The PEGylation pattern was monitored at various pH conditions and molar ratios of reactants to allow optimization of the PEGylation reaction conditions for the production of N-terminally mono-PEGylated octreotide.     

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.     

A solid-phase adsorption method for PEGylation of human serum albumin and staphylokinase: preparation,purification and biochemical characterization

A solid-phase adsorption method was developed to circumvent the disadvantage of the conventional liquid-phase PEGylation, i.e. the heterogeneity of the PEGylated products. The model proteins, human serum albumin (HSA) and staphylokinase (SAK), were adsorbed on the ion exchange chromatography media, followed by PEGylation with succinimidyl carbonate (SC)-mPEG5K and salt elution. Since PEGylation with SC-PEG5K alters the positive charge of the proteins, Q-Sepharose Big Beads and DEAE Sepharose Fast Flow were used for adsorption of HSA and SAK, respectively. Size exclusion chromatography and SDS-PAGE studies demonstrated that solid-phase PEGylation of proteins generate monoPEGylated proteins with the yield of 35–47%. Circular dichroism and intrinsic fluorescence studies showed that solid-phase PEGylation led to little conformational change of the proteins. Solid-phase PEGylation resulted in 35% loss in the biological activity of SAK, which is lower than the liquid-phase PEGylation (70%).     

干扰素a-2b的聚乙二醇修饰

采用分子量为20 kD的单甲氧基聚乙二醇丙醛(mPEG-ALD)修饰重组人干扰素a-2b(IFN a-2b), 建立了修饰反应及分离纯化工艺。考察了修饰反应各因素对单修饰转化率以及单修饰产物体外活性的影响, 获得了优化的修饰反应条件, 即在pH 6.5, 20 mmol/L的磷酸氢二钠-柠檬酸缓冲溶液中, 干扰素a-2b的浓度为4 mg/mL, PEG与IFN a-2b的摩尔比为8:1, 4oC时反应20 h; 在优化的反应条件下, 单修饰PEG-IFN a-2b的转化率达到55%。并且, 采用离子交换层析对修饰产物进行分离纯化, 单修饰产品纯度达到97%, 体外活性保留达到未修饰干扰素a-2b的13.4%, 其在SD大鼠体内的循环半衰期得到了较大的延长, 且具有较好的水溶液稳定性。