Intermolecular interactions during ultrafiltration of pegylated proteins

Recent studies have demonstrated the feasibility of using membrane ultrafiltration for the purification of pegylated proteins; however, the separations have all been performed at relatively low protein concentrations where intermolecular interactions are unimportant. The objective of this study was to examine the behavior at higher PEG concentrations and to develop an appropriate theoretical framework to describe the effects of intermolecular interactions. Ultrafiltration experiments were performed using pegylated α‐lactalbumin as a model protein with both neutral and charged composite regenerated cellulose membranes. The transmission of the pegylated α‐lactalbumin, PEG, and α‐lactalbumin all increase with increasing PEG concentration due to the increase in the solute partition coefficient arising from unfavorable intermolecular interactions in the bulk solution. The experimental results were in good agreement with a simple model that accounts for the change in Gibbs free energy associated with these intermolecular interactions, including the effects of concentration polarization on the local solute concentrations upstream of the membrane. These intermolecular interactions are shown to cause a greater than expected loss of pegylated product in a batch ultrafiltration system, and they alter the yield and purification factor that can be achieved during a diafiltration process to remove unreacted PEG. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:655–663, 2013     

Modification of recombinant asparaginase from Erwinia carotovora with polyethylene glycol 5000

A method for polyethylene conjugation with recombinant asparaginase has been developed to improve therapeutically important properties of enzyme. Methoxy-p-nitrophenyl carbamate of polyethylene glycol with molecular weight 5000 was employed as the modification reagent. Optimization of the pegylation procedure resulted in high level of enzyme modification. Under 4.5 molar excess of the modification reagent more than 10 molecules of methoxy-polyethylene bound per one asparaginase molecular. The modified asparaginase retained 57% of initial activity. A simple and efficient pegylation procedure described in this work can be used for production of asparaginase with improved therapeutic properties.     

聚乙二醇化修饰对蛋白多肽药物药代动力学的影响

聚乙二醇（PEG）化修饰在生物制药领域被认为是改变蛋白多肽类药物生物、理化性质的最佳方法之一。众多研究表明,PEG化后的蛋白多肽在生物体内的药代动力学行为有了很大改变,主要表现在：吸收相半衰期及消除相半衰期延长．血药峰浓度提高,达峰时间滞后,表观分布容积变小,免疫反应性减弱,血浆清除减慢。这些变化极大地改善了蛋白多肽药物在机体内清除快、免疫原性高、有效血药浓度持续时间短、需频繁给药等缺点。聚乙二醇化修饰为生物制药领域开辟了新的天地。     

集成干扰素突变体IIFN72C的构建及其聚乙二醇定点修饰

目的：设计构建集成干扰素突变体IIFN72C并进行聚乙二醇定点修饰,以获得高活性的长效干扰素分子。方法：利用蛋白质分子同源模建,选择在集成干扰素分子IIFN的第72位引入半胱氨酸残基构成集成干扰素突变体IIFN72C。诱导表达后经包涵体变复性和层析纯化,与单甲氧基聚乙二醇（mPEGMAL）定点偶联。修饰产物经纯化后,以SDSPAGE考察其纯度,用WISHVSV系统进行生物活性测定。结果：IIFN72C以包涵体形式表达,表达量占菌体总蛋白的30%以上,比活性与突变前相当;修饰产物大多数为单修饰体,纯化后纯度大于98%,比活性保留约为修饰前的8%。结论：成功设计并表达IIFN72C用于PEG定点修饰,修饰产物活性保留得以提高。     

Asparaginase (native ASNase or pegylated ASNase) in the treatment of acute lymphoblastic leukemia

The discovery of the tumor-inhibitory properties of asparaginase (ASNase) began in the early 1950s with the observation that guinea pig serum-treated lymphoma-bearing mice underwent rapid and often complete regression. About 4000 cases of acute lymphoblastic leukemia (ALL) are diagnosed very year in the US and many more through out the world. The majority of these cases are in children and young adults, making ALL the most common form of malignancy in these age groups. The treatment protocols of ALL are complex and use 6-12 drugs. Consequently, the improvement in the protocol design has improved significantly the success rate for long-term event-free survival in the past 20-30 years, which is now approximately 75% for patients afflicted with the higher risk ALL features and just above this percentage for patients with standard or good features. Despite this success, approximately 15% of patients die from ALL, making leukemic relapse the most common cause of treatment failure in pediatric oncology. ASNases have been the cornerstone of ALL therapies since the late 1970s. Native or pegylated L-asparaginase (ASNase or PEG-ASNase) are highly specific for the deamination of L-asparagine (Asn) to aspartic acid and ammonia. Depletion of Asn leads to a nutritional deprivation and inhibition of protein biosynthesis, resulting in apoptosis in T-lymphoblastic leukemias, which require Asn from external sources. The reactions of the host exposed to repeated ASNase treatments as well as the up-regulation of the mammalian enzymes to overcome the ASN-depletion toxic condition are of significant importance and may make us relearn the lessons on this important antileukemic drug.     

Effects of Polyethylene Glycol on Bovine Intestine Alkaline Phosphatase Activity and Stability

In this study, we evaluated the effects of polyethylene glycol (PEG) on bovine intestine alkaline phosphatase (BIALP) activity and stability. In the hydrolysis of p-nitrophenylphosphate (pNPP) at pH 9.8 at 20 °C, the k _cat?K _m values of BIALP plus 5–15% w/v free PEG with molecular masses of 1, 2, 6, and 20 kDa (PEG1000, PEG2000, PEG6000, and PEG20000 respectively) were 120–140%, 180–300%, 130–170%, and 110–140% respectively of that of BIALP without free PEG (1.8 μM^?1 s^?1), indicating that activation by PEG2000 was the highest. Unmodified BIALP plus 5% PEG2000 and BIALP pegylated with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine exhibited 1.3-fold higher activity on average than that of BIALP without free PEG under various conditions, including pH 7.0–10.0 and 20–65 °C. The temperatures reducing initial activity by 50% in 30-min incubation of unmodified BIALP plus 5% PEG2000 and pegylated BIALP were 51 and 47 °C respectively, similar to that of BIALP without free PEG (49 °C). These results indicate that the addition of PEG2000 and pegylation increase BIALP activity without affecting its stability, suggesting that they can be used in enzyme immunoassay with BIALP to increase sensitivity and rapidity.     

Ultrafiltration characteristics of pegylated proteins

There is growing clinical interest in the use of pegylated recombinant proteins with enhanced stability, half-life, and bioavailability. The objective of this study was to develop a quantitative understanding of the ultrafiltration characteristics of a series of pegylated proteins with different degrees of pegylation. Sieving data were compared with available theoretical models and with corresponding results for the partition coefficient in size exclusion chromatography (SEC). The sieving coefficients of the pegylated proteins depended not only on the protein size and the total molecular weight of the polyethylene glycol (PEG) but also on the number of PEG chains. This is in sharp contrast to the partition coefficient in SEC, which was uniquely determined by the total molecular weight of the PEG and protein. This difference is due to the deformation and/or elongation of the PEG chains caused by the convective flow into the membrane pores, an effect that is not present in SEC. These results provide important insights into the transport and separation characteristics of pegylated proteins.     

Tumor targeting using liposomal antineoplastic drugs

During the last years, liposomes (microparticulate phospholipid vesicles) have been used with growing success as pharmaceutical carriers for antineoplastic drugs. Fields of application include lipid-based formulations to enhance the solubility of poorly soluble antitumor drugs, the use of pegylated liposomes for passive targeting of solid tumors as well as vector-conjugated liposomal carriers for active targeting of tumor tissue. Such formulation and drug targeting strategies enhance the effectiveness of anticancer chemotherapy and reduce at the same time the risk of toxic side-effects. The present article reviews the principles of different liposomal technologies and discusses current trends in this field of research.     

聚乙二醇定点修饰重组人粒细胞集落刺激因子突变体的研究

研究了重组人粒细胞集落刺激因子突变体（rmhG-CSF）的聚乙二醇化修饰、分离纯化和活性鉴定。通过对人重组粒细胞集落刺激因子（rhG-CSF）第1,3,4,5,17位氨基酸进行突变,并在C末端加了一个半胱氨酸,获得了体外活性为原型rhG-CSF 150％以上的重组人粒细胞集落刺激因子突变体（rmhG-CSF）。然后用分子量为20kD的甲氧聚乙二醇马来酸酐（mPEG-Mal）修饰rmhG-CSF,反应混合物经离子交换和凝胶过滤柱纯化,得到纯化的聚乙二醇重组人粒细胞集落刺激因子突变体（PEG-rmhG-CSF）。SDS-PAGE电泳分析表明纯化后的PEG-rmhG-CSF的纯度大于95％,体外活性分析表明PEG-rmhG-CSF活性优于目前临床使用的聚乙二醇重组人粒细胞集落刺激因子（PEG-rhG-CSF,Neulasta^?）,药代动力学研究表明PEG-rmhG-CSF体内半衰期约为14h,比修饰前延长了7倍。     

Nanomedicines in the treatment of chronic hepatitis C--focus on pegylated interferon alpha-2a

Nanotechnology is the application of nanotechnology within medicine. An illustration of this is the use of pegylation as a means of modifying naturally occurring proteins which may have clinical applications, in order to improve the pharmacodynamics of the protein resulting in an effective medication. An example of this is pegylated interferon. The purpose of this review is to examine the chemistry, clinical pharmacology, pharmacokinetics, pharmacodynamics, and clinical studies with 40 kDa pegylated interferon to illustrate the general principles of pegylated biological proteins. The use in clinical practice is reviewed along with the evidence for both efficiacy, safety, and advantages over standard interferon.