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.     

Capillary electrophoretic separation of high-molecular-weight poly(ethylene glycol)-modified proteins

This study was designed to demonstrate the utility of capillary electrophoresis (CE) for separating high-molecular-weight poly(ethylene glycol) (PEG)-conjugated proteins. As a CE method, sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) was applied to analyze interferon alpha (IFN) modified with branched and trimer-structured PEG molecules. Five mono-PEG-IFN conjugates prepared with two branched PEGs (MW 20 and 40 kDa) and three trimer-structured PEGs (MW 23.5, 43.5, and 47 kDa) were purified by cation-exchange chromatography and their masses were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The SDS-CGE method showed high separation capacity by differentiating PEG-IFN conjugates with small differences in molecular size, such as PEG_40K-, PEG_43.5K-, and PEG_47K-IFNs, and it was useful for checking the purity of each mono-PEG-IFN. This study shows that SDS-CGE can well be utilized in the development and quality control of PEGylated proteins prepared with various types of PEG.     

Development of a process to manufacture PEGylated orally bioavailable insulin

To make insulin orally bioavailable, insulin was modified by covalent attachment (conjugation) of a short-chain methoxy polyethylene glycol (mPEG) derivative to the ε-amino group of a specific amino acid residue (LysB(29)). During the conjugation process, activated PEG can react with any of the free amino groups, the N-terminal of the B chain (PheB(1)), the N-terminal of the A chain (GlyA(1)), and the ε-amino group of amino acid (LysB(29)), resulting in a heterogeneous mixture of conjugated products. The abundance of the desired product (Methoxy-PEG(3)-propionyl--insulin at LysB(29):IN-105) in the conjugation reaction can be controlled by changing the conjugation reaction conditions. Reaction conditions were optimized for maximal yield by varying the proportions of protein to mPEG molecule at various values of pH and different salt and solvent concentrations. The desired conjugated molecule (IN-105) was purified to homogeneity using RP-HPLC. The purified product, IN-105, was crystallized and lyophilized into powder form. The purified product was characterized using multiple analytical methods including ESI-TOF and peptide mapping to verify its chemical structure. In this work, we report the process development of new modified insulin prepared by covalent conjugation of short chain mPEG to the insulin molecule. The attachment of PEG to insulin resulted in a conjugated insulin derivative that was biologically active, orally bioavailable and that showed a dose-dependent glucose lowering effect in Type 2 diabetes patients.     

Size-exclusion reaction chromatography (SERC): a new technique for protein PEGylation

A new, widely applicable process that combines reaction and separation in a single unit operation is described. The process, size-exclusion reaction chromatography (SERC), simultaneously allows control of the extent of reactions in which molecular size is altered and the separation of products and reactants. In SERC, a moving reaction zone is formed by injection of reactants onto a size-exclusion chromatography column. Reactants and products are partitioned differently within the mobile phase, resulting in different linear flow rates through the column. The products are therefore removed selectively from the reaction zone, minimizing their residence time in the reaction zone and allowing their separation in the downstream section of the column. For reactions such as protein PEGylation, in which successive addition of PEG groups to the protein results in significant molecular size increases, SERC potentially offers a method by which a dominant final PEGylated protein size can be produced at high yield. The SERC PEGylation of two model proteins, alpha-lactalbumin and beta-lactoglobulin, is demonstrated and results show that simultaneous reaction and separation was obtained.     

Conjugation of Multiple Copies of Polyethylene Glycol to Hemoglobin Facilitated Through Thiolation: Influence on Hemoglobin Structure and Function

PEGylation induced changes in molecular volume and solution properties of HbA have been implicated as potential modulators of its vasoconstrictive activity. However, our recent studies with PEGylated Hbs carrying two PEG chains/Hb, have demonstrated that the modulation of the vasoconstrictive activity of Hb is not a direct correlate of the molecular volume and solution properties of the PEGylated Hb and implicated a role for the surface charge and/or the pattern of surface decoration of Hb with PEG. HbA has now been modified by thiolation mediated maleimide chemistry based PEGylation that does not alter its surface charge and conjugates multiple copies of PEG5K chains. This protocol has been optimized to generate a PEGylated Hb, (SP-PEG5K)₆-Hb, that carries ~six PEG5K chains/Hb – HexaPEGylated Hb. PEGylation increased the O₂ affinity of Hb and desensitized the molecule for the influence of ionic strength, pH, and allosteric effectors, presumably a consequence of the hydrated PEG-shell generated around the protein. The total PEG mass in (SP-PEG5K)₆-Hb, its molecular volume, O₂ affinity and solution properties are similar to that of another PEGylated Hb, (SP-PEG20K)₂-Hb, that carries two PEG20K chains/Hb. However, (SP-PEG5K)₆-Hb exhibited significantly reduced vasoconstriction mediated response than (SP-PEG20K)₂-Hb. These results demonstrate that the enhanced molecular size and solution properties achieved through the conjugation of multiple copies of small PEG chains to Hb is more effective in decreasing its vasoconstrictive activity than that achieved through the conjugation of a comparable PEG mass using a small number of large PEG chains.     

Adsorption of poly(ethylene glycol)-modified ribonuclease A to a poly(lactide-co-glycolide) surface

Protein adsorption is a source of variability in the release profiles of therapeutic proteins from biodegradable microspheres. We employ optical reflectometry and total internal reflection fluorescence to explore the extent and kinetics of ribonuclease A (RNase A) adsorption to spin-cast films of poly(lactide-co-glycolide) (PLG) and, in particular, to determine how covalent grafting of polyethylene glycol (PEG) to RNase A affects adsorption. Adsorption kinetics on PLG surfaces are surface-limited for RNase A but transport-limited for unconjugated PEG homopolymers and for PEG-modified RNase A, indicating that PEG anchors the conjugates to the surface during the transport-limited regime. PEG modification of RNase A decreases the total number of adsorbed molecules per unit area but increases the areal surface coverage because the grafted PEG chains exclude additional surface area. Total internal reflection fluorescence-based exchange measurements show that there is no exchange between adsorbed and solution-phase protein molecules. This indicates an unusually tenacious adsorption. Streaming current measurements indicate that the zeta potential of the PLG surface becomes increasingly negative as the film is exposed to water for several weeks, as expected. Aging of the PLG surface results in increased adsorption of unmodified RNase A but decreased adsorption of unconjugated PEG homopolymers and of PEG-RNase A conjugates, relative to the extent of adsorption on freshly prepared PLG surfaces. Adsorption results correlate well with an increase in the rate, total extent and preservation of bioactivity of RNase A released from PLG microspheres for the PEG-modified version of RNase A.     

白细胞介素－2的PEG化

用自制的氨基ＰＥＧ化试剂ｒＩＬ－２进行化学修饰,研究了试剂浓度,溶液ｐＨ,反应时间等与ＰＥｃａ－ｒＩＬ－２产率及ＩＬ－２活性保持之间的关系,建立了一套获得稳定修饰度的ＰＥＧ－ｒＩＬ－２的方法。研究发现,反应时间跟修饰度关系不大;溶液ｐＨ对修饰度有一定的影响,中性ｐＨ以上反应都可进行;而试剂浓度直接决定修饰度的高低,过量越多,修饰度越高,而生物活性保留也越低;但低度修饰,对活性几乎没有影响,可保留活性在９５％左右。     

PEGylation of protein-based MRI contrast agents improves relaxivities and biocompatibilities

Magnetic resonance imaging (MRI) has emerged as a leading diagnostic technique in clinical and preclinical settings. However, the application of MRI to assess specific disease markers for diagnosis and monitoring drug effect has been severely hampered by the lack of desired contrast agents with high relaxivities, and optimized in vivo retention time. We have reported the development of protein-based MRI contrast agents (ProCA1) by rational design of Gd^3 + binding sites into a stable protein resulting in significantly increased longitudinal (r₁) and transverse (r₂) relaxivities compared to Gd-DTPA. Here, we report a further improvement of protein contrast agents ProCA1 for in vivo imaging by protein modification with various sizes of polyethylene glycol (PEG) chain. PEGylation results in significant increases of both r₁ and r₂ relaxivities (up to 200%), and these high relaxivities persist even at field strengths up to 9.4 T. In addition, our experimental results demonstrate that modified contrast agents have significant improvement of in vivo MR imaging and biocompatibilities including dose efficiency, protein solubility, blood retention time and decreased immunogenicity. Such improvement can be important to the animal imaging and pre-clinical research at high or ultra-high field where there is an urgent need for molecular imaging probes and optimized contrast agent.     

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.     

蛋白药物的聚乙二醇定点修饰策略与最佳位点

聚乙二醇修饰是一种改善蛋白质药物临床药效行之有效的方法。聚乙二醇修饰具有延长蛋白质药物在体内的半衰期、降低免疫原性和延缓蛋白酶降解、提高稳定性和溶解性等优点。而聚乙二醇的定点修饰由于能够获得均一性和高活性保留率的产物,并能提高产率,已经引起了广泛关注。综述了近年来聚乙二醇定点修饰蛋白质药物方面的研究进展,着重介绍了聚乙二醇定点修饰的策略及最佳修饰位点,并对聚乙二醇定点修饰技术的发展趋势进行了展望。