PEGylation Site Research Articles

Exactly defined molecular weight poly(ethylene glycol) allows for facile identification of PEGylation sites on proteins

PEGylation (the covalent attachment of one or more poly(ethylene glycol) (PEG) units to a therapeutic) is a well-established technique in the pharmaceutical industry to increase blood-residence time and decrease immunogenicity. A challenging aspect of PEGylation is the dispersity of PEGylation agents, which results in batch-to-batch variations and analytical limitations. Herein, we present an approach to overcome these limitations by manufacturing a defined molecular weight (dispersity-free) PEGylation agent. We synthesise a defined molecular weight (Mw), linear 5 kDa methoxy-PEG (mPEG) active ester in an efficient and scalable manner using an iterative liquid-phase approach based on Nanostar Sieving. We then perform a comparative study on the random PEGylation and subsequent characterisation of the protein bovine serum albumin (BSA), using both the defined Mw, dispersity-free mPEG active ester, and a commercially available disperse 5 kDa mPEG active ester. We demonstrate that the defined Mw PEG both allows for facile monitoring of chemical modification reactions during the synthesis of the PEGylation agents, and facilitates straightforward identification of the PEGylated fragments within a PEGylated protein via a simple peptide mapping approach using UPLC-MS.

Characterization of PEGylation sites in Neulasta and a biosimilar candidate with a combined fragmentation strategy in mass spectrometry analysis.

In the development of biosimilar products to Neulasta, it is essential to determine the intact molecular mass and confirm precise PEGylation sites. In this study, we applied a combination of techniques, including post-column addition of triethylamine in reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) to determine the intact molecular mass, and in-source fragmentation (ISF) and higher-energy collision dissociation-tandem mass spectrometry (HCD-MS/MS) to identify the PEGylation site. Our results show that both the pegfilgrastim biosimilar candidate and Neulasta lots are mono-PEGylated at the N-terminal end. Furthermore, we show that the combined ISF and HCD-MS/MS method can be used for identifying the PEGylation sites in the diPEGylated variant of pegfilgrastim. The diPEGylated variant has modification sites at the N-terminal end and a lysine at position 35 in the protein sequence.

Therapeutic effect of long-acting FGF21 with controlled site-specific modification on nonalcoholic steatohepatitis

FGF21 plays an active role in the treatment of type 2 diabetes, obesity, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH). However, the short half-life and poor stability of wild-type FGF21 limit its clinical application. Previous studies found that PEGylation can significantly increase the stability of FGF21. However, the uneven distribution of PEGylation sites in FGF21 makes it difficult to purify PEG-FGF21, thereby affecting its yield, purity, and activity. To obtain long-acting FGF21 with controlled site-specific modification, we mutated lysine residues in FGF21, resulting in PEGylation only at the N-terminus of FGF21 (mFGF21). In addition, we modified mFGF21 molecules with different PEG molecules and selected the PEG-mFGF21 moiety with the highest activity. The yield of PEG-mFGF21 in this study reached 1 g/L (purity >99 %), and the purification process was simple and efficient with strong quality controllability. The half-life of PEG-mFGF21 in rats reached 40.5–67.4 h. Pharmacodynamic evaluation in mice with high-fat, high-cholesterol- and methionine and choline deficiency-induced NASH illustrated that PEG-mFGF21 exhibited long-term efficacy in improving liver steatosis and reducing liver cell damage, inflammation, and fibrosis. Taken together, PEG-mFGF21 could represent a potential therapeutic drug for the treatment of NASH.

Impact of the PEG length and PEGylation site on the structural, thermodynamic, thermal, and proteolytic stability of mono-PEGylated alpha-1 antitrypsin.

Conjugation to polyethylene glycol (PEG) is a widely used approach to improve the therapeutic value of proteins essentially by prolonging their body residence time. PEGylation may however induce changes in the structure and/or the stability of proteins and thus on their function(s). The effects of PEGylation on the thermodynamic stability can either be positive (stabilization), negative (destabilization), or neutral (no effect). Moreover, various factors such as the PEG length and PEGylation site can influence the consequences of PEGylation on the structure and stability of proteins. In this study, the effects of PEGylation on the structure, stability, and polymerization of alpha1-antitrypsin (AAT) were investigated, using PEGs with different lengths, different structures (linear or 2-armed) and different linking chemistries (via amine or thiol) at two distinct positions of the sequence. The results show that whatever the size, position, and structure of PEG chains, PEGylation (a) does not induce significant changes in AAT structure (either at the secondary or tertiary level); (b) does not alter the stability of the native protein upon both chemical- and heat-induced denaturation; and (c) does not prevent AAT to fully refold and recover its activity following chemical denaturation. However, the propensity of AAT to aggregate upon heat treatment was significantly decreased by PEGylation, although PEGylation did not prevent the irreversible inactivation of the enzyme. Moreover, conjugation to PEG, especially 2-armed 40 kDa PEG, greatly improved the proteolytic resistance of AAT. PEGylation of AAT could be a promising strategy to prolong its half-life after infusion in AAT-deficient patients and thereby decrease the frequency of infusions.

Assessing site-specific PEGylation of TEM-1 β-lactamase with cell-free protein synthesis and coarse-grained simulation

PEGylation is a broadly used strategy to enhance the pharmacokinetic properties of therapeutic proteins. It is well established that the location and extent of PEGylation have a significant impact on protein properties. However, conventional PEGylation techniques have limited control over PEGylation sites. Emerging site-specific PEGylation technology provides control of PEG placement by conjugating PEG polymers via click chemistry reaction to genetically encoded non-canonical amino acids. Unfortunately, a method to rapidly determine the optimal PEGylation location has yet to be established. Here we seek to address this challenge. In this work, coarse-grained molecular dynamic simulations are paired with high-throughput experimental screening utilizing cell-free protein synthesis to investigate the effect of site-specific PEGylation on the two-state folder protein TEM-1 β-lactamase. Specifically, the conjugation efficiency, thermal stability, and enzymatic activity are studied for the enzyme PEGylated at several different locations. The results of this analysis confirm that the physical properties of the PEGylated protein vary considerably with PEGylation site and that traditional design recommendations are insufficient to predict favorable PEGylation sites. In this study, the best predictor of the most favorable conjugation site is coarse-grained simulation. Thus, we propose a dual combinatorial screening approach in which coarse-grained molecular simulation informs site selection for high-throughput experimental verification.

Stability of a Novel PEGylation Site on a Putative Haemoglobin-Based Oxygen Carrier.

PEGylation of protein sulfhydryl residues is a common method used to create a stable drug conjugate to enhance vascular retention times. We recently created a putative haemoglobin-based oxygen carrier using maleimide-PEG to selectively modify a single engineered cysteine residue in the α subunit (αAla19Cys). However, maleimide-PEG adducts are subject to deconjugation via retro-Michael reactions, with consequent cross-conjugation to endogenous plasma thiols such as those found on human serum albumin or glutathione. In previous studies mono-sulfone-PEG adducts have been shown to be less susceptible to deconjugation. We therefore compared the stability of our maleimide-PEG Hb adduct with one created using a mono-sulfone PEG. The corresponding mono-sulfone-PEG adduct was significantly more stable when incubated at 37°C for 7days in the presence of 1mM reduced glutathione, 20mg/mL human serum albumin, or human serum. In all cases haemoglobin treated with mono-sulfone-PEG retained >90% of its conjugation whereas maleimide-PEG showed significant deconjugation, especially in the presence of 1mM reduced glutathione where <70% of the maleimide-PEG conjugate remained intact. Although maleimide-PEGylation of Hb seems adequate for an oxygen therapeutic intended for acute use, if longer vascular retention is required reagents such as mono-sulfone-PEG may be more appropriate.

Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit.

In order to use a Hemoglobin Based Oxygen Carrier as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the hemoglobin molecule to prevent rapid renal clearance. A common method uses maleimide PEGylation of sulfhydryls created by the reaction of 2-iminothiolane at surface lysines. However, this creates highly heterogenous mixtures of molecules. We recently engineered a hemoglobin with a single novel, reactive cysteine residue on the surface of the alpha subunit creating a single PEGylation site (βCys93Ala/αAla19Cys). This enabled homogenous PEGylation by maleimide-PEG with >80% efficiency and no discernible effect on protein function. However, maleimide-PEG adducts are subject to deconjugation via retro-Michael reactions and cross-conjugation to endogenous thiol species in vivo. We therefore compared our maleimide-PEG adduct with one created using a mono-sulfone-PEG less susceptible to deconjugation. Mono-sulfone-PEG underwent reaction at αAla19Cys hemoglobin with > 80% efficiency, although some side reactions were observed at higher PEG:hemoglobin ratios; the adduct bound oxygen with similar affinity and cooperativity as wild type hemoglobin. When directly compared to maleimide-PEG, the mono-sulfone-PEG adduct was significantly more stable when incubated at 37°C for seven days in the presence of 1 mM reduced glutathione. Hemoglobin treated with mono-sulfone-PEG retained > 90% of its conjugation, whereas for maleimide-PEG < 70% of the maleimide-PEG conjugate remained intact. Although maleimide-PEGylation is certainly stable enough for acute therapeutic use as an oxygen therapeutic, for pharmaceuticals intended for longer vascular retention (weeks-months), reagents such as mono-sulfone-PEG may be more appropriate.

Effect of PEGylation on Host Defense Peptide Complexation with Bacterial Lipopolysaccharide

Conjugation with poly(ethylene glycol) ("PEGylation") is a widely used approach for improving the therapeutic propensities of peptide and protein drugs through prolonging bloodstream circulation, reducing toxicity and immunogenicity, and improving proteolytic stability. In the present study, we investigate how PEGylation affects the interaction of host defense peptides (HDPs) with bacterial lipopolysaccharide (LPS) as well as HDP suppression of LPS-induced cell activation. In particular, we investigate the effects of PEGylation site for KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), a peptide displaying potent anti-inflammatory effects, primarily provided by its N-terminal part. PEGylation was performed either in the N-terminus, the C-terminus, or in both termini, keeping the total number of ethylene groups (n = 48) constant. Ellipsometry showed KYE28 to exhibit pronounced affinity to both LPS and its hydrophobic lipid A moiety. The PEGylated peptide variants displayed lower, but comparable, affinity for both LPS and lipid A, irrespective of the PEGylation site. Furthermore, both KYE28 and its PEGylated variants triggered LPS aggregate disruption. To investigate the peptide structure in such LPS complexes, a battery of nuclear magnetic resonance (NMR) methods was employed. From this, it was found that KYE28 formed a well-folded structure after LPS binding, stabilized by hydrophobic domains involving aromatic amino acids as well as by electrostatic interactions. In contrast, the PEGylated peptide variants displayed a less well-defined secondary structure, suggesting weaker LPS interactions in line with the ellipsometry findings. Nevertheless, the N-terminal part of KYE28 retained helix formation after PEGylation, irrespective of the conjugation site. For THP1-Xblue-CD14 reporter cells, KYE28 displayed potent suppression of LPS activation at simultaneously low cell toxicity. Interestingly, the PEGylated KYE28 variants displayed similar or improved suppression of LPS-induced cell activation, implying the underlying key role of the largely retained helical structure close to the N-terminus, irrespective of PEGylation site. Taken together, the results show that PEGylation of HDPs can be done insensitively to the conjugation site without losing anti-inflammatory effects, even for peptides inducing such effects through one of its termini.

Design and Study of PEG Linkers That Enable Robust Characterization of PEGylated Proteins.

Several PEGylated therapeutic proteins are approved drugs, and more are under development. However, the synthesis and characterization of these bioconjugates, especially heterogeneous mixtures of PEGylated proteins, are challenging. The present study focuses on the development of PEG linkers that can be installed through biocatalytic route and render much simpler and insightful analytical characterization of PEG-protein conjugates. This linker enables traditional peptide mapping assay to determine protein sequence coverage, natural PTMs, and PEG attachment sites. Novel PEG linkers are cleavable during traditional sample preparation, leaving behind reporter amino acids to allow the determination of PEG attachment sites by peptide mapping. Products of transglutaminase-catalyzed bioconjugation of 5K PEG to Interferon α-2b were analyzed, and K31, K134, and K164 were identified as the PEGylation sites; the former two being newly determined sites demonstrates the sensitivity of the approach. In another instance, conjugation sites on Interleukin-2-PEG conjugation were found to be K31, K47, K48, and K75.

Site-specific monoPEGylated interferon alpha2a mediated by microbial transglutaminase

PEGylation is considered one of the most successful techniques to improve the characteristics of protein drugs including to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. One known PEG modification method is to attach PEG to the free amino group, typically at lysine residues or at the N-terminal amino acid with no selectivity, resulting in a heterogeneous product mixture. This lack of selectivity can present problems when a therapeutic PEGylated protein is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic PEGylation of proteins is one route to overcome this limitation. Transglutaminases (TGase) are enzyme candidates for site-specific PEGylation. We use human interferon alpha 2a (IFN α2a) as a test case, and predict that the potential modification residues are Gln101 by computational approach as it contains 12 potential PEGylation sites. IFN α2a was PEGylated by Y shaped PEG40k-NH2 mediated by microbial transglutaminase. Our results show that the microbial transglutaminase mediated PEGylation of IFN α2a was site-specific only at the site of Gln101 in IFN α2a, yielding the single mono-conjugate PEG-Gln101-IFN α2a with a mass of 59 374.66 Da. Circular dichroism studies showed that PEG-Gln101-IFN α2a preserved the same secondary structures as native IFN α2a. As expected, the bioactivity and pharmacokinetic profile in rats of PEG-Gln101-IFN α2a revealed a significant improvement to unmodified IFN α2a, and better than PEGASYS.

A bioengineered arginine-depleting enzyme as a long-lasting therapeutic agent against cancer.

L-Arginine (L-Arg) depletion has attracted great attention in cancer therapy. Although two types of arginine-depleting enzymes, arginine deiminase (ADI) and human arginase I, are undergoing clinical trials, random site of PEGylation, low efficacy of heavy metal as co-factor, and immunogenicity limit the performance of these drugs and cause difficulty in a homogeneous production. Here we screened ten catalytic metal ions and have successfully produced a site-specific mono-PEGylated human arginase I mutant by conjugating the Cys45 residue to PEG-maleimide to minimize the decrease in activity and produce a homogeneous product. The catalytic efficiency trend of metal ion-enriched human arginase I mutant (HAI) was Co2+ > Ni2+ ≫ Mn2+. The overall kcat/KM values of Co-HAI and Ni-HAI were higher than Mn-HAI by ~ 8.7- and ~ 5.2-folds, respectively. Moreover, the results of enzyme kinetics and circular dichroism spectrometry demonstrated that the 20 or 40kDa linear and branched PEG attached on the HAI surface did not affect the enzyme activity and the protein secondary structures. In vitro studies showed that both Co-HAI-PEG20L and Ni-HAI-PEG20L inhibited the growth of eight types of cancer cell lines. The pharmacodynamic study in mice demonstrated that the i.p. administration of Co-HAI-PEG20L at 13mg/kg and Ni-HAI-PEG20L at 15mg/kg was able to maintain a L-Arg level below its detection limit for over 120h after one injection. The body weights of mice could return to normal levels within 5days after injection, showing that the doses were well-tolerated. Therefore, both the Ni-HAI-PEG20L and Co-HAI-PEG20L are promising candidates for cancer therapy. KEY POINTS: • Mono-PEGylation applied on human arginase I mutant (HAI) successfully. • The catalytic efficiency of Co- and Ni-enriched HAI was higher than the wild type. • At least eight types of cancer cell lines were inhibited by Co- and Ni-HAI-PEG20L. • Co- and Ni-HAI-PEG20L were able to achieve weekly depletion of L-Arg. Graphical abstract.

Pegylation and Cell Based Bioassay of Human Interferon-α2b Along its Docking Studies and Effect on Plasma Half-Life.

Interferon-α2b is FDA approved drug for the treatment of chronic HCV and HBV, melanoma, AIDS-related KS, carcinomas, hairy cell leukemia and chronic myelogenous leukemia. However, administration of interferon-α2b to patients takes place thrice in a week due to short in vivo circulation half-life. To extend the circulation half-life of IFN-α2b, it is conjugated with polyethylene glycol (PEG). However, PEGylation may results in reduction of its antiviral and antiproliferative activities but on the other side, it results in prolonged plasma half-life. Human interferon-α2b was PEGylated with linear 20kDa methoxypolyethlene glycol (mPEG) Propionaldehyde (IFN-Ald20K), Y-Shaped 40kDa mPEG-Propionaldehyde (IFNAld40K), linear 20-kDa mPEG-Succinimidyl Succinate (IFN-NHS20K), and Y shaped 40kDa mPEG-Succinimidyl Succinate (IFN-NHS40K). Impact of PEG size, shape and PEGylation site was studied to establish their relationship with antiprolifetaive activities and serum retention time of PEGylated IFN-α2b. RP-HPLC studies showed that larger PEGs (40kDa) increased the hydrodynamic volume and increased the serum retention time while antiproliferative activity in HepG2 cell line was decreased with increase in PEGylated interferon-α2b size. Molecular docking results also dictated the same effect that increase in PEGylated interferon-α2b size results in steric shielding of the receptor-binding site on interferon-α2b. IFN-Ald20K showed highest (45%) biological activity with serum half-life 40 hours while IFN-NHS40K showed least (7%) biological activity with serum halflife 56 hours. Thus, IFN-Ald40K with 12% residual activity and 62 hours of serum half-life proved to be a potent candidate for anticancer and antiviral effect with enhanced serum retention time.

Influence of PEGylation on the Strength of Protein Surface Salt Bridges.

Conjugation of polyethylene glycol (PEGylation) is a well-known strategy for extending the serum half-life of protein drugs and for increasing their resistance to proteolysis and aggregation. We previously showed that PEGylation can increase protein conformational stability; the extent of PEG-based stabilization depends on the PEGylation site, the structure of the PEG-protein linker, and the ability of PEG to release water molecules from the surrounding protein surface to the bulk solvent. The strength of a noncovalent interaction within a protein depends strongly on its microenvironment, with salt-bridge and hydrogen-bond strength increasing in nonpolar versus aqueous environments. Accordingly, we wondered whether partial desolvation by PEG of the surrounding protein surface might result in measurable increases in the strength of a salt bridge near a PEGylation site. Here we explore this possibility using triple-mutant box analysis to assess the impact of PEGylation on the strength of nearby salt bridges at specific locations within three peptide model systems. The results indicate that PEG can increase the nearby salt-bridge strength, though this effect is not universal, and its precise structural prerequisites are not a simple function of secondary structural context, of the orientation and distance between the PEGylation site and salt bridge, or of salt-bridge residue identity. We obtained high-resolution X-ray diffraction data for a PEGylated peptide in which PEG enhances the strength of a nearby salt bridge. Comparing the electron density map of this PEGylated peptide versus that of its non-PEGylated counterpart provides evidence of localized protein surface desolvation as a mechanism for PEG-based salt-bridge stabilization.

Rational surface engineering of an arginine deiminase (an antitumor enzyme) for increased PEGylation efficiency.

Arginine deiminase (ADI) is a therapeutic protein for cancer therapy of arginine-auxotrophic tumors. However, its application as anticancer drug is hampered by its poor stability under physiological conditions in the bloodstream. Commonly, random PEGylation is being used for increasing the stability of ADI and in turn the improved half-life. However, the traditional random PEGylation usually leads to poor PEGylation efficiency due to the limited number of Lys on the protein surface. To boost the PEGylation efficiency and enhance the stability of ADI further, surface engineering of PpADI (an ADI from Pseudomonas plecoglossicida) to increase the suitable PEGylation sites was carried out. A new in silico approach for increasing the PEGylation sites was developed. The validation of this approach was performed on previously identified PpADI variant M31 to increase potential PEGylation sites. Four Arg residues on the surface of PpADI M31 were selected through three criteria and subsequently substituted to Lys, aiming for providing primary amines for PEGylation. Two out of the four substitutions (R299K and R382K) enhanced the stability of PEGylated PpADI in human serum. The average numbers of PEGylation sites were increased from ~12 (tetrameric PpADI M31, starting point) to ~20 (tetrameric PpADI M36, final variant). Importantly, the PEGylated PpADI M36 after PEGylation exhibited significantly improved Tm values (M31: 40°C; M36: 40°C; polyethylene glycol [PEG]-M31: 54°C; PEG-M36: 64°C) and half-life in human serum (M31: 1.9 days; M36: 2.0 days; PEG-M31: 3.2 days; PEG-M36: 4.8 days). These proved that surface engineering is an effective approach to increase the PEGylation efficiency which therefore enhances the stability of therapeutic enzymes. Furthermore, the PEGylated PpADI M36 represents a highly attractive candidate for the treatment of arginine-auxotrophic tumors.

Site specific PEGylation of β-lactoglobulin at glutamine residues and its influence on conformation and antigenicity

β-lactoglobulin (β-LG) is one of the main allergens in milk. Polyethylene glycol (PEG) modification (PEGylation) was found to have the ability to reduce the antigenicity of proteins. To determine the effect of site specific PEGylation on β-LG antigenicity and conformation, we applied 5 kDa methoxy polyethylene glycol-amine (mPEG-NH2) to modify β-LG at glutamine (Gln) residues under the catalysis of transglutaminase. The antigenicity of β-LG was measured using rabbit IgG antibodies by indirect competitive ELISA. The result indicated that the antigenicity of β-LG was decreased from 72.2 μg/mL to 22.7 μg/mL after PEGylation. SDS-PAGE and MALDI-TOF-MS showed that the molecular mass of native β-LG was about 18.3 kDa while the PEGylated β-LG had a molecular mass of 23.4 kDa, which meant that mono-PEGylated β-LG was obtained after PEGylation and purification by cation exchange chromatography. Additionally, the circular dichroism spectrum of the PEGylated β-LG was approximately superimposed on that of β-LG and the secondary structure content of β-LG also had no significant changes after PEGylation, which indicated that the secondary structure of β-LG was preserved. After PEGylation, the intrinsic fluorescence intensity of β-LG decreased from 6361 to 5159 while the surface hydrophobicity increased, which indicated that the tertiary structure of β-LG was slightly changed. PEGylation site analysis result showed that Gln 155 or Gln 159 might be the most possible binding site. In conclusion, the decrease of the antigenicity of β-LG induced by the PEGylation is mainly due to the steric shielding effect of PEG chain rather than conformational changes of β-LG.

Compassionate Use of Ropeginterferon-Alfa-2b/P1101 for Treatment of High Risk Polycythemia Vera and Essential Thrombocythemia Patients Previously Controlled on Pegylated Interferon-Alfa-2a/Pegasys®

Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative neoplasms (MPN) associated with morbidity and mortality resulting from thrombosis or transformation to myelofibrosis and/or secondary acute leukemia. The acquired somatic mutation JAK2V617Fhas been observed in 98% of PV patients, and around 50% of ET patients. Interferon is known to be an effective treatment for chronic MPNs, but unlike hydroxyurea (HU, Hydrea®), it has been shown to target the malignant clone by decreasing JAK2V617Fallelic burden, and in some patients restoring polyclonal hematopoiesis. (Liu et al, Blood. 2003;101:3294-3301). The covalent binding of polyethylene glycol to the interferon molecule, through pegylation, prolongs the half-life by lowering the clearance of interferon, thereby extending the duration of its therapeutic effects, permitting less frequent administration and fewer side-effects. Currently, there are two commercially available pegylated interferons in the U.S., peginterferon alfa-2b (PEG-Intron®, Merck) and peginterferon alfa-2a (Pegasys®, PEG, Roche), both with heterogeneous pegylation with multiple pegylation sites. Ropeginterferon-alfa 2b/P1101 (ROPEG) is a novel longer acting mono-pegylated recombinant proline-interferon alfa-2b which has shown good efficacy in PV and has successfully completed a phase III trial in PV patients comparing it against HU (PROUD-PV study) in Europe (ASH2016 Abstract;Blood. 2016;128:475).Earlier, we had enrolled 53 high risk PV and ET patients to the Myeloproliferative Disorders Research Consortium (MPD-RC) trials #111 (refractory to HU) and #112 (randomized to HU vs PEG)(www.clinicaltrials.gov; NCT01259856 and NCT01259817). After the completion of these trials, PEG was no longer supplied by the study and not adequately covered by insurance for many of our patients. Therefore, ROPEG was procured under an FDA-approved expanded access program and used as a substitute for those controlled on PEG, and these patients are being transitioned from PEG to ROPEG according to the dosing strategy shown in Table 1.Seven patients have been switched onto ROPEG as of writing this abstract (Table 2). After a mean follow-up of roughly 8 weeks (range: 2 - 10 weeks), all have maintained response to therapy and none have had a thrombotic event or new or increased interferon-related side effects thus far. Patient 002 experienced elevated AST/ALT while on PEG, which normalized after four q2 week doses of ROPEG (8 weeks of therapy). Patients 003 and 004 who had persistent fatigue on PEG, saw significant improvement of their symptoms after switching to ROPEG. In patient 005 who has ET, bone marrow cytogenetics at entry to PEG had a paracentric inversion of the long arm of chromosome 3, namely 3q21.3q26.2 (RPN1/MECOM, inv3; diagnostic of AML per WHO criteria), in 16/20 (80%) metaphases. Over the 4 ensuing years while on PEG, this cytogenetic abnormality progressively decreased. FISH assay was set up 2 years ago wherein 86/200 cells (46%) were positive with lowest achieved level of 36/200 cells (18%) without any evidence of emerging AML. Upon transition to ROPEG (2 fortnightly injections of 50 mcg), this response was maintained (23/200 cells scored (11.5%). Of the 3 patients who were JAK2V617Fpositive, one patient (patient 003) showed a decrease in the JAK2V617Fallelic burden (37% to 18%) after two months of ROPEG despite decreasing the dose to 50 mcg (from 100 mcg) 2 weeks after the first injection, while the other two patients had no statistically significant change in their JAK2V617Fallelic burden. Clonality will be followed in female subjects to see if clonal hematopoiesis is suppressed and normalizes with long-term administration of ROPEG.Our early experience suggests ROPEG is a safe and effective alternative to PEG permitting less frequent administration for patients with high risk PV and ET. Notable observations include normalization of hepatic transaminases previously induced by PEG, maintenance or even suppression of one highly deleterious cytogenetic abnormality, maintenance of ET & PV remission, and amelioration of interferon side-effects with lower equivalent doses. In the future, we intend to increase the pool of patients by designing a protocol for newly diagnosed high risk ET and PV with randomization to HU versus ROPEG. DisclosuresNo relevant conflicts of interest to declare.

Kinetic Optimization and Scale-Up of Site-Specific Thiol-PEGylation of Loxenatide from Laboratory to Pilot Scale

PEG-loxenatide is a new once-weekly GLP-1 receptor agonist candidate for the treatment of type 2 diabetes mellitus. Site-specific thiol-PEGylation of loxenatide with Y-shaped mPEG2-MAL of 40 kDa was first optimized using a reaction kinetic model based approach on a laboratory scale. The optimized reaction conditions were further scaled up to a pilot scale, and PEG-loxenatide achieved a high yield and purity. This result demonstrated an efficient and convenient scale-up PEGylation reaction process, which achieved the maximum utilization of the raw materials and the minimum generation of byproducts. Furthermore, PEG-loxenatide was efficiently purified using cation exchange chromatography on a pilot scale. The purified PEG-loxenatide was further desalted, concentrated, and lyophilized to improve its stability. PEGylation site analysis revealed that a single PEG molecule was conjugated to the unique cysteine residue at the C-terminus of loxenatide. This study will provide useful information for the preparatio...

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 5kDa 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 20kDa 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.

The Locational Impact of Site-Specific PEGylation: Streamlined Screening with Cell-Free Protein Expression and Coarse-Grain Simulation.

Although polyethylene glycol (PEG) is commonly used to improve protein stability and therapeutic efficacy, the optimal location for attaching PEG onto proteins is not well understood. Here, we present a cell-free protein synthesis-based screening platform that facilitates site-specific PEGylation and efficient evaluation of PEG attachment efficiency, thermal stability, and activity for different variants of PEGylated T4 lysozyme, including a di-PEGylated variant. We also report developing a computationally efficient coarse-grain simulation model as a potential tool to narrow experimental screening candidates. We use this simulation method as a novel tool to evaluate the locational impact of PEGylation. Using this screen, we also evaluated the predictive impact of PEGylation site solvent accessibility, conjugation site structure, PEG size, and double PEGylation. Our findings indicate that PEGylation efficiency, protein stability, and protein activity varied considerably with PEGylation site, variations that were not well predicted by common PEGylation guidelines. Overall our results suggest current guidelines are insufficiently predictive, highlighting the need for experimental and simulation screening systems such as the one presented here.

Characterization of singly and multiply PEGylated insulin isomers by reversed-phase ultra-performance liquid chromatography interfaced with ion mobility mass spectrometry

Conjugation of poly(ethylene glycol) (PEG) to protein drugs (PEGylation) is increasingly utilized in the biotherapeutics field because it improves significantly the drugs' circulatory half-life, solubility, and shelf-life. The activity of a PEGylated drug depends on the number, size, and location of the attached PEG chain(s). This study introduces a 2D separation approach, including reversed-phase ultra-performance liquid chromatography (RP-UPLC) and ion mobility mass spectrometry (IM-MS), in order to determine the structural properties of the conjugates, as demonstrated for a PEGylated insulin sample that was prepared by random amine PEGylation. The UPLC dimension allowed separation based on polarity. Electrospray ionization (ESI) of the eluates followed by in-source dissociation (ISD) truncated the PEG chains and created insulin fragments that provided site-specific information based on whether they contained a marker at the potential conjugation sites. Separation of the latter fragments by size and charge in the orthogonal IM dimension (pseudo-4D UPLC-ISD-IM-MS approach) enabled clear detection and identification of the positional isomers formed upon PEGylation. The results showed a highly heterogeneous mixture of singly and multiply conjugated isomers plus unconjugated material. PEGylation was observed on all three possible attachment sites (ε-NH2 of LysB29, A- and B-chain N-termini). Each PEGylation site was validated by analysis of the same product after disulfide bond cleavage, so that the PEGylated A- and B- chain could be individually characterized with the same pseudo-4D UPLC-ISD-IM-MS method.