Investigation on PEGylation strategy of recombinant human interleukin-1 receptor antagonist

Although PEGylation is a potential approach to prolong the half-lives and reduce the dosing frequency of therapeutic proteins, conjugation behaviors of polymer have pivotal effects on the remaining bioactivities of the derivatives. In this study, the PEGylation strategy of recombinant human interleukin-1 receptor antagonist was investigated. The random conjugation of polyethylene glycol to amino groups on the protein resulted in a severe loss of activity and only retained 9.8% of the activity. In contrast, the PEGylation at the thiol groups had moderate effects on the bioactivity of protein and 40% of activity was conserved. The results suggested that the thiol-target PEGylation was more beneficial for IL-1ra.     

Relationship between protein structure and methionine oxidation in recombinant human alpha 1-antitrypsin

alpha 1-Antitrypsin is a metastable and conformationally flexible protein that belongs to the serpin family of protease inhibitors. Although it is known that methionine oxidation in the protein's active site results in a loss of biological activity, there is little specific knowledge regarding the reactivity of each of the protein's methionine residues. In this study, we have used peptide mapping to study the oxidation kinetics of each of alpha 1-antitrypsin's methionines in alpha 1-AT((C232S)) as well as M351L and M358V mutants. These kinetic studies establish that Met1, Met226, Met242, Met351, and Met358 are reactive with hydrogen peroxide at neutral pH and that each reactive methionine is oxidized in a bimolecular, rather than coupled, mechanism. Analysis of Met226, Met351, and Met358 oxidation provides insights regarding the structure of alpha 1-antitrypsin's active site that allow us to relate conformation to experimentally observed reactivity. The relationship between solution pH and methionine oxidation was also examined to evaluate methionine reactivity under conditions that perturb the native structure. Methionine oxidation data show that at pH 5, global conformational changes occur that alter the oxidation susceptibility of each of alpha 1-antitrypsin's 10 methionine residues. Between pH 6 and 9, however, more localized conformational changes occur that affect primarily the reactivity of Met242. In sum, this work provides a detailed analysis of methionine oxidation in alpha 1-antitrypsin and offers new insights into the protein's solution structure.     

High-pressure studies of aggregation of recombinant human interleukin-1 receptor antagonist: thermodynamics, kinetics, and application to accelerated formulation studies

Recombinant human interleukin-1 receptor antagonist (IL-1ra) in aqueous solutions unfolds and aggregates when subjected to hydrostatic pressures greater than about 180 MPa. This study examined the mechanism and thermodynamics of pressure-induced unfolding and aggregation of IL-1ra. The activation free energy for growth of aggregates (DeltaG-/+(aggregation)) was found to be 37 +/- 3 kJ/mol, whereas the activation volume (DeltaV-/+(aggregation)) was -120 +/- 20 mL/mol. These values compare closely with equilibrium values for denaturation: The free energy for denaturation, DeltaG(denaturation), was 20 +/- 5 kJ/mol, whereas the partial specific volume change for denaturation, DeltaV(denaturation), was -110 +/- 30 mL/mol. When IL-1ra begins to denature at pressures near 140 MPa, cysteines that are normally buried in the native state become exposed. Under oxidizing conditions, this results in the formation of covalently cross-linked aggregates containing nonnative, intermolecular disulfide bonds. The apparent activation free energy for nucleation of aggregates, DeltaG-/+(nuc), was 42 +/- 4 kJ/mol, and the activation volume for nucleation, DeltaV-/+(nuc),was -175 +/- 37 mL/mol, suggesting that a highly solvent-exposed conformation is needed for nucleation. We hypothesize that the large specific volume of IL-1ra, 0.752 +/- 0.004 mL/g, coupled with its relatively low conformational stability, leads to its susceptibility to denaturation at relatively low pressures. The positive partial specific adiabatic compressibility of IL-1ra, 4.5 +/- 0.7 +/- 10(-12) cm2/dyn, suggests that a significant component of the DeltaV(denaturation) is attributable to the elimination of solvent-free cavities. Lastly, we propose that hydrostatic pressure is a useful variable to conduct accelerated formulation studies of therapeutic proteins.     

Loss of conformational stability in calmodulin upon methionine oxidation.

We have used electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and fluorescence spectroscopy to investigate the secondary and tertiary structural consequences that result from oxidative modification of methionine residues in wheat germ calmodulin (CaM), and prevent activation of the plasma membrane Ca-ATPase. Using ESI-MS, we have measured rates of modification and molecular mass distributions of oxidatively modified CaM species (CaMox) resulting from exposure to H2O2. From these rates, we find that oxidative modification of methionine to the corresponding methionine sulfoxide does not predispose CaM to further oxidative modification. These results indicate that methionine oxidation results in no large-scale alterations in the tertiary structure of CaMox, because the rates of oxidative modification of individual methionines are directly related to their solvent exposure. Likewise, CD measurements indicate that methionine oxidation results in little change in the apparent alpha-helical content at 28 degrees C, and only a small (0.3 +/- 0.1 kcal mol(-1)) decrease in thermal stability, suggesting the disruption of a limited number of specific noncovalent interactions. Fluorescence lifetime, anisotropy, and quenching measurements of N-(1-pyrenyl)-maleimide (PMal) covalently bound to Cys26 indicate local structural changes around PMal in the amino-terminal domain in response to oxidative modification of methionine residues in the carboxyl-terminal domain. Because the opposing globular domains remain spatially distant in both native and oxidatively modified CaM, the oxidative modification of methionines in the carboxyl-terminal domain are suggested to modify the conformation of the amino-terminal domain through alterations in the structural features involving the interdomain central helix. The structural basis for the linkage between oxidative modification and these global conformational changes is discussed in terms of possible alterations in specific noncovalent interactions that have previously been suggested to stabilize the central helix in CaM.     

Pharmacokinetics, safety and immunomodulatory effects of human recombinant interleukin-1 receptor antagonist in healthy humans.

A phase I study of human recombinant interleukin-1 receptor antagonist (IL-1ra) was conducted in healthy males between the ages of 18 and 30. Twenty-five volunteers received a single, 3 h continuous intravenous infusion of doses ranging between 1 mg/kg and 10 mg/kg IL-1ra. At 3 h into the infusion, plasma IL-1ra levels were 3.1 micrograms/ml and 29 micrograms/ml for the 1 mg/kg and 10 mg/kg doses, respectively. Post-infusion plasma IL-1ra levels declined rapidly, exhibiting an initial half-life of 21 min and a terminal half-life of 108 min. Clinical, hematological, biochemical, endocrinological and immunomodulatory effects were monitored over 72 h and compared to those of four subjects receiving a 3 h infusion of saline. There were no clinically significant differences between the drug and saline groups in symptoms, physical examinations, complete blood counts, mononuclear cell phenotypes, blood chemistry profiles, serum iron and serum cortisol levels. Peripheral blood mononuclear cells (PBMC) obtained after completion of the IL-1ra infusion synthesized significantly less interleukin 6 ex vivo than PBMC from saline-injected controls. These data suggest that transient blockade of interleukin 1 receptors is safe and does not significantly affect homeostasis.     

Inhibition of interleukin-1-induced synovitis and articular cartilage proteoglycan loss in the rabbit knee by recombinant human interleukin-1 receptor antagonist

Intra-articular injection of interleukin-1 (IL-1) into the knee joints of rabbits produces a synovitis associated with the loss of proteoglycan from the matrix of articular cartilage. This experimental finding supports the hypothesis that IL-1 is a possible mediator of the pathology of inflammatory joint diseases and suggests that antagonism of IL-1 could offer a therapeutic approach to these diseases. It has recently been reported that culture of human monocytes on adherent IgG stimulates these cells to synthesize a specific inhibitor of IL-1 bioactivity (IL-1ra) that acts as a receptor antagonist with lymphocytes and mesenchymal cells. We have now shown that intravenous injection of IL-1ra into rabbits given an intra-articular injection of recombinant IL-1 beta not only inhibits the entry of leukocytes into the synovial lining and joint cavity but blocks the ability of IL-1 to cause loss of proteoglycan from articular cartilage. This ability of IL-1ra to inhibit IL-1-induced arthritis in the rabbit reveals that this protein has appropriate pharmacokinetic and pharmacodynamic properties and further strengthens the belief that it may be a useful therapeutic agent.     

Temperature dependence of benzyl alcohol- and 8-anilinonaphthalene-1-sulfonate-induced aggregation of recombinant human interleukin-1 receptor antagonist

The critical role played by temperature in ligand-induced protein aggregation was investigated. Recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and the ligands benzyl alcohol and 8-anilinonaphthalene-1-sulfonate (ANS) were used. We investigated aggregation kinetics and the conformation and cysteine reactivity of rhIL-1ra in buffer alone or in the presence of 0.9% (w/v) benzyl alcohol or 4.2 or 21 mM ANS at 25 and 37 degrees C. In buffer, protein aggregation was not detected at 25 degrees C but occurred at 37 degrees C. At 25 degrees C, neither benzyl alcohol nor 4.2 mM ANS enhanced aggregation. However, at 37 degrees C, both compounds greatly accelerated protein aggregation. With 21 mM ANS, rhIL-1ra aggregation was accelerated at both temperatures, but the effect was more pronounced at 37 degrees C than at 25 degrees C. Increasing the temperature from 25 to 37 degrees C caused a minor perturbation in the tertiary structure of rhIL-1ra in buffer but no detectable alteration in secondary structure. Benzyl alcohol enhanced the tertiary structural perturbation at 37 degrees C, but the secondary structure was not affected by the ligand. The reactivity of buried free cysteines of rhIL-1ra was enhanced by benzyl alcohol at 37 degrees C but not at 25 degrees C, consistent with the structural results. Isothermal titration calorimetry documented that the interaction of benzyl alcohol with rhIL-1ra was hydrophobic and that the degree of hydrophobic interactions increased with temperature. At 25 degrees C, the interaction of ANS with rhIL-1ra was electrostatic, but at 37 degrees C, both electrostatic and hydrophobic interactions were important. Taken together, our results support the conclusion that benzyl alcohol and ANS interact hydrophobically with partially unfolded aggregation-prone protein molecules, resulting in temperature-dependent increases in their levels and acceleration of protein aggregation.     

Oxidation of methionine residues in lutropin

Bovine lutropin and its subunits were submitted to oxidation by sodium periodate or chloramine T. Methionine residues were easily oxidized but partial destruction of fucose was observed. After oxidation treatment most of the lutropin exhibits the same elution volume in gel filtration as the native hormone. Sucrose gradient sedimentation or gel filtration experiments show however that the oxidation of isolated subunits is accompanied by aggregation of conformational changes even in the case of porcine beta subunit which contains only one methionine residue, unit and intact alpha subunit exhibit very low residual activity.     

Oxidation of methionine residues in the prion protein by hydrogen peroxide

Reaction of H(2)O(2) with the recombinant SHa(29-231) prion protein resulted in rapid oxidation of multiple methionine residues. Susceptibility to oxidation of individual residues, assessed by mass spectrometry after digestion with CNBr and lysC, was in general a function of solvent exposure. Met 109 and Met 112, situated in the highly flexible amino terminus, and key residues of the toxic peptide PrP (106-126), showed the greatest susceptibility. Met 129, a residue located in a polymorphic position in human PrP and modulating risk of prion disease, was also easily oxidized, as was Met 134. The structural effect of H(2)O(2)-induced methionine oxidation on PrP was studied by CD spectroscopy. As opposed to copper catalyzed oxidation, which results in extensive aggregation of PrP, this reaction led only to a modest increase in beta-sheet structure. The high number of solvent exposed methionine residues in PrP suggests their possible role as protective endogenous antioxidants.     

Organization of the human interleukin-1 receptor antagonist gene IL1HY1

 The interleukin (IL)-1 family of proteins plays an important role in inflammatory and defense mechanisms. The recently characterized IL1HY1 cDNA encodes a new member of the IL-1 receptor antagonist family (IL-1ra). In this report, we describe the complete nucleotide sequence of the human IL1HY1 gene. We sequenced approximately 7600 nucleotides and found four coding exons ranging in size from 55 to 2288 nucleotides. The 5′ untranslated region is formed by one of two alternatively used exons and one invariably present exon which also contains the region encoding the first nine amino acids of the protein. IL1HY1 and IL-1ra intron positions are well conserved within the protein-coding region, providing evidence that these genes arose from a duplication of a primordial IL-1 receptor antagonist gene. Received: 15 October 1999 / Revised: 30 December 1999     

Comparative oxidation studies of methionine residues reflect a structural effect on chemical kinetics in rhG-CSF

The effect of protein conformation on the rate of chemical degradation is poorly understood. To address the role of structure on chemical degradation kinetics, comparative oxidation studies of methionine residues in recombinant human granulocyte colony-stimulating factor (rhG-CSF) were performed. The kinetics of oxidation of methionine residues by hydrogen peroxide (H2O2) in rhG-CSF and corresponding chemically synthesized peptides thereof was measured at different temperatures. To assess structural effects, equilibrium denaturation experiments also were conducted on rhG-CSF, yielding the free energy of unfolding as a function of temperature. A comparison of the relative rates of oxidation of methionine residues in short peptides with those of corresponding methionine residues in rhG-CSF yields an understanding of how protein tertiary structure affects oxidation reactions. For the temperature range that was studied, 4-45 degrees C, the oxidation rate constants followed an Arrhenius equation quite well, suggesting the lack of temperature-induced local structural perturbations that affect chemical degradation rates. One of the four methionine residues, Met 122, exhibited an activation energy significantly different from that of the corresponding peptide. Extrapolation of kinetic data predicts non-Arrhenius behavior around the melting temperature. Three phenomenological models based on different mechanisms are discussed, and an application to shelf life prediction of pharmaceuticals is presented.     

Anion binding and controlled aggregation of human interleukin-1 receptor antagonist

Highly concentrated human recombinant interleukin-1 receptor antagonist (IL-1ra) aggregates at elevated temperature without perturbation in its secondary structure. The protein aggregation can be suppressed depending on the buffer ionic strength and the type of anion present in the sample solution. Phosphate is an approximately 4-fold weaker suppressant than either citrate or pyrophosphate on the basis of the measured protein aggregation rates. This is in agreement with the strength of protein-anion interactions at the IL-1ra single anion-binding site as judged by the estimated dissociation constant values of 2.9 mM, 3.8 mM, and 13.7 mM for pyrophosphate, citrate, and phosphate, respectively. The strength of binding also correlates with the anion size and with the number of ionized groups available per molecule at a given pH. Affinity probing of IL-1ra with methyl acetyl phosphate (MAP) in combination with proteolytic digestion and mass spectral analysis show that an anion-binding site location on the IL-1ra surface is contributed by lysine-93 and lysine-96 of the loop 84-98 as well as by lysine-6 of the unstructured N-terminal region 1-7. The replacement of lysine-93 with alanine by site-directed mutagenesis results in dramatically suppressed IL-1ra aggregation. Furthermore, when the unstructured N-terminal region of IL-1ra is removed by limited proteolysis, a 2-fold increase in the time course of the aggregation lag phase is observed for the truncated protein. An anion-controlled mechanism of IL-1ra aggregation is proposed by which the anion competition for the protein cationic site prevents formation of intermolecular cation-pi interactions and, thus, interferes with the protein asymmetric self-association pathway.     

High altitude increases circulating interleukin-6, interleukin-1 receptor antagonist and C-reactive protein

Hypoxic pulmonary vasoconstriction is associated with but may not be sufficient for the development of high-altitude pulmonary oedema (HAPO). Hypoxia is known to induce an inflammatory response in immune cells and endothelial cells. It has been speculated that hypoxia-induced inflammatory cytokines at high altitude may contribute to the development of HAPO by causing capillary leakage in the lung. We were interested if such an inflammatory response, possibly involved in a later development of HAPO, is detectable at high altitude in individuals without HAPO. We examined the plasma levels of interleukin 6 (IL-6), interleukin 1 receptor antagonist (IL-1ra) and C-reactive protein (CRP) in two independent studies: study A, Jungfraujoch, Switzerland, three overnight stays at 3458 m, n=12; study B: Capanna Regina Margherita, Italy, 3 overnight stays at 3647 m and one overnight stay at 4559 m, n=10. In both studies, probands showed symptoms of acute mountain sickness but no signs of HAPO. At the Jungfraujoch, IL-6 increased from 0.1+/-0.03 pg/ml to 2. 0+/-0.5 pg/ml (day 2, P=0.03), IL-1ra from 101+/-21 to 284+/-73 pg/ml (day 2, P=0.01), and CRP from 1.0+/-0.4 to 5.8+/-1.5 micrograms/ml (day 4, P=0.01). At the Capanna Margherita, IL-6 increased from 0. 5+/-0.2 pg/ml to 2.0+/-0.8 pg/ml (P=0.02), IL-1ra from 118+/-25 to 213+/-28 pg/ml (P=0.02), and CRP from 0.4+/-0.03 to 3.5+/-1.1 micrograms/ml (P=0.03). IL-8 was below the detection limit of the ELISA (<25 pg/ml) in both studies. The increase of IL-6 and IL-1ra in response to high altitude was delayed and preceded the increase of CRP. We conclude that: (1) circulating IL-6, IL-1ra and CRP are upregulated in response to hypobaric hypoxic conditions at high altitude, and (2) the moderate systemic increase of these inflammatory markers may reflect considerable local inflammation. The existence and the kinetics of high altitude-induced cytokines found in this study support the hypothesis that inflammation is involved in the development of HAPO.     

Physiologic role of interleukin-1 receptor antagonist

Recent studies have described the spontaneous development of arthritis or vasculitis in IL-1 receptor antagonist (IL-1Ra) knockout mice bred on specific and different genetic backgrounds. The levels of both secreted and intracellular isoforms of IL-1Ra produced in the rheumatoid joint or in the arterial wall may not be adequate to effectively inhibit the excess amounts of locally produced IL-1. Thus, an imbalance between IL-1 and IL-1Ra may predispose to local inflammatory disease in particular tissues in the presence of other as yet unknown genetically influenced factors.     

Expression and characterization of human glycosylated interleukin-1 receptor antagonist in Pichia pastoris

Interleukin-1 receptor antagonist is an inhibitor of the pro-inflammatory action of interleukin-1. The gene encoding for interleukin-1 receptor antagonist (IL-1ra) was cloned into a Pichia pastoris expression vector pPICzalphaA (Invitrogen, USA) and transformed into P. pastoris strain SMD1168H. Multi-copy selection of the gene produced a high expressing strain of IL-1ra that produced 17mg/L of total secreted purified protein. The IL-1ra produced in P. pastoris was a mixture of glycosylated and non-glycosylated IL-1ra where 70% of the total protein was glycosylated. SP-Sepharose purification allowed for separation of the two expressed forms of IL-1ra, which permits biochemical investigation of glycosylated and non-glycosylated IL-1ra using one expression system. Mass spectrometric analysis revealed the expression of the full-length protein and that the glycosylated IL-1ra contained high mannose glycoforms that ranged from Man(9)GlcNAc(2) to Man(14)GlcNAc(2).     

Oxidation of methionine residues in coagulation factor VIIa

The oxidation of the activated form of recombinant coagulation factor VII (FVIIa) by hydrogen peroxide has been studied. The three predominant oxidation products observed at pH 7.5 have been characterized as methionine sulfoxide derivatives of the parent protein involving two of the four methionine residues of the protein, Met298 and Met306. We conclude that oxidation of FVIIa with hydrogen peroxide only affects methionine residues and selectively oxidizes those which are readily accessible to the solvent. The oxidation process has been studied in the pH range 3.5-9.5. The total rate of oxidation of FVIIa as well as the formation of the three oxidation products is consistent over the pH interval 7.5-9.5. However, under acidic conditions, significant variations have been observed indicating a conformational change of FVIIa. Oxidized FVIIa had the same amidolytic activity as the native protein. The binding to soluble tissue factor (TF) was weaker after oxidation as manifested by a threefold increase in dissociation constant and the amidolytic activity in complex with soluble TF was 80% compared to that of native FVIIa. In complex with lipid surface TF, the rate of factor X activation catalyzed by oxidized FVIIa was also reduced by approximately 20% compared to that of native FVIIa. However, native and oxidized FVIIa appeared to bind lipidated TF with indistinguishable affinities.     

The interleukin-1 receptor antagonist influences interleukin-1 effects in rat and mouse

In this work we have focused on the ability of interleukin-1 to induce an acute phase protein response and a degranulation of polymorphonuclear leukocytes in vivo. The capacity of the interleukin-1 receptor antagonist to influence these events was also investigated. It was shown that interleukin-1 induced an acute phase protein response in rats and mice. In rats alpha(2)-macroglubolin levels were increased in plasma after an interleukin-1 injection whereas alpha(1)-inhibitor-3 decreased in plasma. In the mice plasma amyloid P was increased. The interleukin-1 receptor antagonist blocked the increase of alpha(2)-macroglobulin and plasma amyloid P in a dose dependent way while the effect on the alpha(1)-inhibitor-3 decrease was less pronounced. Interleukin-1 led to polymorphonuclear leukocyte degranulation in vivo as measured by increased cathepsin G plasma levels. The interleukin-1 receptor antagonist could influence the early phase of this degranulation.     

Specific volume and adiabatic compressibility measurements of native and aggregated recombinant human interleukin-1 receptor antagonist: density differences enable pressure-modulated refolding

High hydrostatic pressures have been used to dissociate non-native protein aggregates and foster refolding to the native conformation. In this study, partial specific volume and adiabatic compressibility measurements were used to examine the volumetric contributions to pressure-modulated refolding. The thermodynamics of pressure-modulated refolding from non-native aggregates of recombinant human interleukin-1 receptor antagonist (IL-1ra) were determined by partial specific volume and adiabatic compressibility measurements. Aggregates of IL-1ra formed at elevated temperatures (55 degrees C) were found to be less dense than native IL-1ra and refolded at 31 degrees C under 1,500 bar pressure with a yield of 57%. Partial specific adiabatic compressibility measurements suggest that the formation of solvent-free cavities within the interior of IL-1ra aggregates cause the apparent increase in specific volume. Dense, pressure-stable aggregates could be formed at 2,000 bar which could not be refolded with additional high pressure treatment, demonstrating that aggregate formation conditions and structure dictate pressure-modulated refolding yields.