The effect of simulated space radiation on the N-glycosylation of human immunoglobulin G1.

On a roundtrip to Mars, astronauts are expectedly exposed to an approximate amount of radiation that exceeds the lifetime limits on Earth. This elevated radiation dose is mainly due to Galactic Cosmic Rays and Solar Particle Events. Specific patterns of the N-glycosylation of human Igs have already been associated with various ailments such as autoimmune diseases, malignant transformation, chronic inflammation, and ageing. The focus of our work was to investigate the effect of low-energy proton irradiation on the IgG N-glycosylation profile with the goal if disease associated changes could be detected during space travel and not altered by space radiation. Two ionization sources were used during the experiments, a Van de Graaff generator for the irradiation of solidified hIgG samples in vacuum, and a Tandetron accelerator to irradiate hIgG samples in aqueous solution form. Structural carbohydrate analysis was accomplished by CE with laser induced fluorescent detection to determine the effects of simulated space radiation on N-glycosylation of hIgG1 samples. Our results revealed that even several thousand times higher radiation doses that of astronauts can suffer during long duration missions beyond the shielding environment of Low Earth Orbit, no changes were observed in hIgG1 N-glycosylation. Consequently, changes in N-linked carbohydrate profile of IgG1 can be used as molecular diagnostic tools in space.

Effect of Surfactants on Mechanical, Thermal, and Photostability of a Monoclonal Antibody.

The purpose of this work was to evaluate the effect of commonly used surfactants (at 0.01% w/v concentration) on mechanical, thermal, and photostability of a monoclonal antibody (MAb1) of IgG1 sub-class and to evaluate the minimum concentration of surfactant (Polysorbate 80) required in protecting MAb1 from mechanical stress. Surfactants evaluated were non-ionic surfactants, Polysorbate 80, Polysorbate 20, Pluronic F-68 (polyoxyethylene-polyoxypropylene block polymer), Brij 35 (polyoxyethylene lauryl ether), Triton X-100, and an anionic surfactant, Caprylic acid (1-Heptanecarboxylic acid). After evaluating effect of surfactants and determining stabilizing effect of Polysorbate 80 against mechanical stress without compromising thermal and photostability of MAb1, the minimum concentration of Polysorbate 80 required for mechanical stability was further examined. Polysorbate 80 concentration was varied from 0 to 0.02%. Mechanical stability was evaluated by agitation of MAb1 at 300 rotations per minute at room temperature for 72 h. Samples were analyzed for purity by SEC-HPLC, turbidity by absorbance at 350 nm, visible particles by visual inspection, and sub-visible particles by light obscuration technique on a particle analyzer. All non-ionic surfactants tested showed a similar effect in protecting against mechanical stress and did not exhibit any significant negative effect on thermal and photostability. However, Caprylic acid had a slightly negative effect on mechanical and photostability when compared to the non-ionic surfactants or sample without surfactant. This work demonstrated that polysorbate 80 is better than other surfactants tested and that a concentration of at least 0.005% (w/v) Polysorbate 80 is needed to protect MAb1 against mechanical stress.

Effect of gamma irradiation on the structural stability and functional activity of plasma-derived IgG.

Plasma-originated commercial intravenous immunoglobulin, which is used for a variety of clinical purposes, has been studied to determine the effect of virus-inactivating doses of gamma irradiation on the structural-functional characteristics of the protein. A detailed analysis has been performed in response to a concern that the use of conventional gamma irradiation may damage biologically active proteins. The results demonstrate that although gamma irradiation of the IgG may have some impact on protein structure, the damage can be reduced or even prevented by appropriate irradiation conditions. At the virucidal dose of gamma irradiation (50 kGy) and a temperature of -80 °C, the integrity of the polypeptide chain of immunoglobulin and the secondary structure of IgG can be completely protected, while conformational changes in tertiary structure are significantly minimized to a level that preserves functional activity. The irradiated IgG retains specific antigen-binding properties and F(c)-binding activity, indicating that the conformational integrity of the most important structural regions is not affected by γ-irradiation. These results present strong evidence that gamma irradiation treatment can be effectively implemented for inactivation of pathogens in IgG solutions that are used for intravenous injection.

Light-induced conversion of Trp to Gly and Gly hydroperoxide in IgG1.

The exposure of IgG1 in aqueous solution to light with λ = 254 nm or λ > 295 nm yields products consistent with Trp radical cation formation followed by (α)C-(ß)C cleavage of the Trp side chain. The resulting glycyl radicals either are reduced to Gly or add oxygen prior to reduction to Gly hydroperoxide. Photoirradiation at λ = 254 nm targets Trp at positions 191 (light chain), 309 and 377 (heavy chain) while photoirradiation at λ > 295 nm targets Trp at position 309 (heavy chain). Mechanistically, the formation of Trp radical cations likely proceeds via photoinduced electron or hydrogen transfer to disulfide bonds, yielding thiyl radicals and thiols, where thiols may serve as reductants for the intermediary glycyl or glycylperoxyl radicals.

Effect of UVC Irradiation on the Oxidation of Histidine in Monoclonal Antibodies.

We oxidized histidine residues in monoclonal antibody drugs of immunoglobulin gamma 1 (IgG1) using ultraviolet C irradiation (UVC: 200-280 nm), which is known to be potent for sterilization or disinfection. Among the reaction products, we identified asparagine and aspartic acid by mass spectrometry. In the photo-induced oxidation of histidine in angiotensin II, 18O atoms from H218O in the solvent were incorporated only into aspartic acid but not into asparagine. This suggests that UVC irradiation generates singlet oxygen and induces [2 + 2] cycloaddition to form a dioxetane involving the imidazole Cγ - Cδ2 bond of histidine, followed by ring-opening in the manner of further photo-induced retro [2 + 2] cycloaddition. This yields an equilibrium mixture of two keto-imines, which can be the precursors to aspartic acid and asparagine. The photo-oxidation appears to occur preferentially for histidine residues with lower pKa values in IgG1. We thus conclude that the damage due to UVC photo-oxidation of histidine residues can be avoided in acidic conditions where the imidazole ring is protonated.

Discovery of a Photoinduced Histidine-Histidine Cross-Link in an IgG4 Antibody.

A novel histidine-histidine (His-His) photooxidative cross-link has been identified in an IgG4 antibody. It was formed between the side chain of a histidine residue of the antibody and histidine from the formulation buffer. The structure of the cross-link was elucidated using high performance liquid chromatography (HPLC) hyphenated to tandem mass spectrometry (MS/MS) with higher energy collisional dissociation (HCD). The cross-link was found in multiple conformations, as the location of the oxygen varied. Furthermore, the extent of cross-link formation was shown to correlate with the amount of light the antibody was exposed to as well as the solvent accessibility of each modification site.

Effects of Glycan Structure on the Stability and Receptor Binding of an IgG4-Fc.

A series of well-defined N-glycosylated IgG4-Fc variants were utilized to investigate the effect of glycan structure on their physicochemical properties (conformational stability and photostability) and interactions with an Fc γ receptor IIIA (FcγRIIIA). High mannose (HM, GlcNAc2Man(8+n) [n = 0-4]), Man5 (GlcNAc2Man5), GlcNAc1, and N297Q IgG4-Fc were prepared in good quality. The physical stability of these IgG4-Fc variants was examined with differential scanning calorimetry and intrinsic fluorescence spectroscopy. Photostability was assessed after photoirradiation between 295 and 340 nm (λ max = 305 nm), and HPLC-MS/MS analysis of specific products was performed. The size of glycans at Asn297 affects the yields of light-induced Tyr side-chain fragmentation products, where the yields decreased in the following order: N297Q > GlcNAc1 > Man5 > HM. These yields correlate with the thermal stability of the glycoforms. The HM and Man5 glycoforms display increased affinity for FcγRIIIA by at least 14.7-fold compared with GlcNAc1 IgG4-Fc. The affinities measured for the HM and Man5 IgG4-Fc (0.39-0.52 µM) are similar to those measured for fucosylated IgG1. Dependent on the mechanisms of action of IgG4 therapeutics, such glycoforms may need to be carefully monitored. The nonglycosylated N297Q IgG4-Fc did not present measurable affinity to FcγRIIIA.

Impact of gamma-radiation on antigenic properties of cow's milk beta-lactoglobulin.

This study evaluated the effects of gamma-radiation on the antigenic properties of beta-lactoglobulin in cow's milk. Liquid and lyophilized samples of cow's milk and whey were irradiated with gamma-cells (60Co) at dose levels of 3, 5, and 10 kGy, at room temperature in the presence of air. Effects of treatment on proteins were monitored by Lowry's method, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and enzyme-linked immunosorbent assay. Radiation did not affect the molecular-weight distributions of proteins, but it did reduce their solubility. Furthermore, results showed that irradiation at 10 kGy increased the recognition of milk and whey powders by anti-beta-lactoglobulin (beta-Lg) rabbit immunoglobulin G, with the other samples remaining antigenically stable. These results indicate that gamma-rays do not reduce cow's milk beta-lactoglobulin antigenicity.

Effect of photo-degradation on the structure, stability, aggregation, and function of an IgG1 monoclonal antibody.

Photostability testing of therapeutic proteins is a critical requirement in the development of biologics. Upon exposure to light, pharmaceutical proteins may undergo a change in structure, stability, and functional properties that could have a potential impact on safety and efficacy. In this work, we studied how exposure to light, according to ICH guidelines, leads to photo-oxidation of a therapeutic IgG1 mAb. We also tested the ability of five different excipients to prevent such oxidation. In samples that were exposed to light, we found that the CH2 domain was considerably destabilized but there were no major changes in the overall structure of the protein. Aggregation of the protein was observed because of light exposure. Mass spectrometry identified that light exposure oxidizes two key methionine residues in the Fc region of the protein. In terms of function, a loss in binding to the neonatal Fc receptor, decreased antibody-dependent cell-mediated cytotoxicity and cell proliferation activities of the protein were seen. Combined analysis of the photo-oxidation effects on the structure, stability, aggregation, and function of the mAb has identified the underlying unifying mechanism. Among the sugars and amino acids tested, methionine was the most effective in protecting mAb against photo-oxidation.

Self-perpetuating mechanisms of immunoglobulin G aggregation in rheumatoid inflammation.

When human IgG is exposed to free radical generating systems such as ultraviolet irradiation, peroxidizing lipids, or activated human neutrophils, characteristic auto-fluorescent monomeric and polymeric IgG is formed (excitation [Ex], 360 nm, emission [Em], 454 nm). 1 h ultraviolet irradiation of IgG results in the following reductions in constituent amino acids; cysteine (37.0%), tryptophan (17.0%), tyrosine (10.5%), and lysine (3.6%). The fluorescent IgG complexes, when produced in vitro, can stimulate the release of superoxide from normal human neutrophils. In the presence of excess unaltered IgG, further fluorescent damage to IgG occurs. Measurement and isolation of fluorescent monomeric and polymeric IgG by high performance liquid chromatography, from in vitro systems and from fresh rheumatoid sera and synovial fluid, indicates that identical complexes are present in vivo; all these fluorescent complexes share the property of enhancing free radical production from neutrophils. The results described in this study support the hypothesis that fluorescent monomeric and aggregated IgG may be formed in vivo by oxygen-centered free radicals derived from neutrophils, and that in rheumatoid inflammation this reaction may be self-perpetuating within the inflamed joint.

Effect of ambient light on IgG1 monoclonal antibodies during drug product processing and development.

Photostability studies are standard stress testing conducted during drug product development of various pharmaceutical compounds, including small molecules and proteins. These studies as recommended by ICH Q1B are carried out using no less than 1.2× 10(6)lux-hours in the visible region and no less than 200Wh/m(2) in UV light. However, normal drug product processing is carried out under fluorescent lamps that emit white light almost exclusively in the >400nm region with a small UV quotient. We term these as ambient or mild light conditions. We tested several IgG1 monoclonal antibodies (mAbs 1-5) under these ambient light conditions and compared them to the ICH light conditions. All the mAbs were significantly degraded under the ICH light but several mAbs (mAbs 3-5) were processed without impacting any product quality attributes under ambient or mild light conditions. Interestingly we observed site-specific Trp oxidation in mAb1, while higher aggregation and color change were observed for mAb2 under mild light conditions. The recommended ICH light conditions have a high UV component and hence may not help to rank order photosensitivity under normal protein DP processing conditions.

Converting relapsing remitting to secondary progressive experimental allergic encephalomyelitis (EAE) by ultraviolet B irradiation.

We induced experimental allergic encephalomyelitis (EAE) in SJL/J mice, an animal model for multiple sclerosis (MS), using myelin oligodendrocyte glycoprotein (MOG)(92-106) peptide, following ultraviolet (UV) irradiation. While all control mice developed relapsing-remitting (RR)-EAE, UV irradiation induced secondary progressive (SP)-EAE in some of the mice. Although mild demyelination was observed with T cell infiltration in RR-EAE, large demyelinating lesions developed in SP-EAE with massive macrophage and neutrophil infiltration and immunoglobulin deposition, but with little T cell infiltration. UV irradiation induced higher anti-MOG antibody responses. In SP-EAE, lymphoproliferative responses and interferon-gamma production were decreased without alteration of interleukin-4.

Spectroscopic studies on IgG aggregate formation.

Spectroscopic techniques have been used to examine the effects of IgG of heating, irradiation by ultraviolet light and exposure to glutaraldehyde. Relatively few changes were observed in treated IgG which remained unaggregated but several significant size-dependent changes were observed in aggregated IgG. These results suggest that IgG aggregate formation by cross-linking with glutaraldehyde involves the least perturbation of the basis IgG structure, whereas aggregate formation by heating involves the most, with ultraviolet irradiation occupying an intermediate position.

Immunogenic and antigenic epitopes of immunoglobulin--XX. Denaturation of human IgG3 by free radicals.

It has previously been demonstrated that exposure of polyclonal IgG to free radicals results in denaturation evidenced by aggregation, auto-fluorescence and destruction of cysteine, proline and aromatic amino acids. In the present study we have used a panel of monoclonal antibodies (McAb) to epitopes expressed on the IgG3 heavy chain to detect changes in antigenicity. When IgG3 was exposed to u.v. irradiation, as a source of free radicals, subclass specific epitopes were rapidly lost whilst other epitopes were unaffected. Prolonged exposure resulted in further denaturation and a progressive loss of expression of further epitopes. The IgG3 subclass specific McAb are specific to epitopes localized to the hinge region of IgG3. Thus, this exposed cysteine and proline rich region is shown to be particularly vulnerable to free radical attack; however, prolonged exposure results in structural alterations throughout the heavy chain.

The effects of oxygen free radicals on the carbohydrate moiety of IgG.

The CH2-linked glycoform of rheumatoid IgG is abnormal in having a reduced galactose content. This has been postulated to be a synthetic defect due to a decrease in the level of rheumatoid B cell galactosyltransferase. However, more recent work has indicated that agalactosylation may be common to chronic inflammatory diseases. In this work we have investigated the effect of oxygen free radicals (OFRs), which are generated by activated phagocytic cells at inflammatory sites, on the carbohydrate moiety of IgG. Radiolytically generated peroxy (ROO.) and hydroxyl radicals (OH.) but not superoxide anion radicals (O2.-) were found to destroy galactose on IgG. After OH. attack, this was associated with an increase in the availability of N-acetylglucosamine, possibly due to its presence as a terminal residue. These results suggest that the agalactosylation associated with chronic inflammation may not only be synthetic in nature, but may also be a consequence of post-synthetic degradation by OFRs.

The C1q binding activity of IgG is modified in vitro by reactive oxygen species: implications for rheumatoid arthritis.

IgG can be denatured in vitro by reactive oxygen species (ROS). Native IgG activates the complement cascade through C1q. Using a modified ELISA, C1q binding activity of rheumatoid IgG has been compared to IgG denatured by neutrophil-derived ROS. The C1q binding activity of rheumatoid synovial fluid IgG is greater than the corresponding serum IgG (P < 0.01). Denaturation of IgG by activated polymorphs or the Fenton reaction decreased its C1q binding activity (P < 0.01). In vitro exposure of IgG to OH. and ROO. increased its interaction with C1q (P < 0.01). Hypochlorous acid had no effect. ROS-induced alteration to IgG-C1q binding activity may promote the inflammatory response in rheumatoid arthritis.

Autoantibodies and immunoglobulins among atomic bomb survivors.

The purpose of this study was to determine if exposure to atomic bomb radiation affects immune responsiveness, such as the occurrence of autoantibodies and levels of immunoglobulins. Rheumatoid factor, antinuclear antibody, antithyroglobulin antibody, anti-thyroid-microsomal antibody and immunoglobulin levels (IgG, IgM, IgA and IgE) were measured among 2,061 individuals exposed to atomic bomb radiation in Hiroshima and Nagasaki whose estimated doses ranged from 0 to 5.6 Gy. The prevalence and titers of rheumatoid factor were found to be increased in the individuals exposed to higher radiation doses. The IgA level in females and the IgM level in both sexes increased as radiation dose increased, although the effects of radiation exposure were not large. No effect of radiation was found on the prevalence of antinuclear antibody, antithyroglobulin antibody and anti-thyroid-microsomal antibody or on the levels of IgG and IgE.

Radiation-induced surface IgG modulation: protein kinase C involvement.

Although radiation-induced loss of surface IgG (s-IgG) expression on murine B cells is known to be dependent on intact energy metabolism and integrity of the cytoskeleton, the exact mechanism of this radiation effect is not known. Evidence reported here shows that inhibition of protein kinase C (PKC) by H-7 impairs the radiation-induced s-IgG modulation, whereas addition of HA-1004, which preferently inhibits c-AMP-dependent protein kinase, shows only minor effects. On the other hand PMA, a PKC activator, mimics the radiation effect, and H-7 but not HA-1004 inhibits the PMA-induced loss of s-IgG expression. Therefore it is suggested that PKC is involved in the modulation of s-IgG induced by irradiation on B cells. The possibility of membrane participation in this event is discussed.

Influence of continuous, very low-dose gamma-irradiation on the mouse immune system.

PURPOSE: To investigate whether continuous, very low-dose gamma-irradiation (10 cGy/year) modifies immune parameters in mice. MATERIAL AND METHODS: C57BL/6 female mice, 4 weeks old, were irradiated for 24 months and compared with control mice living in the same room. B- and T-cell subsets were evaluated by flow cytometry before and after stimulation with lectins; subclasses of immunoglobulins were determined by ELISA 2, 4, 6, 8, 12, 18 and 24 months after the beginning of the irradiation. RESULTS: No difference was found in the percentage of CD4(+) and CD8(+) cells in the thymus and the spleen, or in the reactivity of T-cells to lectins. While the number of B-cells in the spleen remained unchanged, a significant decrease of IgG1, IgG2b and IgG2a was observed after respectively 12, 18 and 24 months of irradiation. CONCLUSION: The parameters of cellular immunity studied were not affected by this chronic low-dose of irradiation, but this dose rate is probably too low to induce the hormetic effect previously described. Further investigations are necessary to assess whether the decline of immunoglobulin secretion is indicative of a lower rate of infectious diseases or a defect in B-cell function.

The protective activity of Thymex L against radiotherapeutically-induced cellular immunodepression in lung cancer patients.

In order to prevent the radiotherapeutically-induced aggravation of initial immunodeficiency, a thymic preparation (Thymex L) was given to lung cancer patients simultaneously with irradiation. The parameters of both cellular and humoral nonspecific immunity were evaluated in two groups of patients: one was treated with radiotherapy only (60 Gy in 30 fractions); the other one received Thymex L (100 mg 3 times a week, total dose 1800 mg, i.m.) simultaneously with radiotherapy. The significant decrease of B and T cell number, and decreased lymphoproliferative response to PHA were found in all patients before therapy; the number and phagocyting capacity of blood monocytes, as well as the concentrations of circulating IgG, IgA and immunocomplexes, were all significantly increased. Immediately after irradiation the patients had even lower number of T and B cells, diminished reactivity to PHA and higher number of mononuclear phagocytes when compared to the values before therapy. In patients treated with Thymex L, the number of B and T cells and PHA-induced proliferative response were significantly higher than in those treated with radiotherapy only. No effect of this therapy was seen on active T cells, on high number and function of mononuclear phagocytes and on elevated concentrations of serum immunoglobulins and immune complexes. Our results indicate that Thymex L can successfully prevent the harmful effect of radiation therapy on cellular immunity in a majority of lung cancer patients.