采用多种抗原测定静注人免疫球蛋白(pH4)中抗体Fc段生物学活性的初探

目的初步探讨采用多种抗原测定静注人免疫球蛋白(IVIG)(pH4)中抗体的Fc段生物学活性,了解IVIG中抗体的Fc段生物学活性。方法采用补体活化的经典途径中免疫复合物激活补体的方法,将不同浓度的麻疹病毒、风疹病毒、乙肝表面抗原(HBsAg)、破伤风类毒素、脑膜炎球菌P64k外膜蛋白和白喉类毒素6种抗原分别致敏人O型血红细胞形成红细胞-抗原结合物;然后,6种致敏红细胞分别与IVIG孵育,与特异性抗体形成红细胞-抗原-抗体复合物;最后,此复合物与补体反应,在541 nm波长处读取吸光值,并绘制溶血反应动力学曲线,分别计算IVIG中针对上述6种抗原IgG的Fc段生物学活性。采用此方法,用6种抗原致敏的红细胞测定IVIG的Fc段生物学活性10次,验证此方法的重复性。结果麻疹病毒、风疹病毒、HBsAg、破伤风类毒素和脑膜炎球菌P64k外膜蛋白致敏的红细胞分别与供试品和补体反应后,测定的溶血反应动力学曲线较平缓,而白喉类毒素致敏的红细胞与供试品和补体反应后,测定的溶血反应动力学曲线下降明显,呈典型的"S"型曲线。计算结果显示,IVIG中针对此六种抗原的抗体Fc段生物学活性相对于参考品均大于80%。Fc段生物学检测方法重复性较好。结论采用多种抗原分别致敏红细胞,可以用来检测IVIG中多种抗体的Fc段生物学活性,为深入了解IVIG制品中的多种抗体的Fc段生物学活性奠定了基础。     

IL-2 and GM-CSF are regulated by DNA demethylation during activation of T cells, B cells and macrophages

High-dose intravenous immunoglobulin (IVIG) preparations are currently used for the treatment of autoimmune diseases such as immune thrombocytopenic purpura (ITP). Although the mechanisms of IVIG efficacy remain enigmatic, some clinical and laboratory studies suggest that interaction of the Fc domain of IgG, especially the Fc domain of dimeric IgG, with its receptors (Fc gamma receptors; FcγRs) plays an essential role. In this study, IVIG was dimerized with chemical crosslinkers to augment its therapeutic efficacy. Dimerized IVIG was found to have a much higher affinity for FcγRs than monomeric IVIG. In a mouse ITP model, chemically dimerized IVIG abrogated the decrease in platelet numbers in the blood that was caused by an anti-platelet antibody at a dose that was one tenth of the required dose of IVIG. These results suggest that chemical dimerization of IVIG should greatly improve the efficacy of IVIG therapy of ITP.     

Functional integrity of intravenous immunoglobulin following irradiation with a virucidal dose of gamma radiation.

Although intravenous immunoglobulins (IVIG) and other plasma therapeutics have had a relatively good safety record, improved methods for viral clearance are constantly being evaluated and incorporated into new manufacturing processes. Gamma irradiation has been used routinely to assure sterility of healthcare products and medical devices, but it has not been applied successfully as a viral inactivation method for biologics. We examine whether virucidal doses of gamma irradiation (50 kGy) can be delivered to a manufacturing intermediate form of IVIG, a fractionated plasma paste, with negligible effect on structural and functional integrity of purified IgG product. Immunoglobulins from paste were examined for radiation-induced damage by SDS-PAGE and ELISAs utilizing viral antigens specific for rubella, CMV and mumps. Fc domain integrity was assessed by immunoblotting, quantitatively comparing the binding of irradiated and non-irradiated materials to cell surface Fcgamma receptors, and by employing quantitative RT-PCR to study the kinetics of accumulation of mRNA for the immune modulatory cytokines IL-1alpha, IL-1beta, IL-4, IL-8, IFNgamma, and TNFalpha. The results demonstrate that Fab and Fc domains of IVIG remain essentially intact and functional after gamma irradiation to virucidal doses, suggesting that this method could be used to enhance the safety of IVIG products.     

A close look at human IgG sialylation and subclass distribution after lectin fractionation

Polyspecific human IgG preparations are indicated for the treatment of primary immunodeficiency disorders associated with defects in humoral immunity. In addition, intraveneous IgG (IVIG) is used to treat patients with autoimmune and systemic inflammatory diseases. Lectin chromatography on Sambucus nigra agglutinin stood at the cradle of the hypothesis that the anti‐inflammatory properties depend on sialylation of the N‐glycans in the Fc region of IgG. A detailed analysis of fractions obtained by lectin chromatography revealed that binding of IVIG is essentially mediated by Fab glycosylation. Moreover, experiments with a monoclonal antibody from a human cell line and IVIG Fc fragments indicated that at least two sialic acids in the Fc region of an antibody are required for lectin binding. Such glycoforms contain either two monosialylated glycans or a disialylated glycan and constitute 1% or less of the total human IgG. Arguably this small proportion holds the entire anti‐inflammatory potency. A new mass spectrometric quantification method of IgG subclass ratio revealed that the IVIG Fc preparation essentially consists of IgG1. This observation may be relevant when studying the effect of human Fc in murine models of inflammation because mouse IgG subclasses differ substantially in their interaction with receptors.     

激活补体试验检测静注人免疫球蛋白Fc段生物学活性的优化

优化激活补体试验的条件,完善静注人免疫球蛋白Fc段生物学活性的检测方法。方法采用补体活化经典途径的原理,分别探讨致敏红细胞密度和贮存时间对人免疫球蛋白Fc段生物学活性检测结果的影响;比较手工法和微孔板分光光度法检测Fc段生物学活性的检测结果。结果在致敏红细胞A541 nm=1.3和致敏红细胞贮存5 d时,检测结果较稳定。微孔板分光光度法检测优于手工法。结论完善了人免疫球蛋白Fc段生物学活性的检测方法,检测结果准确、重复性好。     

静注人免疫球蛋白中白喉抗体效价对其Fc段生物学活性检测结果影响的分析

目的分析静注人免疫球蛋白(IVIG)中白喉抗体效价对其Fc段生物学活性检测的影响。方法检测20批IVIG的白喉抗体效价水平和Fc段生物学活性结果,进一步探讨白喉抗体效价与Fc段生物学活性的关系。结果20批IVIG中白喉抗体效价水平均符合中国药典的要求,在3~60 HAU/g之间,其中有两批IVIG的白喉抗体效价水平相对较高,其他18批IVIG的白喉抗体效价水平变化趋势不明显。20批IVIG的Fc段生物学活性均较高,在60%~140%之间。白喉抗体效价水平高者,其Fc段生物学活性并非高,反之亦然。结论 IVIG的Fc段生物学活性与其白喉抗体效价水平无明显的相关性。     

Human intravenous immunoglobulin preparation and virus inactivation by pasteurization and solvent detergent treatment

Human intravenous immunoglobulin (IVIG) solutions were prepared by two different methods and compared to each other. The crude immunoglobulin fraction obtained from Cohn-Oncley fractionation of plasma was further purified and subjected to virus inactivation, either by polyethylene glycol precipitation and pasteurization at 60 degrees C for 10 hours, or by ion exchange chromatography and solvent/detergent treatment. The final preparations, formulated in 5% immunoglobulin solutions were characterized by in vitro analyses of biochemical and biological properties and compared with the samples of other manufacturer's IVIG solution products. The critical properties evaluated in this study were purity, molecular intactness, and the biological functions such as Fc function and anticomplementary activity. Virus inactivation and removal by processing steps and by deliberate virucidal steps, as described above, were tested on various human pathogenic viruses, such as human immunodeficiency and experimental model viruses. The tested viruses were successfully inactivated and removed. We conclude that the intravenous immunoglobulins prepared by two different methods, as described above, provide an equivalent viral safety and quality.     

Analyzing titers of antibodies against bacterial and viral antigens,and bacterial toxoids in the intravenous immunoglobulins utilized in Taiwan

Intravenous immunoglobulin (IVIG) manufactured from human plasma contains IgG as the primary ingredient, and is used for indications such as immunodeficiency syndrome. Available IVIGs in Taiwan are either manufactured from Taiwanese or North American plasma. The effectiveness of the national immunization program of Taiwan can be evaluated by analyzing and comparing IVIG antibody titers that are induced through the corresponding vaccines (tetanus, diphtheria, and pertussis, measles, rubella, hepatitis A, hepatitis B and varicella). Both enzyme-linked immunosorbent assay (ELISA) and the in vitro neutralization test demonstrated that all IVIGs provide adequate clinical protection against diphtheria and tetanus toxins. ELISA results further revealed that plasma of Taiwanese subjects contains higher levels of pertussis toxin and filamentous hemagglutinin antibodies, when compared to foreign IVIGs. This may be related to the later adoption of acellular pertussis vaccine in Taiwan. Antibodies titers against measles, rubella, hepatitis A, and varicella-zoster virus were otherwise low. Low titers of hepatitis B surface antigen antibodies are present in Taiwanese plasma IVIG, indicating immune memory decline or loss. In conclusion, our results show that Taiwanese IVIG contains varying titers of vaccine-induced antibodies, and serves as a guide for future amendments to Taiwan's immunization program.     

Both the Fab and Fc domains of IgG are essential for ROS emission from TNF-α-primed neutrophils by IVIG

Intravenous immunoglobulin (IVIG) is currently a very important therapeutic used for not only infectious diseases, but also for autoimmune diseases such as idiopathic thrombocytopenic purpura (ITP). Untoward reactions of IVIG have been thought to result from complement activation by aggregated IgG in IVIG. In addition, the aggregates have been known to activate neutrophils, which may result in the untoward reactions. However, the effect and mechanism of IVIG on neutrophils remain unclear. In this study, we investigated the activation of neutrophils by IVIG in terms of their reactive oxygen species (ROS) emission to elucidate the mechanisms. IVIG-induced ROS emission from purified neutrophils was remarkably augmented by TNF-α priming of the cells. The ROS emission from TNF-α-primed neutrophils occurred by activation with whole gammaglobulin (GG) molecules, but not F(ab')(2), Fc, or a mixture of F(ab')(2) and Fc. ROS emission by GG was inhibited by the F(ab')(2) fragment and an inhibitory antibody against FcγRIII. These results suggest that binding of IVIG to not only surface antigen(s), but also FcγRIII on neutrophils, is involved in IVIG-induced ROS emission from TNF-α-primed neutrophils, and contribute to the untoward reactions of IVIG.     

Correlations between pharmacokinetics of IgG antibodies in primates vs. FcRn-transgenic mice reveal a rodent model with predictive capabilities

Transgenic mice expressing human neonatal Fc receptor (FcRn) instead of mouse FcRn are available for IgG antibody pharmacokinetic (PK) studies. Given the interest in a rodent model that offers reliable predictions of antibody PK in monkeys and humans, we set out to test whether the PK of IgG antibodies in such mice correlated with the PK of the same antibodies in primates. We began by using a single research antibody to study the influence of: (1) different transgenic mouse lines that differ in FcRn transgene expression; (2) homozygous vs. hemizygous FcRn transgenic mice; (3) the presence vs. absence of coinjected high-dose human intravenous immunoglobulin (IVIG), and (4) the presence vs. absence of coinjected high-dose human serum albumin (HSA). Results of those studies suggested that use of hemizygous Tg32 mice (Tg32 hemi) not treated with IVIG or HSA offered potential as a predictive model for PK in humans. Mouse PK studies were then done under those conditions with a panel of test antibodies whose PK in mice and primates is not significantly affected by target binding, and for which monkey or human PK data were readily available. Results from the studies revealed significant correlations between terminal half-life or clearance values observed in the mice and the corresponding values reported in humans. A significant relationship in clearance values between mice and monkeys was also observed. These correlations suggest that the Tg32 hemi mouse model, which is both convenient and cost-effective, can offer value in predicting antibody half-life and clearance in primates.     

静注人免疫球蛋白IgG含量测定中的影响因素

为了进一步分析静注人免疫球蛋白(IVIG)IgG含量测定中的影响因素,分别以不同的稀释缓冲液、不同的稀释度、不同的稳定剂对IVIG的IgG含量进行测定。结果表明以不同的稀释缓冲液、不同的稳定剂测定的同批IgG含量无显著性差异;而用不同的稀释度测定的同批IgG含量有差异。因此可见,IVIG制品中采用的不同稳定剂对IgG含量测定影响不大;可以用不同的稀释度测定的均值作为该批IVIG制品的IgG含量。     

International collaborative study to assess candidate reference preparations to control the level of anti-D in IVIG for use in Europe and the United States.

Regulatory requirements to control the level of anti-D in intravenous immunoglobulin (IVIG) products with European and United States (US) licences are to be introduced. A reference preparation of IVIG containing anti-D at 0.0475 IU/ml and having a nominal titre of 8 using the proposed direct haemagglutination reference method was deemed suitable to define the anti-D limit. This preparation, code 02/228, and a negative control IVIG preparation, code 02/226, were established by the World Health Organization as International Reference Reagents (IRRs). As stocks of the IRRs are limited, new larger fill stocks of positive and negative reference preparations, codes 04/132 and 04/140, respectively, were produced. The results from an international collaborative study involving 16 laboratories showed that preparations 04/132 and 04/140 are indistinguishable from the corresponding IRRs 02/228 and 02/226, respectively, using the proposed direct haemagglutination reference method. Stocks of 04/132 and 04/140 have been shared with the European Directorate for the Quality of Medicines (re-coded as 23613 and 23614, respectively) and with the Center for Biologics Evaluation and Research of the United States Food and Drug Administration (re-coded as CBER Lots 1B and 1N-b, respectively) for use as European and US Biological Reference Preparations, respectively.     

Combined estimation of tetanus and diphtheria antitoxin in human sera by the in vitro Toxin-Binding Inhibition (ToBI) test

The use of the principle of inhibition of toxin binding to an antitoxin coated immunoassay plate as described in a previous paper for tetanus antitoxin titration, was adapted for the estimation of diphtheria antitoxin in human sera. With a few modifications, a Toxin-Binding Inhibition (ToBI) test was developed which could be used for a combined estimation of both tetanus and diphtheria antitoxin levels. The application of streptavidin-biotinylated peroxidase complex when using small serum samples (less than 50 microliters) is discussed. Antitoxin titres (both diphtheria and tetanus) of 0.002 IU ml-1 were detectable by the ToBI test, this being far below the level considered to be protective in man. Sera from 140 adults with different vaccination histories were titrated for both tetanus and diphtheria antitoxin. Good correlations were found between the estimates obtained by the ToBI test and those obtained by the toxin-neutralization (TN) test in mice (tetanus antitoxin) and those obtained in the in vitro neutralization test in VERO cells (diphtheria antitoxin). It is concluded that the ToBI test is a simple and reliable alternative to the functional models currently in use for the estimation of diphtheria and tetanus antitoxin levels. In addition, the ToBI test eliminates the need for laboratory-animal or cell-culture facilities and can be performed with small quantities of serum as required in field trials.     

Immunogenicity test of tetanus component in adsorbed vaccines by toxin binding inhibition test

Samples from 20 lots of diphtheria-tetanus (adult use dT) vaccine and from 20 lots of diphtheria-tetanus-pertussis (DTP) vaccine were used to standardize and validate the in vitro toxin binding inhibition (ToBI) test for the immunogenicity test of the tetanus component. The levels of tetanus antitoxin obtained by ToBI test were compared to those obtained using the toxin neutralization (TN) test in mice routinely employed to perform the quality control of the tetanus component in adsorbed vaccines. The results ranged from 1.8 to 3.5 IU/ml for dT and 2 to 4 IU/ml for DTP by ToBI test and 1.4 to 3 IU/ml for dT and 1.8 to 3.5 IU/ml for DTP by TN in mice. These results were significantly correlated. From this study, it is concluded that the ToBI test is an alternative to the in vivo neutralization procedure in the immunogenicity test of the tetanus component in adsorbed vaccines. A substantial refinement and a reduction in use of animals can be achieved.     

Human neutrophils and eosinophils have structurally distinct Fc gamma receptors

Human Fc gamma receptors were isolated from surface radioiodinated granulocytes and eosinophils by using repetitive affinity chromatography on human IgG-Sepharose columns. Analysis by SDS-polyacrylamide gel electrophoresis demonstrated that cell preparations containing eosinophils possessed a 43,000 Mr Fc gamma-binding macromolecule. Nylon wool-filtered cells from patients with eosinophilia and cell cultures derived from normal donors provided highly purified eosinophil preparations that expressed only the 43,000 Mr Fc gamma receptor. Granulocyte populations yielded the 52,000 to 68,000 Mr Fc gamma receptor characteristic of neutrophils as well as the Fc gamma-binding macromolecules apparently derived from eosinophils. The 43,000 Mr Fc gamma receptor of the eosinophil and the 31,000 and 34,000 Mr fragments that appear to be derived from it were able to rebind selectively to human IgG1-Sepharose, Fc gamma 1-Sepharose, IgG3-Sepharose, and Fc gamma 3-Sepharose. In contrast, the 52,000 to 68,000 Mr Fc gamma receptor from neutrophils could rebind only to IgG1-Sepharose and Fc gamma 1-Sepharose. The results demonstrate that the Fc gamma receptor of human eosinophils is distinct in structure from the neutrophil Fc gamma receptor and that these Fc gamma receptors, at least in their solubilized states, differ in specificity for human IgG3.     

The determination of the potency of human tetanus immunoglobulins by an enzyme-linked immunosorbent assay

A micro enzyme-linked immunosorbent assay has been adapted to the quantitation of specific tetanus antibodies in commercial preparations of human immunoglobulins. The results of the assay are compared with those obtained from the same samples tested by seroneutralization tests in vivo. Statistical analysis of the data shows good correlation between the titres obtained with the two tests. Results obtained by indirect haemagglutination are also reported. It is proposed that all interested laboratories perform the described immunoenzymatic method in vitro for a given period in parallel with the in vivo test to gain sufficient experience of this technique with a view to its use as an alternative to the in vivo test.     

Purification of intravenous immunoglobulin G from human plasma--aspects of yield and virus safety

Plasma-derived intravenous immunoglobulin (IVIG) preparations have been successfully applied for the prophylactic prevention of infectious diseases in immunodeficient patients. In addition to its replacement therapy of primary and secondary antibody deficiencies, IVIG has found increased use in autoimmune and inflammatory diseases. IVIG has become the major plasma product on the global blood product market. The world wide consumption nearly tripled between 1992 and 2003, from 19.4 to 52.6 tons. Classical manufacturing processes of IVIG, but also new strategies for purification are discussed with respect to practicability and yield. Ethanol fractionation is still the basis for most IVIG processes, although isolation and purification of immunoglobulin G (IgG) by chromatography has gained ground. The efficiency of virus inactivation methods and virus removal techniques in terms of logarithmic reduction factors are analyzed, but also the IgG losses are taken into consideration. Some of these methods also have the ability to separate prions. High pathogen safety and high yields have become the dominant goals of the plasma fractionation industry.     

Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity.     

Analysis and functional consequences of increased Fab-sialylation of intravenous immunoglobulin (IVIG) after lectin fractionation

It has been proposed that the anti-inflammatory effects of intravenous immunoglobulin (IVIG) might be due to the small fraction of Fc-sialylated IgG. In this study we biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA) lectin fractionation. Two main IgG fractions isolated by elution with lactose (E1) or acidified lactose (E2) were analyzed for total IgG, F(ab')(2) and Fc-specific sialic acid content, their pattern of specific antibodies and anti-inflammatory potential in a human in vitro inflammation system based on LPS- or PHA-stimulated whole blood. HPLC and LC-MS testing revealed an increase of sialylated IgG in E1 and more substantially in the E2 fraction. Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region. This indicates preferential binding of the Fab sialic acid to SNA. ELISA analyses of a representative range of pathogen and auto-antigens indicated a skewed antibody pattern of the sialylated IVIG fractions. Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2); again these effects were Fab- but not Fc-dependent. Our results show that SNA fractionation of IVIG yields a minor fraction (approx. 10%) of highly sialylated IgG, wherein the sialic acid is mainly found in the Fab region. The tested anti-inflammatory activity was associated with Fab not Fc sialylation.     

Different IVIG Glycoforms Affect In Vitro Inhibition of Anti-Ganglioside Antibody-Mediated Complement Deposition

Intravenous immunoglobulin (IVIG) is the first line treatment for Guillain–Barré syndrome and multifocal motor neuropathy, which are caused by anti-ganglioside antibody-mediated complement-dependent cytotoxicity. IVIG has many potential mechanisms of action, and sialylation of the IgG Fc portion reportedly has an anti-inflammatory effect in antibody-dependent cell-mediated cytotoxicity models. We investigated the effects of different IVIG glycoforms on the inhibition of antibody-mediated complement-dependent cytotoxicity. Deglycosylated, degalactosylated, galactosylated and sialylated IgG were prepared from IVIG following treatment with glycosidases and glycosyltransferases. Sera from patients with Guillain–Barré syndrome, Miller Fisher syndrome and multifocal motor neuropathy associated with anti-ganglioside antibodies were used. Inhibition of complement deposition subsequent to IgG or IgM autoantibody binding to ganglioside, GM1 or GQ1b was assessed on microtiter plates. Sialylated and galactosylated IVIGs more effectively inhibited C3 deposition than original IVIG or enzyme-treated IVIGs (agalactosylated and deglycosylated IVIGs). Therefore, sialylated and galactosylated IVIGs may be more effective than conventional IVIG in the treatment of complement-dependent autoimmune diseases.