Analysis of sialic acid levels in Chinese intravenous immunoglobulins by high‐performance liquid chromatography with fluorescence detection

Intravenous immunoglobulin (IVIg) is increasingly used for the treatment of autoimmune and systemic inflammatory diseases with both licensed and off‐label indications. Recent studies indicated that IVIg‐mediated immunomodulation and anti‐inflammation are closely associated with the IgG sialylation, especially with IgG crystallizable fragment (Fc) sialylation. The sialic acid levels of the IgG molecules and Fc fragments in 12 IVIg preparations from six Chinese manufacturers were evaluated. The Fc fragments were derived from the papain digestion of IVIg, followed by affinity and size exclusion chromatography. The sialic acid levels in Fc fragments and IVIg preparations were determined by high‐performance liquid chromatography with fluorescence detection, after the sialic acid residues were released from the proteins. The results showed that the sialic acid levels in Chinese IVIg preparations ranged from 0.875 (mol/mol IgG) to 1.085 (mol/mol IgG), and the sialic acid levels in Fc fragments were from 0.321 (mol/mol Fc) to 0.361 (mol/mol Fc). Furthermore, the sialic acid levels of IVIg preparations and Fc fragments from different Chinese manufactures were significantly different. These findings will contribute to an increased understanding of Chinese IVIg preparations and the relationship between the sialic acid levels in IVIg preparations and their clinical efficacy in future clinical studies.     

Phospho‐l‐tyrosine‐agarose chromatography: Adsorption of human IgG and its proteolytic fragments

The behavior of human immunoglobulin G (IgG) and antigen‐binding fragment (Fab fragment) adsorption onto phospho‐l ‐tyrosine immobilized on agarose (P‐Tyr‐agarose) was evaluated by pseudoaffinity chromatography. The effects of buffer systems MES, MOPS, Bis–Tris, Tris–HCl and sodium phosphate (NaP) and pH on IgG adsorption were studied and high purity values were obtained (96%, based on ELISA analysis of albumin, transferrin and immunoglobulins A, G and M) when IgG was purified from human plasma diluted in 10 mmol L^?1 NaP buffer at pH 6.0. The capture of IgG by the P‐Tyr‐agarose was also promising, since 91% of the IgG was adsorbed when plasma was diluted in 25 mmol L^?1 MES buffer at pH 5.5, recommending its use for IgG depletion from human plasma under this condition. The experimental data on IgG adsorption kinetics were in agreement with the pseudo‐second‐order model. The adsorption isotherm data were well described by the Langmuir–Freundlich model with the value of parameter n being <1 (0.72), indicating negative cooperativity. Selectivity was achieved on P‐Tyr‐agarose from digested human IgG in HEPES 25 mmol L^?1 buffer at pH 7.0 where Fab fragments were obtained in eluted fractions without Fc fragments (but with uncleaved IgG) with 86.2% recovery.     

Characterization of the Fc receptors of the murine leukemia L1210

A glycoprotein extract prepared from the plasma membranes of L1210 cells was passed over columns of Sepharose 4B to which either heat-aggregated human IgG or F(ab')2 fragments has been coupled. The intact IgG column bound 35.7 percent of the applied counts, whereas the F(ab')2 columns bound 2.8 percent. The bound glycoproteins were eluted with citrate buffer (pH 3.2) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three peaks with apparent molecular weights of 65,000 45,000, and 28,000 daltons were identified and purified by electroelution from polyacrylamide gels. The isolated proteins were able to bind to the same sub-classes of mouse IgG myeloma proteins as the intact L1210 cells, indicating that these molecules are related to L1210 surface Fc receptors. Amino acid analyses of the 3 proteins were markedly similar suggesting that the observed molecular heterogeneity might be due to carbohydrate differences. Neuraminidase digestion of the isolated proteins resulted in mobility shifts on polyacrylamide gel electrophoresis which were consistent with the interpretation that either the isolated proteins have considerably different sialic acid contents, or that removal of the sialic acid results in disaggregation of an Fc receptor molecule.     

Optimization and development of a high-performance liquid chromatography-based one-site immunometric assay with chemiluminescence detection

Various practical and theoretical considerations were examined in the creation and optimization of a high-performance liquid chromatography (HPLC)-based one-site immunometric assay. This method used an HPLC analyte analog column and post-column chemiluminescence detection. The specific analyte chosen as the model for this study was l-thyroxine (also known as T₄). In this technique, a sample containing thyroxine was first combined with an excess of anti-T₄ antibody Fab fragments that had earlier been conjugated with chemiluminescent acridinium ester labels. After incubation, the mixture was injected onto a column that contained immobilized T₄. The amount of thyroxine in the original sample was then determined by measuring the labeled Fab fragments that appeared in the non-retained fraction, or the decrease in excess Fab fragments that were bound to and later eluted from the column. Items considered in creating this assay included the preparation of acridinium ester-labeled Fab fragments, the detection of these fragments with a post-column reactor, and the creation of a suitable immobilized analog column for capturing excess labeled Fab fragments. The final method could measure T₄ in standards at clinically-relevant concentrations and provided a response within 1.5 min of sample injection, following a 20-45 min incubation with the labeled Fab fragments. Possible applications of this method include its use in clinical chemistry and the screening of proteomic or combinatorial libraries.     

Oriented immobilized anti‐hIgG via Fc fragment‐imprinted PHEMA cryogel for IgG purification

Antibodies are used in many applications, especially as diagnostic and therapeutic agents. Among the various techniques used for the purification of antibodies, immunoaffinity chromatography is by far the most common. For this purpose, oriented immobilization of antibodies is an important step for the efficiency of purification step. In this study, F_c fragment‐imprinted poly(hydroxyethyl methacrylate) cryogel (MIP) was prepared for the oriented immobilization of anti‐hIgG for IgG purification from human plasma. Non‐imprinted poly(hydroxyethyl methacrylate) cryogel (NIP) was also prepared for random immobilization of anti‐hIgG to compare the adsorption capacities of oriented (MIP/anti‐hIgG) and random (NIP/anti‐hIgG) cryogel columns. The amount of immobilized anti‐hIgG was 19.8 mg/g for the NIP column and 23.7 mg/g for the MIP column. Although the amount of immobilized anti‐hIgG was almost the same for the NIP and MIP columns, IgG adsorption capacity was found to be three times higher than the NIP/anti‐hIgG column (29.7 mg/g) for the MIP/anti‐hIgG column (86.9 mg/g). Higher IgG adsorption capacity was observed from human plasma (up to 106.4 mg/g) with the MIP/anti‐hIgG cryogel column. Adsorbed IgG was eluted using 1.0 m NaCl with a purity of 96.7%. The results obtained here are very encouraging and showed the usability of MIP/anti‐hIgG cryogel prepared via imprinting of Fc fragments as an alternative to conventional immunoaffinity techniques for IgG purification. Copyright © 2012 John Wiley & Sons, Ltd.     

Well-oriented ZZ–PS-tag with high Fc-binding onto polystyrene surface for controlled immobilization of capture antibodies

The site specificity and bioactivity retention of antibodies immobilized on a solid substrate are crucial requirements for solid phase immunoassays. A fusion protein between an immunoglobulin G (IgG)-binding protein (ZZ protein) and a polystyrene-binding peptide (PS-tag) was constructed, and then used to develop a simple method for the oriented immobilization of the ZZ protein onto a PS support by the specific attachment of the PS-tag onto a hydrophilic PS. The orientation of intact IgG was achieved via the interaction of the ZZ protein and the constant fragment (Fc), thereby displayed the Fab fragment for binding antigen. The interaction between rabbit IgG anti-horseradish peroxidase (anti-HRP) and its binding partner HRP was analyzed. Results showed that the oriented ZZ–PS-tag yielded an IgG-binding activity that is fivefold higher than that produced by the passive immobilization of the ZZ protein. The advantage of the proposed immunoassay strategy was demonstrated through an enzyme-linked immunosorbent assay, in which monoclonal mouse anti-goat IgG and HRP-conjugated rabbit F(ab′)₂ anti-goat IgG were used to detect goat IgG. The ZZ–PS-tag presented a tenfold higher sensitivity and a wider linear range than did the passively immobilized ZZ protein. The proposed approach may be an attractive strategy for a broad range of applications involving the oriented immobilization of intact IgGs onto PS supports, in which only one type of phi-PS (ZZ–PS-tag) surface is used.     

Probing the orientation of surface-immobilized immunoglobulin G by time-of-flight secondary ion mass spectrometry

Static time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a powerful surface analysis technique for the characterization of protein films because of its chemical selectivity and surface sensitivity. In this study, static ToF-SIMS and principal component analysis (PCA), a multivariate data analysis method, were combined to probe the orientation of surface-immobilized immunoglobulin G (IgG). IgG orientation can enhance its ability to detect its antigen in immunoassay techniques. The IgG used in this work is the mouse monoclonal anti-human chorionic gonadotropin (anti-hCG). Anti-hCG films on different well-defined substrates have been studied using its F(ab')2 and Fc fragments as references. Atomic force microscopy was used to characterize these protein films before static ToF-SIMS analysis. The results from PCA of ToF-SIMS spectra were related to the antibody primary amino acid composition and its three-dimensional structure.     

大孔及涂敷硅球作基质的组氨酸拟亲和色谱纯化人免疫球蛋白G

研究用分子组氨酸和配体、大孔硅球为基质的拟亲和色谱分离纯化人免疫蛋白Ｇ（ＩｇＧ），认为键合组氨酸具有半抗原体质而与ＩｇＧ发生免疫亲和作用，以色谱组份重新进样验证了色谱柱对ＩｇＧ亲和专一性，并用包敷Ｄｅｘｔｒａｎ大孔硅球作基质的拟亲和色谱纯化人血清中的ＩｇＧ，减少了色谱峰拖尾，缩短了分离时间。     

Matrix-assisted laser desorption/ionization mass spectrometry, enzymatic digestion, and molecular modeling in the study of nonenzymatic glycation of IgG

The glycation-induced functional change of immunoglobulins is of particular interest. The glycation levels of IgG in 10 healthy subjects and 20 diabetic patients with different degrees of metabolic control were studied by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. It reveals the number of glucose molecules that have condensed on the protein, which range from 1 to 5 for healthy subjects, from 5 to 9 for well controlled diabetic patients, and from 10 to 25 for poorly controlled ones. The identification of the most favored glycation sites has been obtained by MALDI analysis of standard and in vitro glycated IgG and plasma protein fraction of a healthy subject after digestion with papain, releasing Fab and Fc fragments of the molecule. Both experiments, as well as molecular modeling of the whole protein, confirm that the most of glucose molecules have condensed on the Fab fragment of IgG, suggesting that the immune deficiency observed in diabetic patients may be explained at the molecular level by a more effective glycation of the Fab fragment, thus inhibiting the process of molecular recognition between antibody and antigen.     

Characterization of therapeutic antibody fragmentation using automated capillary western blotting as an orthogonal analytical technique

Fragmentation in protein‐based molecules continues to be a challenge during manufacturing and storage, and requires an appropriate control strategy to ensure purity and integrity of the drug product. Electrophoretic and chromatographic methods are commonly used for monitoring the fragments. However, size‐exclusion chromatography often suffers from low resolution of low molecular weight fragments. Electrophoretic methods like CE‐SDS are not compatible with enriching fragments for additional characterization tests such as MS. These limitations may result in inadequate control strategy for monitoring and characterizing fragments for protein‐based molecules. Capillary western blotting was used in this study as an orthogonal method for characterization of fragments in an IgG1 antibody under reduced conditions. To achieve a comprehensive mapping of various fragments generated by thermal stress, capillary western profiles were generated using recognition antibodies for IgG kappa (κ) light chain, Fc, and Fab regions that enabled unambiguous fragment identification. Additionally, three different enzymatic digestion methods (IdeS, PNGase F, and IgdE) were applied coupled with capillary western blotting for clip identifications. Finally, complementary data collected using traditional chromatographic and electrophoretic methods allowed to establish a comparison of analytical profiles with an added benefit of fragment identification offered by capillary western profiling. In addition to various Fc and Fab‐related low molecular weight fragments, a non‐reducible thio‐ether linked 75 kDa HL fragment was also identified.     

Selectivity of monolithic supports under overloading conditions and their use for separation of human plasma and isolation of low abundance proteins

Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These two proteins frequently interfere with detection, determination and purification of low abundance proteins that can be potential biomarkers and biomarker candidates for various diseases. Some low abundance plasma proteins such as clotting factors and inhibitors are also important therapeutic agents. In this paper, the characterization of ion-exchange monolithic supports under overloading conditions was performed by use of sample displacement chromatography (SDC). If these supports were used for separation of human plasma, the composition of bound and eluted proteins in both anion- and cation-exchange mode is dependent on column loading. Under overloading conditions, the weakly bound proteins such as HSA in anion-exchange and IgG in cation-exchange mode are displaced by stronger binding proteins, and this phenomenon was not dependent on column size. Consequently, small monolithic columns with a column volume of 100 and 200 μL are ideal supports for high-throughput screening in order to develop new methods for separation of complex mixtures, and for sample preparation in proteomic technology.     

蛋白A连续床亲和毛细管色谱柱的制备及性能考察

人免疫球蛋白 G(HIg G)是一种重要的生物大分子 ,是人血浆中的主要成分之一 ,通常采用免疫学的方法测定 .蛋白 A(Protein A)与免疫球蛋白 (HIg G)的 Fc区之间具有很强的特异性亲和作用 ,因而固载蛋白 A的亲和介质可用于免疫球蛋白及单克隆抗体的分离、纯化和分析测定[1～ 3 ] .根据固定相存在形式的不同 ,毛细管色谱柱主要有开管、填充和连续床柱 3种方式 .连续床具有相比高、易制备 (一步合成 )、孔径易控制、不需烧塞子和易改性等优点 .连续床与其它常用的亲和介质 (如球型凝胶颗粒、灌流色谱基质 [4、 5] 、膜介质 [6,7] 等 )相比具…     

Scanning force microscopy and fluorescence microscopy of microcontact printed antibodies and antibody fragments

Unlabeled primary immunoglobulin G (IgG) antibodies and its F(ab')2 and Fc fragments were attached to oxygen-plasma-cleaned glass substrates using either microcontact printing (MCP) or physical adsorption during bath application from dilute solutions. Fluorescently labeled secondary IgGs were then bound to surface-immobilized IgG, and the relative surface coverage was determined by measuring the fluorescence intensity. Results indicated that the surface coverage of IgG increased with increasing protein solution concentration for both MCP and bath-applied IgG and that a greater concentration of IgG was transferred to a glass substrate using MCP than during physisorption during bath applications. Scanning force microscopy (SFM) showed that patterned MCP IgG monolayers were 5 nm in height, indicating that IgG molecules lie flat on the substrate. After incubation with a secondary IgG, the overall line thickness increased to around 15 nm, indicating that the secondary IgG was in a more vertical orientation with respect to the substrate. The surface roughness of these MCP patterned IgG bilayers as measured by SFM was observed to increase with increasing surface coverage. Physisorption of IgG to both unmodified patterned polydimethylsiloxane (PDMS) stamps and plasma-cleaned glass substrates was modeled by Langmuir adsorption kinetics yielding IgG binding constants of K(MCP) = 1.7(2) x 10(7) M(-1) and K(bath) = 7.8(7) x 10(5) M(-1), respectively. MCP experiments involving primary F(ab')2 and Fc fragments incubated in fluorescently labeled fragment-specific secondary IgGs were carried out to test for the function and orientation of IgG. Finally, possible origins of MCP stamping defects such as pits, pull outs, droplets, and reverse protein transfer are discussed.     

Proteolysis of whole cell extracts with immobilized enzyme columns as part of multidimensional chromatography

This paper describes the efficacy of immobilized trypsin columns in the digestion of cellular extracts that contained thousands of proteins. Effectiveness of proteolysis was evaluated with extracts of Escherichia coli by size-exclusion chromatography. Immobilized trypsin columns were operated in either the continuous-flow or stopped-flow mode at temperatures ranging from ambient to 37 degrees C with incubation times of 0-2 h. The results of these studies indicate that reduced and alkylated extracts of proteins from E. coli can be digested in 20 min when the enzyme column is operated at elevated temperature. The advantage of immobilized enzyme columns is that they can be easily incorporated into multidimensional separation systems for automated proteomics.     

Automated signature peptide approach for proteomics

This paper addresses the issue of automating the multidimensional chromatographic, signature peptide approach to proteomics. Peptides were automatically reduced and alkylated in the autosampler of the instrument. Trypsin digestion of all proteins in the sample was then executed on an immobilized enzyme column and the digest directly transferred to an affinity chromatography column. Although a wide variety of affinity columns may be used, the specific column used in this case was a Ga(III) loaded immobilized metal affinity chromatography (IMAC) column. Ga(III)-IMAC is known to select phosphorylated peptides. Phosphorylated peptides selected by the affinity column from tryptic digests of milk were automatically transferred to a reversed-phase liquid chromatography (RPLC) column. Further fractionation of tryptic peptides on the RPLC column was achieved with linear solvent gradient elution. Effluent from the RPLC column was electrosprayed into a time-of-flight mass spectrometer. The entire process was controlled by software in the liquid chromatograph. With slight modification, it is possible to add multiple columns in parallel at any of the single column positions to further increase throughput. Total analysis time in the tandem column mode of operation was under 2 h.     

Sepharose derivatives containing citric acid as affinity ligand. Purification of fumarase

Six Sepharose derivatives, in which citrate was immobilized via methylene carbons, were prepared by coupling of the alpha- and beta-isomers of citrylpolymethylenediamine to Sepharose. The purification of fumarase from pig heart was dependent on the length of the spacer arm, but not on the isomeric configuration of the immobilized citrate. Gels having six methylene carbons had the largest adsorption capacity for the enzyme and therefore were the most suitable for use in affinity columns for its purification. Affinity chromatography with these gels was followed by hydrophobic interaction chromatography on an octamethylenediamine-Sepharose column.     

Synthesis of high-capacity immunoaffinity sorbents with oriented immobilized immunoglobulins or their Fab' fragments for isolation of proteins

Two methods for synthesizing high-capacity immunoaffinity sorbents on Sepharose and Separon HEMA E-1000 are described. The first is the oriented immobilization of monovalent immunoglobulin Fab fragments on a maleimide derivative of Sepharose via the formation of a covalent bond between the SH group of the Fab fragment at the C-terminus of the molecule and the maleimide covalently coupled to Sepharose. The second method is based on the oxidation of the immunoglobulin carbohydrate component, located in the Fc fragment, by periodate with subsequent immobilization of the derivatives on hydrazide derivatives of Sepharose or Separon. Sorbents for the isolation of monoclonal antibodies from the culture supernatants and the elongation factor EF-G from a crude extract of Escherichia coli cells were obtained. These sorbents are characterized by a high capacity, minimal non-specific sorption and high stability.     

Separation of isomeric 2-aminopyridine derivatized N-glycans and N-glycopeptides of human serum immunoglobulin G by using a zwitterionic type of hydrophilic-interaction chromatography

Isomeric oligosaccharides and isomeric glycopeptides are sometimes difficult to separate on normal-phase (NP) and reversed-phase (RP) columns. A zwitterionic type of hydrophilic-interaction chromatography column with sulfobetaine groups (called ZIC-HILIC column) was first applied to the separation of 2-aminopyridine derivatized (PA) N-glycans and tryptic peptides of human serum immunoglobulin G (IgG). It is shown that the ZIC-HILIC column has high capability for structural recognition of isomeric N-glycans as well as high selectivity for glycopeptides. The former feature (i.e., structural recognition) was proven by sufficient separation of neutral PA N-glycan isomers, which are usually difficult to separate on NP and RP columns. In addition, it is noteworthy that IgG glycopeptides consisting of isomeric N-glycans and the same peptide sequences can be sufficiently separated on a ZIC-HILIC column. The latter feature (i.e., selectivity) was also demonstrated by easily separating two peptide groups with/without N-glycans. Thus, we note that the ZIC-HILIC column is highly promising for a simple analysis of N-glycans and N-glycopeptide samples.     

Enhancement of the sensitivity of surface plasmon resonance biosensor with colloidal gold labeling technique

In our experiment colloidal gold was used to enhance the signal obtained from the surface-plasmonresonance biosensor. Fab′ fragments of human IgG molecules were immobilized on the gold surface and reacted with the sheep-anti-human IgG(SAH-IgG) molecules in solution. It was found that when the colloidal gold labeling technique was introduced the sensitivity of surface plasmon resonance instruments increased by a factor of about 300.     

Fast and efficient separation of immunoglobulin M from immunoglobulin G using short monolithic columns

Certain diagnostic, analytical and preparative applications require the separation of immunoglobulin G (IgG) from immunoglobulin M (IgM). In the present work, different ion-exchange methacrylate monoliths were tested for the separation of IgG and IgM. The strong anion-exchange column had the highest dynamic binding capacity reaching more than 20mg of IgM/ml of support. Additionally, separation of IgM from human serum albumin, a common contaminant in immunoglobulin purification, was achieved on the weak ethylenediamino anion-exchange column, which set the basis for the IgM purification method developed on convective interaction media (CIM) supports. Experiments also confirmed flow independent characteristics of the short monolithic columns.