Preparation and properties of recombinant Clostridium ramosum IgA proteinase. Isolation of Fc-SC and Fab fragments of human secretory IgA.

Immunoglobulin A (IgA) proteinase from Clostridium ramosum is the enzyme which cleaves IgA of both subclasses; in contrast, the other bacterial proteinases cleave only IgA1 proteins. Previous reports characterized the activity of proteinase naturally secreted by C. ramosum specific for the normal human serum IgA of IgA1 and IgA2m(1) subclasses and also for secretory IgA (SIgA). Its amino acid sequence was determined, and the recombinant proteinase which cleaved IgA of both subclasses was prepared. Here we report the optimized expression, purification, storage conditions and activity testing against purified human milk SIgA. The recombinant C. ramosum IgA proteinase isolated in the high degree of purity exhibited almost complete cleavage of SIgA of both subclasses. The proteinase remained active upon storage for more than 10 month at -20 °C without substantial loss of enzymatic activity. Purified SIgA fragments are suitable for studies of all antigen-binding and Fc-dependent functions of SIgA involved in the protection against infections with mucosal pathogens.

Generation and characterization of a high-affinity chimeric anti-OX40 antibody with potent antitumor activity.

OX40 is a costimulatory molecule that belongs to the tumor necrosis factor receptor (TNFR) superfamily. OX40 agonist-based combinations are emerging as promising candidates for novel cancer immunotherapy. Clinical trials have shown that OX40 agonist antibodies could lead to better results in cancer patients. Using a hybridoma platform and three different types of immunization strategies, namely recombinant protein, DNA, and overexpressing cells, we identified a chimeric anti-OX40 antibody (mAb035-hIgG1 from DNA immunization) that shows excellent binding specificity, and slightly stronger activation of human memory CD4+ T cells and similar potent antitumor activity compared with BMS 986178, an anti-OX40 antibody currently being evaluated for the treatment of solid tumors. This paper further systematically investigates the antigen-specific immune response, the number of binders, epitope bins, and functional activities of antibodies among different immunization strategies. Interestingly, we found that different immunization strategies affect the biological activity of monoclonal antibodies.

Magnetic bead extraction with acid dissociation immunoassay for the determination of serum CD80-Fc fusion protein.

Background: To detect concentrations of subtherapeutic doses of the CD80-Fc fusion protein FPT155 in serum in Phase I studies, a highly sensitive assay was developed. Materials & methods: FPT155 was purified from human serum using magnetic beads coupled to cytotoxic T-lymphocyte-associated antigen-4. After washing away the serum components, FTP155 was released by acid dissociation and neutralization. The eluted drug was quantified in an ELISA using cytotoxic T-lymphocyte-associated antigen-4 as a capture reagent and biotinylated anti-human Fc for detection. The assay was validated with a calibration range of 5-40 ng/ml and a dilutional integrity of up to 100,000 ng/ml. Conclusion: A highly sensitive assay to determine serum concentrations of FPT155 using readily available reagents was developed. The results were in conformity with theoretical calculations.

Validation of the Production of Antibodies in Different Formats in the HEK 293 Transient Gene Expression System.

Mammalian cells are the most commonly used production system for therapeutic antibodies. Protocols for the expression of recombinant antibodies in HEK293-6E cells in different antibody formats are described in detail. As model, antibodies against Kallikrein-related peptidase 7 (KLK7) were used. KLK7 is a key player in skin homeostasis and represents an emerging target for pharmacological interventions. Potent inhibitors can not only help to elucidate physiological and pathophysiological functions but also serve as a new archetype for the treatment of inflammatory skin disorders. Phage display-derived affinity-matured human anti-KLK7 antibodies were converted to scFv-Fc, IgG, and Fab formats and transiently produced in the mammalian HEK293-6E system. For the production of the corresponding antigen-KLK7-the baculovirus expression vector system (BEVS) and virus-free expression in Hi5 insect cells were used in a comparative approach. The target proteins were isolated by various chromatographic methods in a one- or multistep purification strategy. Ultimately, the interaction between anti-KLK7 and KLK7 was characterized using biolayer interferometry. Here, protocols for the expression of recombinant antibodies in different formats are presented and compared for their specific features. Furthermore, biolayer interferometry (BLI), a fast and high-throughput biophysical analytical technique to evaluate the kinetic binding constant and affinity constant of the different anti-KLK7 antibody formats against Kallikrein-related peptidase 7 is presented.

An improved protocol for large scale production of high purity 'Fc' fragment of human immunoglobulin G (IgG-Fc).

We describe a simplified approach for purification and characterization of human 'IgG-Fc' fragment used widely as immunochemical tool and for therapeutic purposes. The 'Fc' fragment was purified from human IgG in a 3-stage column chromatography. The purified 'Fc' fragment appeared as a dimer glycoprotein with an apparent molecular mass of 52,981 Dalton (Ultraflex MALDI TOF/TOF). The Size-exclusion HPLC profile of the purified 'Fc' fragment of human IgG matched that of a commercially procured reference 'Fc' fragment material. The purity of the 'Fc' fragments was >99% by SDS-PAGE and size-exclusion HPLC. The results of Western blotting, immunoelectrophoresis, and mass spectrometry analysis indicate a high purity of the 'Fc' fragment. Peptide mass fingerprint analysis of the purified 'Fc' protein yielded peptides that partially match the known database sequences of FCG3B_HUMAN (Uniprot ID: O75015). This method of purification of the 'Fc' fragment is suitable for achieving high purity level of 'Fc' fragment protein. With this purification approach, the cost of the purified 'Fc' fragment of human IgG is significantly reduced as compared with the current market price of IgG-Fc fragment protein in international market. The purified 'IgG-Fc' fragment protein was found to be negative for major viral markers.

Purification of Fab and Fc using papain immobilized cryogel bioreactor separator system.

The continuous bed bioreactor systems have been used for the production of protein therapeutics, such as IgG, using immobilized enzyme in biopharmaceutical applications. We developed macroporous poly(hydroxyethyl methacrylate-co-glycidyl methacrylate) cryogel-based bioreactor matrix using sodium dodecyl sulfate as surfactans in the presence of ethylene glycol dimethacrylate as cross linking agent by bulk polymerization. The developed polyGMA immobilized bioreactor with papain enzyme was used for specific fragmentation of immunoglobulin G. The catalysis efficiency for immobilized enzyme were investigated in comparison with free enzyme. The immobilized papain displayed broad catalytic activity over a variety of conditions, with maximal activity around pH 7.0 and 70 °C. The Michaelis-Menten kinetic constant (Km), the maximum reaction velocity (Vmax), and the catalytic efficiency (kcat) for free enzyme were 0.1097 mg/mL, 29.9 mg/mL/min, and 92.01 1/min, respectively, whereas for immobilized enzyme, Km, Vmax, and kcat values were 0.1078 mg/mL, 30.53 mg/mL/min, and 94.3 1/min, respectively. In a further step, after digestion, remarkable digestion products of bioreactor, Fab and Fc fragments, produced with immobilized papain bioreactors were analyzed in two ways by SDS-PAGE and reversed-phase HPLC; it was demonstrated that papain immobilized bioreactor successfully used for the digestion of human IgG with high activity. Therefore, the polyGMA cryogel immobilized with papain exhibited a very effective matrix for the bioreactor which can be considered as an alternative bioreactor matrix with great promise in biopharmaceutical applications.

Highly sensitive HPLC analysis and biophysical characterization of N-glycans of IgG-Fc domain in comparison between CHO and 293 cells using FcγRIIIa ligand.

Quality control of monoclonal antibodies is challenging due in part to the diversity of post-translational modifications present. The regulation of the N-glycans of IgG-Fc domain is one of the key factors to maintain the safety and efficacy of antibody drugs. The FcγRIIIa affinity column is an attractive tool for the precise analysis of the N-glycans in IgG-Fc domain. We used the mutant FcγRIIIa, which is produced in Escherichia coli and is therefore not glycosylated, as an affinity reagent to analyze the N-glycans of monoclonal antibodies expressed in Expi293 and ExpiCHO cells. The monoclonal antibodies expressed in these cells showed very different chromatograms, because of differences in terminal galactose residues on the IgG-Fc domains. We also carried out kinetic and thermodynamic analyses to understand the interaction between monoclonal antibodies and the mutant FcγRIIIa. Expi293 cell-derived monoclonal antibodies had higher affinity for the mutant FcγRIIIa than those derived from ExpiCHO cells, due to slower off rates and lower binding entropy loss. Collectively, our results suggest that the FcγRIIIa column can be used to analyze the glycosylation of antibodies rapidly and specifically.

An easy and simple separation method for Fc and Fab fragments from chicken immunoglobulin Y (IgY).

Antigen-binding (Fab) and crystallizable (Fc) fragments are the active components of yolk immunoglobulin (IgY), which have been widely used in the pharmaceutical field. However, the common purification methods for the Fab and Fc fragments use combinations of multi-columns are complex and time-consuming. The objective of this study was to improve the separation efficiency of the Fab and Fc fragments from the hydrolyzed IgY and increase the purity of the isolated Fab and Fc fragments. Natural IgY was hydrolyzed using papain for 6 hr and then treated with 45% saturated ammonium sulfate to remove small molecular-weight-peptides. The fraction containing Fab and Fc fragments was loaded on a DEAE-Sepharose ion exchange column and the Fab fraction was washed out first with 10 mM Tris-HCl buffer (pH 7.6). Then, the Fc fraction bound to the DEAE Sepharose was eluted with 10 mM Tris-HCl buffer (pH 7.6) containing 0.21 M NaCl. The purity of the two fragments was 88.7% and 90.1%, respectively. The results of Western blotting and MS analyses indicated that this method purified Fab and Fc fractions with high purity. This method is easy and simple compared with other methods, and the active fragments separated can be easily used.

Absolute quantitation of high abundant Fc-glycopeptides from human serum IgG-1.

Absolute quantitation of IgG-1 Fc-glycosylation, which is crucial for the clinical practice of glyco-biomarkers and quality control of biopharmaceuticals, has been hindered by the lack of glycopeptide standards. In this study, eleven high abundant IgG-1 Fc-glycopeptides with definite peptide sequences and glycoforms were purified from commercial IgG protein by using two-dimensional hydrophilic interaction liquid chromatographic system. Based on the acquired glycopeptide standards, an absolute quantitation strategy was developed to determine the concentrations of 11 target IgG-1 glycopeptides from pooled human sera. A wide range of Fc-glycopeptide concentrations from 0.60 to 17.61 nmol mL-1 was achieved with excellent accuracy and reproducibility from pooled human sera IgG-1. Compared to conventional relative quantitation, this strategy provides more accurate distribution profiles of 11 high abundant Fc-glycopeptides and degree of glycosylation from pooled human sera IgG-1.

Choice of Host Cell Line Is Essential for the Functional Glycosylation of the Fc Region of Human IgG1 Inhibitors of Influenza B Viruses.

Abs are glycoproteins that carry a conserved N-linked carbohydrate attached to the Fc whose presence and fine structure profoundly impacts on their in vivo immunogenicity, pharmacokinetics, and functional attributes. The host cell line used to produce IgG plays a major role in this glycosylation, as different systems express different glycosylation enzymes and transporters that contribute to the specificity and heterogeneity of the final IgG-Fc glycosylation profile. In this study, we compare two panels of glycan-adapted IgG1-Fc mutants expressed in either the human endothelial kidney 293-F or Chinese hamster ovary-K1 systems. We show that the types of N-linked glycans between matched pairs of Fc mutants vary greatly and in particular, with respect, to sialylation. These cell line effects on glycosylation profoundly influence the ability of the engineered Fcs to interact with either human or pathogen receptors. For example, we describe Fc mutants that potently disrupted influenza B-mediated agglutination of human erythrocytes when expressed in Chinese hamster ovary-K1, but not in human endothelial kidney 293-F cells.

Homogenous Scavenging Resolves Low-Purification Yield/Selectivity Caused by Secondary Binding of Protein-A to Antigen-Binding Antibody Fragments.

Antigen-binding fragments of antibodies are biotechnologically useful agents for decorating drug delivery systems, for blocking cell-surface receptors in cell culture, for recognizing analytes in biosensors, and potentially as therapeutics. They are typically produced by enzymatic digestion of full antibodies and isolated from the undesirable fragment crystallizable (Fc) by affinity chromatography using Protein-A columns. However, while Protein-A has a strong "classical" interaction with Fc fragments, it can also more weakly bind to an "alternative" site on the heavy chain variable region of antigen-binding fragments. As such, purifying small amounts of antibody fragments by Protein-A chromatography can result in low yield. Moreover, loading larger amounts of antibody fragments onto a Protein-A column can result in poor separation, because of competition of Fc and antigen-binding fragments for immobilized Protein-A. This study demonstrates that Protein-A-based homogeneous scavenging resolves this issue by precisely controlling the stoichiometry of Protein-A to Fc fragments, something that is not possible for conventional flow-type systems, such as affinity chromatography.

Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development.

Heavy chain (Hc) heterodimers represent a majority of bispecific antibodies (bsAbs) under clinical development. Although recent technologies achieve high levels of Hc heterodimerization (HD), traces of homodimer contaminants are often present, and as a consequence robust purification techniques for generating highly pure heterodimers in a single step are needed. Here, we describe two different purification methods that exploit differences in Protein A (PA) or Protein G (PG) avidity between homo- and heterodimers. Differential elution between species was enabled by removing PA or PG binding in one of the Hcs of the bsAb. The PA method allowed the avidity purification of heterodimers based on the VH3 subclass, which naturally binds PA and interferes with separation, by using a combination of IgG3 Fc and a single amino acid change in VH3, N82aS. The PG method relied on a combination of three mutations that completely disrupts PG binding, M428G/N434A in IgG1 Fc and K213V in IgG1 CH1. Both methods achieved a high level of heterodimer purity as single-step techniques without Hc HD (93-98%). Since PA and PG have overlapping binding sites with the neonatal Fc receptor (FcRn), we investigated the effects of our engineering both in vitro and in vivo. Mild to moderate differences in FcRn binding and Fc thermal stability were observed, but these did not significantly change the serum half-lives of engineered control antibodies and heterodimers. The methods are conceptually compatible with various Hc HD platforms such as BEAT® (Bispecific Engagement by Antibodies based on the T cell receptor), in which the PA method has already been successfully implemented.

Expression of Human VH Single Domains as Fc Fusions in Mammalian Cells.

Single-domain antibodies represent an emerging class of antibody fragments with promising therapeutic and diagnostic potential. As a result, multiple strategies have been developed in order to improve their biophysical and/or biological properties. In particular, the fusion of single-domain antibodies to the Fc part of an IgG molecule has become a common protein engineering approach toward this aim. Here, we describe a detailed protocol for a streamlined laboratory-scale production of VH single-domain antibodies as Fc fusions in mammalian cells. Firstly, DNA sequence encoding VH domain of interest fused to an IgG Fc is synthesized as a double-stranded gene fragment. Secondly, the DNA fragment is directly assembled into a restriction enzyme-digested vector in an assembly reaction. Finally, vector carrying the VH-Fc-fusion construct is introduced into suspension-adapted mammalian cells for transient expression of the Fc chimeric fusion. One-week post-transfection, the expressed Fc-fusion protein is purified using protein A/G affinity chromatography. Using this protocol, we were able to clone, express, and purify milligrams of isolated anti-HER2 VH domain as a mouse IgG2c Fc fusion in less than 2 weeks. This protocol can be readily modified to express proteins of interest other than VH domains as Fc fusions.

Measurement of Neutral and Sialylated IgG N-Glycome at Asn-297 by CE-LIF to Assess Hypogalactosylation in Rheumatoid Arthritis.

Modulations in immunoglobulin G (IgG) N-glycosylation have been observed in many human diseases including chronic inflammatory diseases such as rheumatoid arthritis and also cancer. In this chapter, we describe how to determine hypogalactosylation for clinical samples, namely the sample preparation of IgG N-glycans at Asn-297 as well as the measurement of neutral and sialylated N-glycans by capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF).This semiautomated protocol describes the isolation polyclonal antibodies from serum, the separation of IgG-Fc glycopeptides from IgG antigen-binding fragment by pepsin digestion. Afterward, enzymatically released IgG-Fc N-glycans are cleaned up using a polyaromatic adsorbent resin followed by carbon purification. Sialic acids are then derivatized prior to glycan labeling. As a result, the agalactosylated N-glycan A2 does not co-migrate with sialylated N-glycans, which refines the measurement of hypogalactosylation by CE-LIF.

The preparation and therapeutic roles of scFv-Fc antibody against Staphylococcus aureus infection to control bovine mastitis.

Staphylococcus aureus-induced bovine mastitis causes significant losses to the dairy industry and available vaccines do not confer adequate protection. As a more attractive alternative, we propose the use of antibody (Ab) therapy. In our previous study, we constructed a bovine single-chain variable fragment (scFv) Ab phage display and successfully obtained scFvs that bound to S. aureus antigens with high affinity. Here, we describe a novel Ab against S. aureus (scFv-Fc Ab). To construct the scFv-Fc Ab, the scFv Ab was genetically fused to the Fc fragment of a bovine IgG1 Ab. Western blot analysis showed that the bovine scFvs-Fc Abs were successfully expressed with horseradish peroxidase-conjugated goat-anti-bovine IgG (Fc) Ab in Escherichia coli cells. The purified bovine scFvs-Fc Abs had good binding activity to S. aureus and effectively inhibited the bacterial growth in culture medium and bovine scFvs-Fc Abs enhanced phagocytosis of S. aureus by neutrophils isolated from peripheral blood in a dose-dependent manner. In the experiment of bovine scFvs-Fc Abs for the treatment of S. aureus-induced bovine mastitis, the total effective percentage reached 82% (9/11). These novel bovine scFvs-Fc Abs may be useful as therapeutic candidates for the prevention and treatment of S. aureus-induced bovine mastitis.

Expression of 5T4 extracellular domain fusion protein and preparation of anti-5T4 monoclonal antibody with high affinity and internalization efficiency.

5T4, a membrane protein, is overexpressed in many tumor tissues but rarely expressed in normal tissues. Here, CHO-5T4+ cells were generated and served as the antigen to immunize mice. Hybridoma techniques were employed to produce monoclonal antibodies (mAbs). The recombinant protein of human IgG Fc-fused extracellular domain of 5T4 (5T4 ECD-Fc) was obtained from transient expression in HEK293F cells. The fusion protein 5T4 ECD-Fc and CHO-5T4+ cells were respectively utilized to screen anti-5T4 antibodies that could bind to the native antigen. In preliminary screening, three hundred and fifty mAbs were obtained. Via surface plasmon resonance and flow cytometry screening, seven anti-5T4 mAbs stood out. Among them, H6 showed a high affinity (KD = 1.6 × 10-11 M) and internalization percentage (36% for 1 h and 80% for 4 h). The molecular weight and isoelectric point of H6 were determined by LC-MS and iCIEF. Moreover, the specific reactivity of H6 was demonstrated by western blotting, flow cytometry, and immunohistochemistry, respectively. In conclusion, we produced human recombinant protein of 5T4 extracellular domain and developed high-affinity internalizing monoclonal antibodies which may be applied in the 5T4-targeting ADC therapy and basic research.

An engineered Staphylococcal Protein A based ligand: Production, characterization and potential application for the capture of Immunoglobulin and Fc-fusion proteins.

In antibody purification processes, affinity chromatography has been used with Staphylococcus aureus protein A (SpA) as the main ligand. In this work, we present a novel Staphylococcal Protein A (AviPure thereafter), a synthetic ligand analogue based on native SpA B domain, with a molecular weight of approximately 14 kDa. The binding affinity of mAbs to AviPure was evaluated using Surface Plasmon Resonance (SPR) and affinity chromatography methods. The equilibrium dissociation constant (KD) between the AviPure and mAbs was systematically measured using 1:1 (Langmuir) model and found to be 4.7 × 10-8 M, with constant of dissociation at kd ≤ 1.0 × 10-3 s-1 and ka being 3.1 × 104 M-1 s-1. When immobilized on Sepharose, the AviPure ligand density was 429 nmol/g moist weight resin and was able to effectively bind immunoglobulin and Fc fragment samples with higher affinity and the most effective flow rate when using ligand - Sepharose beads was at 75 cm/h giving the dynamic binding capacity of 53 mg/mL and 91% recovery of IgG. Suitable ligands used in affinity purification should have a KD ≤ 10-6 M and a dissociation rate (ka) averaging 10-3 M-1 s-1 with the kd ranging between 103 - 108 M-1. Therefore, the AviPure ligand can be used as an alternative to the standard protein A ligand in the purification of mAbs and Fc-fused proteins.

Combined detection of the ActRII-Fc fusion proteins Sotatercept (ActRIIA-Fc) and Luspatercept (modified ActRIIB-Fc) in serum by means of immunoaffinity purification, tryptic digestion, and LC-MS/MS.

Therapeutic proteins are a continuously growing class of pharmaceuticals and comprise several drug candidates with potential performance-enhancing properties. In particular, activin receptor competitors, such as the ActRII-Fc fusion proteins Sotatercept (ActRIIA-Fc) and Luspatercept (modified ActRIIB-Fc), have the potential for being misused as doping agents in sports as they were found to inhibit negative regulators of late-stage erythropoiesis. Within this study, ammonium sulfate precipitation, immunoaffinity purification, tryptic digestion, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were employed to develop an assay for the combined detection of Sotatercept and Luspatercept in doping control serum samples. The assay was optimized, comprehensively characterized, and found to be fit-for-purpose for application to sports drug testing. It complements existing tests for ActRII-Fc fusion proteins and expands the range of available detection methods for novel protein therapeutics.

Updated protocols for the detection of Sotatercept and Luspatercept in human serum.

We recently published two protocols for the detection of Sotatercept (ACE-011, ACVR2A-Fc) and Luspatercept (ACE-536, ACVR2B-Fc) in human serum. Both methods used covalently immobilized antibodies on agarose beads for immunoprecipitation and SAR-PAGE/Western blotting for detection. Disadvantages were the relatively high amount of antibody required per sample (10 µg) and the need of a secondary antibody for the final detection. The updated protocols overcome these limitations by antigen-antibody complex formation in solution followed by capture of the complex with anti-antibody-coated magnetic beads. They also omit the secondary antibody incubation step by usage of biotinylated primary antibodies, which can be directly incubated with streptavidin-HRP. Thus, the new protocols are faster, simpler, and cheaper and offer comparable sensitivities.

Mechanisms of precipitate formation during the purification of an Fc-fusion protein.

Protein precipitates that arise during bioprocessing can cause manufacturing challenges, but they can also aid in clearance of host-cell protein (HCP) and DNA impurities. Such precipitates differ from many protein precipitates that have been studied previously in their heterogeneous composition, particularly in the presence of high concentrations of the product protein. Here, we characterize the precipitates that form after neutralization of protein A purified and viral-inactivated material of an Fc-fusion protein produced in Chinese hamster ovary cells. The physical growth of precipitate particles was observed by optical microscopy, transmission electron microscopy, dynamic light scattering, and small-angle and ultra-small-angle X-ray scattering to characterize the precipitate microstructure and growth mechanism. The precipitate microstructure is well-described as a mass fractal with fractal dimension approximately 2. The growth is governed by a diffusion-limited aggregation mechanism as indicated by a power-law dependence on time of the size of the principal precipitate particles. Optical microscopy shows that these primary particles can further aggregate into larger particles in a manner that appears to be promoted by mixing. Absorbance experiments at varying pH and salt concentrations reveal that the growth is largely driven by attractive electrostatic interactions, as growth is hindered by an increase in ionic strength. The solution conditions that resulted in the most significant particle growth are also correlated with the greatest removal of soluble impurities (DNA and HCPs). Proteomic analysis of the precipitates allows identification of O ( 100 ) unique HCP impurities, depending on the buffer species (acetate or citrate) used for the viral inactivation. Most of these proteins have pI values near the precipitation pH, supporting the likely importance of electrostatic interactions in driving precipitate formation.