Use of thiophilic adsorption chromatography for the one-step purification of a bacterially produced antibody F(ab) fragment without the need for an affinity tag

Thiophilic adsorption chromatography (TAC) was employed for the purification of a recombinant F(ab) fragment of the antibody IN-1 from the periplasmic protein fraction of Escherichia coli. Adsorption of the F(ab) fragment to the T-gel was achieved at a high concentration of ammonium sulfate and turned out to be independent of the presence of a His(6) tag or Strep tag or of the human or murine nature of the C(H)1 and C(L) domains (subclass IgG1/kappa). Elution was effected by means of a decreasing salt gradient, yielding fractions with the correctly assembled, heterodimeric F(ab) fragment at high purity. Interestingly, the single substitution of an alanine residue with phenylalanine in the CDR-L1 of the F(ab) fragment significantly enhanced the retention on the column so that quantitative elution necessitated prolonged application of a low-salt buffer. Our findings suggest that TAC is generally suitable for the isolation of bacterially produced F(ab) fragments and support the notion that aromatic side chains play an important role in the interaction with the affinity matrix. This method should prove valuable in the production of proteins for in vivo applications as might be the case for the F(ab) fragment of the antibody IN-1, which promotes axonal regeneration in the central nervous system.     

A generic strategy for subcloning antibody variable regions from the scFv phage display vector pCANTAB 5 E into pASK85 permits the economical production of F(ab) fragments and leads to improved recombinant immunoglobulin stability

Apart from the decisive sensitivity and specificity of immunosensors, the employed antibodies essentially contribute to additional key factors like fabrication costs for sensor chips and sensor stability. A production scheme for recombinant antibody fragments has been optimised with respect to these particular issues of biosensor development. The phagemid vector pCANTAB 5 E is widely used for the selection of antibody fragments from corresponding libraries. However, large-scale production of the selected single-chain F(v) (scFv) fragments is substantially restricted by the high cost for the inducer IPTG and the anti-E-tag antibody. The latter is needed in significant amounts for the purification of the recombinant protein. A generic strategy was established for subcloning scFv variable regions from pCANTAB 5 E into the plasmid pASK85 for the expression of F(ab) fragments. pASK85 bears coding sequences for murine constant domains including a His(6) tag at the carboxyl-terminal end of the constant heavy chain domain. The anti-s-triazine antibody K47H served as a model system in this study. Biosynthesis of the F(ab) fragment in a high cell density fermenter was induced by addition of anhydrotetracycline. The F(ab) fragment was subsequently purified from the periplasmic extract in a single step by immobilized metal affinity chromatography (IMAC). A yield of 100 microg/lxOD(550) purified F(ab) fragment was obtained employing a standard fermentation scheme. The sensitivity and cross-reactivity of the F(ab) was comparable to the parent scFv when assayed by enzyme immunoassay. However, the F(ab) fragment exhibited significantly improved long-term stability.     

Mimic of a large‐scale diafiltration process by using ultra scale‐down rotating disc filter

Ultra scale‐down (USD) approach is a powerful tool to predict large‐scale process performance by using very small amounts of material. In this article, we present a method to mimic flux and transmission performance in a labscale crossflow operation by an USD rotating disc filter (RDF). The Pellicon 2 labscale system used for evaluation of the mimic can readily be related to small pilot and industrial scale. Adopted from the pulsed sample injection technique by Ghosh and Cui (J Membr Sci. 2000;175:5‐84), the RDF has been modified by building in inserts to allow the flexibility of the chamber volume, so that only 1.5 mL of processing material is required for each diafiltration experiment. The reported method enjoys the simplicity of dead‐end mode operation with accurate control of operation conditions that can mimic well the crossflow operation in large scale. Wall shear rate correlations have been established for both the labscale cassette and the USD device, and a mimic has been developed by operating both scales under conditions with equivalent averaged shear rates. The studies using E. coli lysate show that the flux vs. transmembrane pressure profile follows a first‐order model, and the transmission of antibody fragment (Fab′) is independent of transmembrane pressure. Predicted flux and transmission data agreed well with the experimental results of a labscale diafiltration where the cassette resistance was considered. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

An engineered IN-1 F(ab) fragment with improved affinity for the Nogo-A axonal growth inhibitor permits immunochemical detection and shows enhanced neutralizing activity

The myelin axonal growth inhibitor NI-220/250 (Nogo-A) has attracted considerable attention in elucidating the mechanisms that account for the lack of plasticity in the adult central nervous system. The cognate monoclonal antibody IN-1, which was obtained prior to the molecular characterization of its Nogo-A antigen, has played a crucial role in this respect. However, this murine IgM/kappa antibody does not only provide an inappropriate format for in vivo studies, its low antigen affinity has also hampered the thorough structure-function analysis of its neutralizing effect toward the Nogo-A inhibitor on a molecular basis. We describe here the affinity maturation of a bacterially produced functional IN-1 F(ab) fragment via protein engineering. A soluble fragment of Nogo-A derived from the central exon 3 of its gene, which was prepared by secretion into the periplasm of Escherichia coli, served as a target in these experiments. After repeated cycles of site-directed random mutagenesis and screening, the mutant II.1.8 of the IN-1 F(ab) fragment was obtained, carrying five side chain substitutions within CDR-L3. Its dissociation constant for the complex with the recombinant Nogo-A fragment was determined in surface plasmon resonance measurements as approximately 1 microM. The affinity of the unmutated IN-1 F(ab) fragment was 8-fold lower. The engineered F(ab) fragment appeared to be well suited for the specific detection of Nogo-A in immunochemical assays and for the histochemical staining of myelin-rich tissue sections. Most importantly, its concentration-dependent neutralizing effect on the Nogo-A inhibitory activity was significantly enhanced in cell culture. This study confirms Nogo-A to be the antigen of the IN-1 antibody and it demonstrates increased potential of the engineered F(ab) fragment as a reagent for promoting axonal regeneration in vivo.     

A versatile noninvasive method for adsorber quantification in batch and column chromatography based on the ionic capacity

Within the Quality by Design (QbD) framework proposed by the International Conference on Harmonisation (ICH), high‐throughput process development (HTPD) and mechanistic modeling are of outstanding importance for future biopharmaceutical chromatography process development. In order to compare the data derived from different column scales or batch chromatographies, the amount of adsorber has to be quantified with the same noninvasive method. Similarly, an important requirement for the implementation of mechanistic modeling is the reliable determination of column characteristics such as the ionic capacity Λ for ion‐exchange chromatography with the same method at all scales and formats. We developed a method to determine the ionic capacity in column and batch chromatography, based on the adsorption/desorption of the natural, uv‐detectable amino acid histidine. In column chromatography, this method produces results comparable to those of classical acid?base titration. In contrast to acid?base titration, this method can be adapted to robotic batch chromatographic experiments. We are able to convert the adsorber volumes in batch chromatography to the equivalent volume of a compressed column. In a case study, we demonstrate that this method increases the quality of SMA parameters fitted to batch adsorption isotherms, and the capability to predict column breakthrough experiments. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:666–677, 2016     

Adsorption of IgG onto hydrophobic teflon. Differences between the F(ab) and F(c) domains

The effect of differences in the degree of hydrophobicity of protein patches/fragments on the adsorption behaviour of the protein is investigated. The adsorption isotherm of a monoclonal mouse anti-human immunoglobulin G (isotype 2b) onto hydrophobic Teflon particles is measured using a depletion method. The adsorption-induced denaturation of the immunoglobulin as a function of the adsorbed amount is studied by differential scanning calorimetry, and the corresponding rearrangements in the secondary structure of the whole IgG molecule and its F(ab) and F(c) fragments are determined by circular dichroism spectroscopy. The effects of adsorption on the F(ab) and F(c) fragments in the intact IgG molecule occur independently. Adsorption of the whole IgG molecule leads to denaturation of the F(ab) fragments, whereas the F(c) fragment remains unperturbed; adsorption of the isolated fragments results in structural changes in both F(ab) and F(c). The surface hydrophobicity of the isolated fragments was studied by HPLC. These experiments support the hypothesis that differences in the degree of denaturation between F(ab) and F(c) are due to the higher degree of hydrophobicity of the F(ab) fragment. The adsorption-induced changes in the secondary structure are more prominent for the isolated fragments as compared to intact IgG. This is ascribed to the higher flexibility of the isolated fragment, as compared to the fragment in the whole molecule.     

A potential new radiopharmaceutical for parathyroid imaging: Radiolabeled parathyroid-specific monoclonal antibody—I. Evaluation of 125I-labeled antibody in a nude mouse model system

Radioiodinated BB5-G1, a parathyroid-specific monoclonal antibody, and its F(ab′)₂ and Fab fragments were characterized using a nude mouse model system. Blood clearance studies indicated that the most slowly clearing species was the ¹²⁵I-BB5-G1 intact antibody, while the most rapidly clearing one was the ¹²⁵I-Fab fragment. ¹²⁵I-F(ab′)₂ retained its capacity to localize in the human parathyroid tissue implants with the uptake at 24 h being similar to that observed with the intact antibody. Poor localization was observed with the Fab fragment. These results suggest that BB5-G1 or its F(ab′)₂ fragment may be useful for parathyroid imaging.     

Conjugation of methotrexate to IgG antibodies and their F(ab)2 fragments and the effect of conjugated methotrexate on tumor growth in vivo

Summary Methotrexate (MTX) was first conjugated to antibovine serum albumin IgG (antiBSA) or its F(ab)₂ fragment to define conditions for retention of drug and antibody activity. With identical drug: protein molar ratios, incorporation in the F(ab)₂ fragment was lower than in intact antiBSA, an observation consistent with analysis of the number of lysine residues (22 in F(ab)₂ compared to 40 in antiBSA). In either case, up to approximately 10 mol MTX could be incorporated per mol protein, with recovery of 70% of the protein. At an incorporation ratio of 6 mol MTX per mol protein, MTX-antiBSA retained 100% of antibody activity and MTX-F(ab)₂antiBSA retained 75%. MTX-antiBSA and MTX-F(ab)₂antiBSA were equally potent in vitro inhibitors of dihydrofolate reductase. Conjugates prepared from antiEL4 IgG (AELG) and from F(ab)₂AELG significantly increased survival in EL4 lymphoma-bearing mice compared with mice receiving equal amounts (5 mg MTX/kg) of free MTX, MTX linked to the F(ab)₂ fragment of normal rabbit IgG, or a simple mixture of MTX and F(ab)₂AELG. MTX-AELG at this dose level produced longer survival than MTX-F(ab)₂AELG (0.0052AELG MTX linked to the F(ab)₂ fragment of AELG - MTX-F(ab)₂antiBSA MTX linked to the F(ab)₂ fragment of antiBSA - MTX-F(ab)₂NRG MTX linked to the F(ab)₂ fragment of NRG - MTX-NRG MTX linked to NRG - NHS N-hydroxysuccinimide - NRG normal rabbit IgG - PBS 0.01 M sodium phosphate (pH 7.1) containing 0.45 M sodium chloride - TAA tumor-associated antigen - t_1/2 half-life     

F(ab′)2 fragment of a gp41 NHR-trimer-induced IgM monoclonal antibody neutralizes HIV-1 infection and blocks viral fusion by targeting the conserved gp41 pocket

Using a recombinant protein N46FdFc that mimics the HIV-1 gp41 N-helix trimer to immunize mice, we identified the first IgM monoclonal antibody 18D3 that specifically bound to the conserved gp41 pocket. Its F(ab′)₂ fragment potently inhibited HIV-1 Env-mediated cell–cell fusion and neutralized infection by laboratory-adapted and primary HIV-1 isolates with different subtypes and tropism, including the T20-resistant variants. This F(ab′)₂ fragment can be used to develop a bispecific broad neutralizing monoclonal antibody or HIV-1 inactivator as a novel immunotherapeutic for treatment and prevention of HIV-1 infection.     

A monoclonal antibody against hinge-cleaved IgG restores effector function to proteolytically-inactivated IgGs in vitro and in vivo

We report a chimeric monoclonal antibody (mAb) directed to a neo-epitope that is exposed in the IgG lower hinge following proteolytic cleavage. The mAb, designated 2095–2, displays specificity for IdeS-generated F(ab’)₂ fragments, but not for full-length IgG or for closely-related F(ab’)₂ fragments generated with other proteases. A critical component of the specificity is provided by the C-terminal amino acid of the epitope corresponding to gly-236 in the IgG1 (also IgG4) hinge. By its ability to bind to IdeS-cleaved anti-CD20 mAb, mAb 2095–2 fully restored antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against WIL2-S cells to the otherwise inactive anti-CD20 IgG1 F(ab’)₂ fragment. Similarly, 2095–2 reinstated ADCC against MDA-MB-231 cells to an anti-CD142 IgG1 F(ab’)₂ fragment. mAb 2095–2 was also capable of eliciting both CDC and ADCC to IgG4 F(ab’)₂ fragments, an IgG subclass that has weaker ADCC and CDC when intact relative to intact IgG1. The in vitro cell-based efficacy of 2095–2 was extended to the in vivo setting using platelets as a cell clearance surrogate. In a canine model, the co-administration of 2095–2 together with IdeS-generated, platelet-targeting anti-CD41/61 F(ab’)₂ fragment not only restored platelet clearance, but did so at a rate and extent of clearance that exceeded that of intact anti-CD41/61 IgG at comparable concentrations. To further explore this unexpected amplification effect, we conducted a rat study in which 2095–2 was administered at a series of doses in combination with a fixed dose of anti-CD41/61 F(ab’)₂ fragments. Again, the combination, at ratios as low as 1:10 (w/w) 2095–2 to F(ab’)₂, proved more effective than the anti-CD41/61 IgG1 alone. These findings suggest a novel mechanism for enhancing antibody-mediated cell-killing effector functions with potential applications in pathologic settings such as tumors and acute infections where protease activity is abundant.     

Antibody directed against the 142-148 sequence of the myosin heavy chain interferes with myosin-actin interaction.

It has been reported recently that the isolated and renatured 23-kDa N-terminal fragment of rabbit skeletal muscle myosin binds tightly to F-actin in an ATP-dependent manner [Muhlrad, A. (1989) Biochemistry 28, 4002-4010]. The binding to actin is of electrostatic nature and may involve a positively charged cluster of residues on the 23-kDa fragment stretching from Arg-143 to Arg-147. An octapeptide containing this positive cluster was synthesized and coupled to BSA through a cysteine residue added to the N-terminus of the peptide. Polyclonal antibody was raised against the BSA-coupled peptide in rabbits which recognized the N-terminal 23-kDa fragment of rabbit skeletal myosin subfragment 1, and a peptide comprised of residues 122-204 of the 23K fragment in Western blots. The purified antibody [IgG and F(ab)] inhibited the actin-activated ATPase activity of S1 without affecting its Mg2(+)- and K+(EDTA)-modulated ATPase activity. Both IgG and F(ab) decreased the binding of S1 to F-actin in a sedimentation assay, and actin inhibited the binding of both IgG and F(ab) to S1 in a competitive binding assay. The cysteine thiol of the synthetic octapeptide was labeled by the fluorescent thiol reagent monobromobimane, and the labeled peptide was found to bind to actin in a sedimentation assay. The results support the possibility that the positively charged Arg-143 to Arg-147 stretch of residues on the 23-kDa fragment participates in actin binding of myosin and may represent an essential constituent of the actin-S1 interface.     

Labeling proteins with fluorine-18 using N-succinimidyl 4-[18F]fluorobenzoate

Two methods were investigated for the no-carrier-added synthesis of N-succinimidyl 4-[¹⁸F]fluorobenzoate (S[¹⁸F]FB). The first, an attempted nucleophilic aromatic substitution by [¹⁸F]fluoride on N-succinimidyl 4-nitrobenzoate was unsuccessful. The second method involved three steps; [¹⁸F]fluoride for trimethylammonium substitution on 4-formyl-N,N,N-trimethylanilinium triflate, oxidation to 4-[¹⁸F]fluorobenzoic acid, followed by reaction with N-hydroxysuccinimide and dicyclohexylcarbodiimide to form S[¹⁸F]FB. Total synthesis and purification time was 100 min and the overall radiochemical yield was 25% (decay corrected). A monoclonal antibody F(ab′)₂ fragment could be labeled in 40–60% yield by reaction with S[¹⁸F]FB for 15–20 min. The tissue distribution in normal mice and in vitro tumor binding of the antibody F(ab′)₂ labeled by reaction with S[¹⁸F]FB were comparable to those observed for the fragment after radioiodination using N-succinimidyl 4-[¹²⁵I]iodobenzoate.     

Epitope mapping of the neuronal growth inhibitor Nogo-A for the Nogo receptor and the cognate monoclonal antibody IN-1 by means of the SPOT technique

Nogo-A is a potent inhibitor of axonal outgrowth in the central nervous system of adult mammals, where it is expressed as a membrane protein on oligodendrocytes and in myelin. Here we describe an attempt to identify linear peptide epitopes in its sequence that are responsible for the interaction either with the Nogo receptor (NgR) or with the neutralizing monoclonal antibody IN-1. Analysis of an array of immobilized overlapping 15 mer peptides covering the entire amino acid sequence of human Nogo-A (1192 residues) revealed a single epitope with prominent binding activity both towards the recombinant NgR and the IN-1 F(ab) fragment. Further truncation and substitution analysis yielded the minimal epitope sequence 'IKxLRRL' (x not equal to P), which occurs within the so-called Nogo66 region (residues 1054-1120) of Nogo-A. The bacterially produced Nogo66 fragment exhibited binding activity both for the recombinant NgR and for the IN-1 F(ab) fragment on the Western blot as well as in ELISA. Unexpectedly, the synthetic epitope peptide and the recombinant Nogo66 showed cross-reactivity with the 8-18C5 F(ab) fragment, which is directed against myelin oligodendrocyte glycoprotein (MOG) as a structurally unrelated target. On the other hand, the recombinant N-terminal domain of Nogo-A (residues 334-966) was shown to specifically interact on the Western blot and in an ELISA with the IN-1 F(ab) fragment but not with the recombinant NgR, which is in agreement with previous results. Hence, our data suggest that there is a distinct binding site for the Nogo receptor in the Nogo66 region of Nogo-A, whereas its interaction with NgR is less specific than anticipated before. Although there probably exists a non-linear epitope for the neutralizing antibody IN-1 in the N-terminal region of Nogo-A, which is likely to be accessible from outside the cell, a previously postulated second binding site for NgR in this region (called Nogo-A-24) remains elusive.     

Pharmacokinetics of a radioiodinated monoclonal antibody F(ab′)2 fragment in a xenograft model with circulating antigen

The pharmacokinetics of ¹³¹I-labeled OC 125 F(ab′)₂ antibody fragment were investigated in athymic mice bearing OVCAR-3 ovarian carcinoma xenografts, a model in which the CA 125 antigen is present in serum. Nine antibody doses between 0.1 and 650 μg were studied. Optimal tumor to normal tissue ratios were obtained at 100–200 μg of F(ab′)₂. At most antibody doses, the pre-injection level of circulating CA 125 appeared to influence the localization of ¹³¹I activity in tumor, liver and spleen.     

广西甜茶苷柱纯化工艺研究

以含70%的广西甜茶粗提取物为原料,通过柱纯化与重结晶对甜茶苷进行提纯,从而制备高纯度的甜茶苷单体。以聚酰胺和LSA-10大孔吸附树脂树脂为吸附剂乙醇为洗脱剂进行柱纯化。实验表明,比较聚酰胺与大孔吸附树脂的吸附与脱附能力,皆为后者强;比较不同吸附材料处理所得甜茶苷单体的纯度,则是聚酰胺所吸附的样纯度高,特别是25%乙醇的处理样。     

Infarcted heart uptake and biodistribution of radiolabelled anti-myosin monoclonal antibody in rat and dog myocardial infarct models

A new mouse monoclonal antibody that recognizes α- and β-heavy chains of human atrial and ventricular myosin and β-heavy chain of human slow skeletal muscle myosin was obtained. The ¹²⁵I- and ¹¹¹In-labelled antibody, and its F(ab′)₂ and Fab fragments localize in isoproterenol induced infarcted rat heart, with the F(ab′)₂ fragment showing the highest uptake. Comparison with ^99mTc-pyrophosphate uptake in infarcted dog heart, induced by selective obstruction of a coronary artery, suggest that the ¹¹¹In-labelled F(ab′)₂ localizes specifically in infarcted myocardium only.     

Expanded bed protein A affinity chromatography of a recombinant humanized monoclonal antibody: process development, operation, and comparison with a packed bed method.

We show that expanded bed protein A affinity chromatography using Streamline rProtein A media is an efficient method for purifying a recombinant humanized monoclonal antibody from unclarified Chinese hamster ovary cell culture fluid and that it provides purification performance comparable to using a packed bed. We determined that the dynamic capacity of the expanded bed media is related to flow rate (measured in column volumes per hour) by a power function, which allows a high capacity at a low flow rate. At 250 cm h-1 with a 25 cm bed height (10 column volumes h-1), the dynamic capacity is 30 g l-1. The yield and purity (measured by the amount of host cell proteins, DNA, SDS-PAGE, and turbidity) of the antibody purified by expanded bed is comparable to the yield and purity obtained on a standard packed bed method using Prosep A media.     

Interaction of GroE with an all-beta-protein.

Molecular chaperones are involved in protein folding both in vivo and in vitro. The Escherichia coli chaperone GroEL interacts with a number of nonnative proteins. A common structural motif of nonnative proteins, which is recognized by GroEL, has not yet been identified. In order to study the role of beta-sheet secondary structure on the interaction of nonnative proteins with GroEL, we used the F(ab) fragment of a monoclonal antibody as a model substrate protein. Here we show that GroEL interacts functionally with this all-beta-protein during reactivation. Antibody fragments refold spontaneously in good yield from the guanidine-denatured state. Functional refolding to the native state is inhibited transiently by GroEL, but there is no complete folding arrest in the absence of Mg-ATP and GroES. The yield of these unspecifically released GroEL-bound F(ab) fragments corresponds to that of the spontaneous reactivation in the absence of chaperones. However, the refolding kinetics in the presence of GroEL are considerably slower. The addition of Mg-ATP to the GroEL.F(ab) complex results in an immediate release of bound substrate protein and a significant increase in the amount of reconstituted antibody fragments compared to spontaneous reactivation. GroES is not essential for functional GroEL-mediated refolding of the F(ab) fragment but affects the reactivation yield to a small extent. Interestingly, stimulation of the GroEL-mediated F(ab) refolding depends primarily on the binding and not on hydrolysis of adenosine triphosphates. Previous results indicate the binding of alpha-helices to GroEL. The results presented in this paper suggest that beta-sheet secondary structural elements are recognized by GroEL. We therefore conclude that the interaction of a nonnative protein with GroEL depends mainly on the nature of the early folding intermediate but not on a specific element of secondary structure.     

Exploitation of the adsorptive properties of depth filters for host cell protein removal during monoclonal antibody purification

Depth filtration has been widely used during process scale clarification of cell culture supernatants for the removal of cells and cell debris. However, in addition to their filtration capabilities, depth filters also possess the ability to adsorb soluble species. This aspect of depth filtration has largely not been exploited in process scale separations and is usually ignored during cell culture harvest development. Here, we report on the ability of depth filters to adsorptively remove host cell protein contaminants from a recombinant monoclonal antibody process stream and characterize some of the underlying interactions behind the binding phenomenon. Following centrifugation, filtration through a depth filter prior to Protein A chromatographic capture was shown to significantly reduce the level of turbidity observed in the Protein A column eluate of the monoclonal antibody. The Protein A eluate turbidity was shown to be linked to host cell protein contaminant levels in the Protein A column load and not to the DNA content. Analogous to flowthrough chromatography in which residence time/bed height and column loading are key parameters, both the number of passes through the depth filter and the amount of centrifuge centrate loaded on the filter were seen to be important operational parameters governing the adsorptive removal of host cell protein contaminants. Adsorption of proteins to the depth filter was shown to be due to a combination of electrostatic and hydrophobic adsorptive interactions. These results demonstrate the ability to employ depth filtration as an integrative unit operation combining filtration for particulate removal with adsorptive binding for contaminant removal.