Purification Of Antibodies Research Articles

Application of microfluidic technology in antibody screening.

Specific antibodies are widely used in the biomedical field. Current screening methods for specific antibodies mainly involve hybridoma technology and antibody engineering techniques. However, these technologies suffer from tedious screening processes, long preparation periods, high costs, low efficiency, and a degree of automation, which have become a bottleneck for the screening of specific antibodies. To overcome these difficulties, microfluidics has been developed as a promising technology for high-throughput screening and high purity of antibody. In this review, we provide an overview of the recent advances in microfluidic applications for specific antibody screening. In particular, hybridoma technology and four antibody engineering techniques (including phage display, single B cell antibody screening, antibody expression, and cell-free protein synthesis) based on microfluidics have been introduced, challenges, and the future outlook of these technologies are also discussed.

Plasmapheresis for a Female with Cryptogenic New-Onset Refractory Status Epilepticus Not Benefiting from Monoclonal Antibody Therapy

Monoclonal antibodies are pure antibodies that react to a specific epitope. Plasmapheresis is a treatment that separates and eliminates disease-causing substances before replacing the blood with plasma. Plasmapheresis has insufficient evidence for treating new-onset refractory status epilepticus (NORSE). Sequential plasmaphereses gradually improved a female cryptogenic NORSE patient who did not benefit from monoclonal antibody treatment.

Comparison of anti-peptide and anti-protein antibody-based purification techniques for detection of SARS-CoV-2 by targeted LC-MS/MS

The COVID-19 pandemic has necessitated exploration of alternative testing methods for detection of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) to ensure clinical laboratories can continue to provide critical testing results. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is established in many clinical laboratories due its high specificity and sensitivity, making it a logical alternative methodology. However, matching the sensitivity of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) remains challenging, which forced utilization of antibody-based enrichment prior to targeted LC-MS/MS analysis. When utilizing antibody purification techniques, investigators must decide whether to enrich the target protein or peptides, but there are few studies comparing the two approaches to assist in this decision-making process. In this work, we present a comparison of intact protein and peptide antibody-based purification for LC-MS/MS based detection of SARS-CoV-2. We have found that protein purification yields more intense LC-MS/MS signals, but is also less specific, yielding higher noise and more background when compared to peptide purification techniques. Therefore, when using traditional data analysis techniques, the enrichment technique that provides superior sensitivity varies for individual peptides and no definitive overall conclusion can be made. These observations are corroborated when using a novel machine learning approach to determine positive/negative test results, which yielded superior sensitivity when using protein purification, but better specificity and area under the ROC curve when performing peptide purification.

Purification and determination of antibody drugs in bio-samples by EGFR/cell membrane chromatography method

With the rapid development of therapeutic monoclonal antibody drugs, it is increasingly difficult to meet clinical needs using traditional antibody purification techniques. In this study, epidermal growth factor receptor (EGFR)-SNAP-tag was expressed in HEK293 cells. Then we captured the EGFR-SNAP-tag from the cell membrane suspension onto a O6-benzylguanine-modified silica gel to prepare a new EGFR stationary phase as a bioactive material, which could specifically recognize its antibody in bio-samples. The EGFR stationary phase was systematically characterized via scanning electron microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, and fourier transform infrared spectroscopy. Then we used EGFR stationary phase to establish a new EGFR cell membrane chromatography (CMC) model. The EGFR/CMC-online-ion exchange chromatography (IEC)/high performance liquid chromatography (HPLC) was established for the efficient purification and specific identification of cetuximab, nituzumab, and panizumab from cell culture medium and human serum. The results show that the EGFR stationary phase prepared by one-step immobilized technique can maintain biological activity and stability like EGFR in cell membrane. The EGFR/CMC-online-IEC/HPLC method has a high specificity, accuracy and sensitivity. Therefore, it will present a valuable method for the purification, identification, and analysis of monoclonal antibody drugs.

Affinity Sedimentation and Magnetic Separation With Plant-Made Immunosorbent Nanoparticles for Therapeutic Protein Purification.

The virus-based immunosorbent nanoparticle is a nascent technology being developed to serve as a simple and efficacious agent in biosensing and therapeutic antibody purification. There has been particular emphasis on the use of plant virions as immunosorbent nanoparticle chassis for their diverse morphologies and accessible, high yield manufacturing via plant cultivation. To date, studies in this area have focused on proof-of-concept immunosorbent functionality in biosensing and purification contexts. Here we consolidate a previously reported pro-vector system into a single Agrobacterium tumefaciens vector to investigate and expand the utility of virus-based immunosorbent nanoparticle technology for therapeutic protein purification. We demonstrate the use of this technology for Fc-fusion protein purification, characterize key nanomaterial properties including binding capacity, stability, reusability, and particle integrity, and present an optimized processing scheme with reduced complexity and increased purity. Furthermore, we present a coupling of virus-based immunosorbent nanoparticles with magnetic particles as a strategy to overcome limitations of the immunosorbent nanoparticle sedimentation-based affinity capture methodology. We report magnetic separation results which exceed the binding capacity reported for current industry standards by an order of magnitude.

Virus clearance by activated carbon for therapeutic monoclonal antibody purification

In our previous study, activated carbon (AC) was employed in the purification process of therapeutic monoclonal antibody (mAb) as a replacement for Protein A affinity chromatography. In addition, we established an innovative column-free flow-through purification process using AC filter. In these investigations, the effective clearance of impurities (high-molecular-weight species, low-molecular-weight species, host cell proteins, and DNA) was observed compared to the conventional Protein A platform purification process. In this study, virus removal capability of our established AC process (AC filter) was investigated using two model viruses, Murine Leukemia Virus (MuLV) and Minute Virus of Mice (MVM) with the combination of two filtration methods (single-pass filtration and re-circulation filtration) using three kinds of mAbs. We found effective clearance of both MuLV and MVM (>3 log reduction factor, LRF) in all mAbs. Not only filtration method but also re-circulation duration didn’t affect LRF, and >3 LRF of virus removal could be achieved by only single-pass filtration. From these results, it is expected that AC will be a promising candidate for the virus removal unit operation for mAb purification processes.

More Than Meets the Kappa for Antibody Superantigen Protein L (PpL).

Immunoglobulin superantigens play an important role in affinity purification of antibodies and the microbiota-immune axis at mucosal areas. Based on current understanding, Staphylococcal Protein A (SpA), Streptococcal Protein G (SpG) and Finegoldia Protein L (PpL) are thought to only bind specific regions of human antibodies, allowing for selective purification of antibody isotypes and chains. Clinically, these superantigens are often classified as toxins and increase the virulence of the producing pathogen through unspecific interactions with immune proteins. To perform an in-depth interaction study of these three superantigens with antibodies, bio-layer interferometry (BLI) measurements of their interactions with a permutation panel of 63 IgG1 variants of Pertuzumab and Trastuzumab CDRs grafted to the six human Vκ and seven human VH region families were tested. Through this holistic and systemic analysis of IgG1 variants with various antibody regions modified, comparisons revealed novel PpL–antibody interactions influenced by other non-canonical antibody known light-chain framework regions, whereas SpA and SpG showed relatively consistent interactions. These findings have implications on PpL-based affinity antibody purification and design that can guide the engineering and understanding of PpL-based microbiota-immune effects.

Dry-compression packing of hydroxyapatite nanoparticles within a flat cuboid chromatography device and its use for fast protein separation

We describe and discuss a simple dry-compression technique for preparing a flat cuboid chromatography device containing a shallow packed-bed of crystalline hydroxyapatite nanoparticles. We then discuss the use of this device for fast protein separation in the bind-and-elute mode. Such separation could be carried out at quite low pressures, making it possible to use inexpensive low pressure chromatography systems. In the flow rate range examined in this study, the pressure-drop across the device increased linearly with flow rate, indicating negligible media compaction during use. Using this device, binary protein mixtures could be separated in about a minute. Contrary to that observed in most packed-bed chromatographic separations, the width of the flow through and eluted peaks decreased with increase in flow rate. Therefore, both productivity and purity could be simultaneously increased by increasing flow rate. The suitability of this device for preparative protein separations was demonstrated by carrying out purification of a monoclonal antibody (Trastuzumab) from mammalian cell culture supernatant. This study opens up the possibility of developing dry-compression based flat cuboid packed-bed chromatography devices for fast preparative protein separation.

Rapid monitoring of high-mannose glycans during cell culture process of therapeutic monoclonal antibodies using lectin affinity chromatography.

We developed a simple high‐performance liquid chromatography assay to monitor high‐mannose glycans in monoclonal antibodies by monitoring terminal alpha‐mannose as a surrogate marker. Analysis of glycan data of therapeutic monoclonal antibodies by 2‐aminobenzamide assay showed a linear relationship between high mannose and terminal mannose of Fc glycans. Concanavalin A has a strong affinity to alpha‐mannose in glycans of typical therapeutic monoclonal antibodies. To show that terminal mannose binds specifically to Concanavalin A column, exoglycosidase‐treated monoclonal antibodies were serially blended with untreated monoclonal antibodies. Linear responses of terminal‐mannose binding to the column and comparable data trending with high mannose levels by 2‐aminobenzamide assay confirmed that terminal‐mannose levels measured by the Concanavalin A column can be used as a surrogate for the prediction of high‐mannose levels in monoclonal antibodies. The assay offers a simple, fast, and specific capability for the prediction of high‐mannose content in samples compared with traditional glycan profiling by 2‐aminobenzamide or mass spectrometry‐based methods. When the Concanavalin A column was coupled with protein A column for purification of antibodies from cell culture samples in a fully automated two‐dimensional analysis, high‐mannose data could be relayed to the manufacturing team in less than 30 min, allowing near‐real‐time monitoring of high‐mannose levels in the cell culture process.

Optimization of anion exchange purification for the large-scale production of plasmid DNA for gene therapy and DNA vaccine applications

Watch the video or read the poster to learn: Considerations for pDNA purification How to prepare and implement a DoE approach to identify optimal conditions for various chromatography mediaHow to optimize recovery and impurity removal in the pDNA capture stepHow to achieve a scalable and flexible pDNA purification processDr Jenny England is a Staff Scientist in the Applications and Innovation group in Purification and Pharma Analytics at Thermo Fisher Scientific. Jenny is a biophysicist by training and earned her PhD from Georgetown University. After graduate school, she did a post doc at the National Cancer Institute that focused on structure-based drug design for protein kinase complexes. Jenny currently leads the application group for process development of the POROS resin products for antibody, mRNA, plasmid, and viral vector purification. Additionally, Jenny evaluates new and emerging technologies that can be applied to solve unmet customer needs in the bioproduction workflow.

Mode and dosage time in polyethylene glycol precipitation process influences protein precipitate size and filterability

Precipitation has gained interest as alternative to the costly protein A chromatography for monoclonal antibody purification. Traditional precipitation processes are based on direct addition of precipitant in a single dose, with limited control on co-precipitation of impurities and not considering batch-to-batch variations. We propose a gradual dosage of polyethylene glycol to prevent co-precipitation and control resulting floc size. We used focused beam reflectance measurement to demonstrate that the PEG6000 dosage time and the final concentration significantly changes the particle size distribution (PSD). We demonstrated that gradual and stepwise precipitant addition was superior to conventional batch PEG precipitation, improving product yield and purity by a factor of 4 for HCP removal, for samples pre-treated with CaCl2 and caprylic acid. We studied the 3D structure of the precipitates by fractal dimension and showed that precipitates exhibited different compactness and density depending on the dosage time, resulting in different filterability in tangential flow filtration and depth filtration. To switch from batch to continuous PEG addition, the 3D structure of precipitates needs to be considered due to its high impact on the resulting process performance. Focused beam reflectance measurement (FBRM) and fractal dimension can be used to adjust the precipitation methodology to improve the product quality attributes and inform about the design of further purification steps.

Development of the ELISA Test System for Quantitative Determination of IgG to the Varizella zoster virus in Human Serum and Assessment of its Diagnostic Efficiency

Relevance. The introduction of Varicella vaccine prophylaxis explains the need to develop a methodology for monitoring the vaccination effectiveness and the intensity of population immunity. This problem can be solved using quantitative immunoassay methods. Aim. Development of an enzyme-linked immunosorbent assay for the concentration of class G immunoglobulins (AB) to Varicella zoster virus (VZV) determining and assessing its functional characteristics and diagnostic efficiency. Materials and methods. Recombinant antigen GE VZV. WHO International Standard for Antibodies to VZV W1044. Blood serum samples from healthy people and patients with Chickenpox and Herpes zoster, blood serum samples containing IgG antibodies to herpes simplex viruses of the first and second types, cytomegalovirus, Epstein-Barr virus. Anti-VZV ELISA (IgG) reagent kit (Euroimmun, Germany). Indirect enzyme-linked immunosorbent assay. Immunization of animals with recombinant antigen GE, isolation, and purification of specific antibodies. Conjugation of monoclonal antibodies to human IgG with antibodies to antigen GE and with horseradish peroxidase. Results. An enzyme-linked immunosorbent assay in «an indirect» format has been developed to determine the specific antibodies to VZV concentration (IU/ml) in human serum/plasma. An artificial calibrator for determining the concentration of AB-VZV had been synthesized and standardized according to the International WHO-standard W1044. The main functional characteristics of the developed enzyme-linked immunosorbent assay are determined in accordance with GOST 51352-2013. The diagnostic kit was tested on blood serum samples from children with chickenpox (n = 43), adults with Herpes zoster (n = 158), healthy individuals (n = 781). The diagnostic sensitivity of the test system was 85%, the diagnostic specificity was 87% according to the ROC analysis. The absence of cross-reactivity of the test system was shown on samples with serological markers of other herpesvirus infections (n = 94). Comparative trials of the developed test system and its commercial analog, the Anti-VZV ELISA (IgG) reagent kit, did not reveal statistically significant differences between their functional characteristics. Conclusions. The developed test system for determining of the AB-VZV concentration in human serum/plasma in terms of its functional characteristics meets the GOST requirements, is characterized by high diagnostic efficiency, can be used to monitor the effectiveness of vaccine prophylaxis and strength of population immunity, as well as to assess the immune response in chickenpox and Herpes zoster.

Antibody Purification Using Affinity Chromatography.

Antibodies are an integral part of many biological assays and biotherapeutics. However, the sources from which antibodies are derived frequently contain other contaminants which may interfere with assays or cause adverse reactions if administeredin vivo. Therefore, a means of isolating these antibodies from their source at high levels of purity is critical. Affinity chromatography is currently one of the most widely applied methods for the purification of antibodies. This method relies on specific and reversible, interactions between antibody structures, or recombinant tags fused to these structures, and ligands immobilized on solid support matrices, generally within a column. Herein, common chromatographic methods applied to antibody purification are described. These include the purification of IgG, and its recombinant forms, through protein A, protein G and immobilized metal affinity chromatography.

Profiling Protein-DNA Interactions Cell-Type-Specifically with Targeted DamID.

Targeted DamID (TaDa) is a means of profiling the binding of any DNA-associated protein cell-type specifically, including transcription factors, RNA polymerase, and chromatin-modifying proteins. The technique is highly sensitive, highly reproducible, requires no mechanical disruption, cell isolation or antibody purification, and can be performed by anyone with basic molecular biology knowledge. Here, we describe the TaDa method and downstream bioinformatics data processing.

Affinity Purification of PfEMP1-Specific Antibodies from Human Blood.

Acquired immunity against Plasmodium falciparum infections relies heavily on IgG antibodies specific for PfEMP1 proteins expressed on the surface of infected erythrocytes. Purified human antibodies can be used, for example, to study the interactions between specific PfEMP1 proteins and receptors expressed by human endothelial cells, and to identify which IgG antibodies play a functional role in natural acquired immunity.This chapter describe how to affinity purify PfEMP1-specific human antibodies on an affinity column coupled with PfEMP1 protein. We include ELISA-based methods for identification of human plasma samples reactive against PfEMP1, and for testing of affinity purified IgG antibodies prior to their use in more advanced procedures.

Expression, purification and characterisation of a human anti-CDK4 single-chain variable fragment antibody

BackgroundCyclin-dependent kinase 4 (CDK4) when hyperactivated drives development and maintenance of most tumour types, thus prompting its use as an essential cancer treatment target and a diagnostic tool. Target-binding molecules, such as single-chain variable fragment (scFv) antibodies, hold tremendous potential for use in a wide range of cancer diagnostic and therapeutic applications.ResultsA human anti-CDK4 scFv antibody (AK2) derived from a human phage display library was expressed in soluble form in Escherichia coli and shown to be secreted into the culture supernatant. Next, soluble AK2 within culture supernatant was successfully purified using affinity chromatography then was shown, using enzyme-linked immunosorbent assays, to bind to recombinant human CDK4 with high affinity and specificity. Further analyses of AK2 interactions with intracellular components demonstrated that AK2 recognised and interacted specifically with endogenous CDK4 and thus could be useful for detection of CDK4 within tumour cells.ConclusionsA novel anti-CDK4 scFv antibody that can recognise and interact specifically with recombinant human CDK4 and endogenous CDK4 in tumour cells was expressed and purified successfully. These results suggest that the anti-CDK4 scFv antibody may serve as a new and promising tool for achieving CDK4-targeted diagnosis, prognosis and treatment of numerous types of cancers.

Purification of monoclonal antibody using cation exchange z2 laterally-fed membrane chromatography – A potential alternative to protein A affinity chromatography

Protein A affinity chromatography, which is the standard method for purifying monoclonal antibodies (mAbs) is expensive and involves elution at acidic pH, which could degrade the mAb. Moreover, it cannot be used to fractionate charge variants or to remove mAb aggregates. In this study, we examine the purification of Trastuzumab by cation-exchange z2 laterally-fed membrane chromatography (or z2LFMC). It has been shown that z2LFMC is suitable for carrying out high-speed, high-resolution protein purification. The results discussed in this paper demonstrate that the purity of Trastuzumab obtained by the z2LFMC process was comparable to that obtained by protein A chromatography (i.e., >95% with both methods), while the recovery was significantly greater with z2LFMC (90.4% as opposed to 81.7%). Also, the z2LFMC process was faster, did not require acidic pH conditions for elution, and the mAb was eluted in 2.24 bed volumes as opposed to 2.74 bed volumes with protein A chromatography. The mAb productivity obtained with the z2LFMC process was more than 3 times higher than that with the column-based purification process. The z2LFMC device is significantly cheaper than its equivalent protein A affinity column, and earlier studies have shown it to be suitable for separating mAb charge variants and aggregates. Therefore, the z2LFMC process could be evaluated as a cheaper and more efficient alternative to protein A affinity chromatography.

Application of novel mixed mode chromatography (MMC) resins having a hydrophobic modified polyallylamine ligand for monoclonal antibody purification

Polyallylamine (PAA) has been utilized as a salt tolerant anion exchange chromatography ligand in downstream processing of biopharmaceuticals. We have developed novel MMC resins based on PAA polymer ligand partially modified with hydrophobic butyl or phenyl group. The resulting hydrophobic modified PAA ligand reduced HCP level to 12% (21–23 ppm) under 6 mS/cm in a flow-through polishing step of mAb, while not modified PAA ligand showed only 79% (145 ppm). We also found that structure of hydrophobic groups in the ligand mainly influenced on mAb yield. That is 25% increase of phenyl group modification ratio reduces mAb yield from 95% to 90%. On the other hand, modification with butyl group kept mAb yield more than 95%. The optimized ligand structure displayed a wide operational conductivity range. Extended purification studies of mAb using the MMC resin in the flow-through polishing step were carried out under optimized pH and conductivity condition as determined in a DOE study. The study revealed that the MMC resin was effective for developing one-step flow-through polishing workflow for mAb purification. In addition, the MMC flow-through polishing step could be directly coupled with a specified CEX chromatography step to efficiently remove mAb aggregates from 2.3% to <1.0% to achieve a biopharmaceutical-grade quality and a high yield of mAb (>93%) with a high loading capacity around 1000 mg/mL-resin. This new MMC resin will be useful in future mAb manufacturing platforms comprising of a robust and cost-effective flow-through polishing step.

Expression, purification and characterization of anti-FGF7 domain antibody identified using phage display technique

Background: Fibroblast growth factors (FGFs) are involved in angiogenesis, wound healing and embryonic development. However, one of the causes of cancer cell growth in fibroblast-dependent cancers is FGF7 secreted by fibroblasts. Therefore, antibodies against FGF7 can be used for treatment of these types of cancers. Methods: In previous studies, a phage displaying single domain antibody, D53, against human FGF7 has been identified using the phage display technique. In the present study, D53 was produced and purified in its isolated form. ELISA experiment was performed to evaluate the binding of D53 to FGF7. The mode of interaction of D53-FGF7 was explored using docking study and molecular dynamics (MD) simulations. Results: The expression and purification processes were verified using western blotting and SDS-PAGE analyses. ELISA experiment showed that D53 is able to recognize and bind FGF7. Docking study and MD simulations indicated that compared to dummy VH, D53 has more affinity towards FGF7. Conclusion: The findings in the current study can be useful for generation and development of FGF7 inhibitors with potential use in fibroblast-dependent cancers.

Peptide Affinity Chromatography Applied to Therapeutic Antibodies Purification.

The interest in therapeutic monoclonal antibodies (mAbs) has significantly grown in the pharmaceutical industry, exceeding 100 FDA mAbs approved. Although the upstream processing of their industrial production has been significantly improved in the last years, the downstream processing still depends on immobilized protein A affinity chromatography. The high cost, low capacity and short half-life of immobilized protein A chromatography matrices, encouraged the design of alternative short-peptide ligands for mAb purification. Most of these peptides have been obtained by screening combinatorial peptide libraries. These low-cost ligands can be easily produced by solid-phase peptide synthesis and can be immobilized on chromatographic supports, thus obtaining matrices with high capacity and selectivity. Furthermore, matrices with immobilized peptide ligands have longer half-life than those with protein A due to the higher stability of the peptides. In this review the design and synthesis of peptide ligands, their immobilization on chromatographic supports and the evaluation of the affinity supports for their application in mAb purification is described.