Purification Of Antibodies Research Articles

Sumateran wild boar (Sus scrofa vittatus) meat antibody production as immunodiagnostic reagent candidate.

Aim:Meat authentication gives significance values in view of religious, food safety, public health, quality assurance, and legal concern. Most of the meat authentication is based on molecular assay; a simpler method to authenticate meat is needed to develop. An immunoassays technique may offer a solution for simpler test. The aim of our current study was to develop a polyclonal antibody of Sus scrofa vittatus (Sumateran wild boar) as an immunodiagnostic reagent candidate.Materials and Methods:Three male New Zealand white rabbits were used in this study for antibody production. Antigen used was meat extract of Sumateran wild boar, each rabbit was immunized with meat extract antigen (0.5 mg/ml) emulsified in Freund’s complete adjuvant at a 1:1 (v/v) ratio as much as 1 ml at subcutaneous route. Booster was carried out 3 times with interval time of 14 days, using meat extract antigen emulsified in Freund’s incomplete adjuvant at a 1:1 (v/v) ratio. Serum samples were taken every week, start from 1 week after the first immunization up to 1 week after the third booster. Antibody purification was performed using ammonium sulfate precipitation and Protein A. The presence of specific antibody was determined using agar gel precipitation test and enzyme-linked immunosorbent assay, while purified specific IgG was characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis method.Results:Specific antibody was detected at 14 days after the first immunization and still detected until 2 weeks after the third booster. Highest absorbance of specific antibody was detected 1 week after the third booster.Conclusion:The present study demonstrated that specific antibody of Sumateran wild boar is favorable to be produced in rabbit and showed that antibody produced is applicable to detect Sumateran wild boar meat antigen in immunodiffusion assay, indicating that it is promising as a reagent candidate in immunodiagnostic assay/kit.

Expression of the recombinant single chain variable fragments recognizing blood antigen fused with thioredoxin in <i> Escherichia coli </i>

The technology of recombinant single chain variable fragments (scFvs) expression has been used in research, diagnosis and treatment of diseases. In the previous study, we studied the expression of a recombinant single chain variable fragment recognizing blood A antigen (antiA-scFv) in E. coli. However, the protein was insoluble form resulting in difficulty for purification, refolding and activity assesment. Here, we present the study on fused expression of the recombinant scFv -specific blood A antigen with thioredoxin (Trx) in the expression vector pET32a (+). The results showed that the Trx/antiA-scFv fusion protein was expressed with molecular weight of 49 kDa in a soluble form reaching 40% of the total recombinant protein. This result facilitates the optimal condition of soluble protein expression, purification and bioactivity determination of the antiA-scFv recombinant antibody.

Deamidation Can Compromise Antibody Colloidal Stability and Enhance Aggregation in a pH-Dependent Manner.

Monoclonal antibodies must be both chemically and physically stable to be developed into safe and effective drugs. Although there has been considerable progress in separately understanding the molecular determinants of antibody chemical and physical stability, it remains poorly understood how defects in one property (e.g., chemical stability) impact the other property (e.g., physical stability). Here, we have investigated the impact of a common chemical modification (deamidation) on the physical stability of two monoclonal antibodies as a function of pH (from pH 3.8 to 7.4). Interestingly, we find that deamidation has significant, antibody-specific impacts on physical stability at low pH values that are common during antibody purification. Deamidation causes increases in self-association and/or aggregation at low pH (3.8), and a key contributor to this behavior appears to be deamidation-dependent increases in antibody hydrophobicity at low pH. Our findings highlight pH-dependent impacts of deamidation on antibody colloidal stability and aggregation, which are important to understand in order to improve the development and production of potent antibody therapeutics with high chemical and physical stabilities.

Optimizing high throughput antibody purification by using continuous chromatography media

The ability to engineer monoclonal antibodies (mAbs) with high specificity made mAbs the fastest growing segment in the drug market. mAbs represent 8 of the top 20 selling drugs with combined sales of more than 57 billion US$ per year. The ability to purify large numbers of mAbs with sufficient yields for initial screening campaigns has direct impact on the timelines of a project. Automated liquid handling (ALH)-based mAb purification platforms have been used to facilitate the production of large numbers of mAbs. However, the ongoing pressure to de-risk potential lead molecules at an early development stage by including bio-physical characterization of mAbs has further increased the demand to produce sufficient quantities from limited sample volumes. A bottleneck so far has been the limited dynamic binding capacity of these systems, which is partly due to the binding properties of commonly used Protein A affinity matrices. The present publication suggests that by using a Protein A matrix optimized for continuous chromatography applications the yields of ALH-based but also standard lab-scale mAb purifications can be significantly increased without the need to change established protocols.

Protein A chromatography: Challenges and progress in the purification of monoclonal antibodies.

Antibodies for therapeutic use are being continuously approved and their demand has been steadily growing. As known, the golden standard for monoclonal antibody (mAb) purification is Protein A affinity chromatography, a technology that has gained high interest because of its great performance and capabilities. The main concerns are the elevated resins costs and their limited lifetime compared to other resins (e.g. ion exchange chromatography). Great efforts have been carried out to improve purification conditions, such as resin characterization and designing alkali/acid stable resins with a longer lifetime. Modification of Protein A ligands and alternative formats such as monoliths membranes and microshperes have been tested to increase the purification performance. New technology has been proposed to improve the large-scale separation; in addition, alternative ligands have been suggested to capture mAbs instead of Protein A ligand; however, most of the information is locked by pharmaceutical companies. This mini review summarizes and describes the advances, results, and impact on the Protein A chromatography purification processing.

Bacterially Derived Antibody Binders as Small Adapters for DNA-PAINT Microscopy.

Current optical super-resolution implementations are capable of resolving features spaced just a few nanometers apart. However, translating this spatial resolution to cellular targets is limited by the large size of traditionally employed primary and secondary antibody reagents. Recent advancements in small and efficient protein binders for super-resolution microscopy, such as nanobodies or aptamers, provide an exciting avenue for the future; however, their widespread availability is still limited. To address this issue, here we report the combination of bacterial-derived binders commonly used in antibody purification with DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy. The small sizes of these protein binders, relative to secondary antibodies, make them an attractive labeling alternative for emerging superresolution techniques. We present here a labeling protocol for DNA conjugation of bacterially derived proteins A and G for DNA-PAINT, having assayed their intracellular performance by targeting primary antibodies against tubulin, TOM20, and the epidermal growth factor receptor (EGFR) and quantified the increases in obtainable resolution.

Ionic Liquid Aqueous Two-Phase Systems From a Pharmaceutical Perspective.

Aqueous Two-Phase Systems (ATPSs) have been extensively studied for their ability to simultaneously separate and purify active pharmaceutical ingredients (APIs) and key intermediates with high yields and high purity. Depending on the ATPS composition, it can be adapted for the separation and purification of cells, nucleic acids, proteins, antibodies, and small molecules. This method has been shown to be scalable, allowing it to be used in the milliliter scale for early drug development to thousands of liters in manufacture for commercial supply. The benefits of ATPS in pharmaceutical separations is increasingly being recognized and investigated by larger pharmaceutical companies. ATPSs use identical instrumentation and similar methodology, therefore a change from traditional methods has a theoretical low barrier of adoption. The cost of typical components used to form an ATPS at large scale, particularly that of polymer-polymer systems, is the primary challenge to widespread use across industry. However, there are a few polymer-salt examples where the increase in yield at commercial scale justifies the cost of using ATPSs for macromolecule purification. More recently, Ionic Liquids (ILs) have been used for ATPS separations that is more sustainable as a solvent, and more economical than polymers often used in ATPSs for small molecule applications. Such IL-ATPSs still retain much of the attractive characteristics such as customizable chemical and physical properties, stability, safety, and most importantly, can provide higher yield separations of organic compounds, and efficient solvent recycling to lower financial and environmental costs of large scale manufacturing.

Adsorption Characteristics of Human Immunoglobulin G on Five New Tetrapeptide Biomimetic Affinity Resins

Alternative antibody-binding affinity ligands are expected to support robust time- and cost-effective antibody purification with rapid improvement of upstream productivity and demand on downstream process optimization. Five new tetrapeptide biomimetic affinity ligands (FYRH, HWRH, YHRI, HYRF, and FHRA) were screened by modeling the molecular interaction mechanism between tetrapeptide ligands and the Fc fragment of IgG with molecular dynamics simulation and the flexible docking method, and the corresponding five resins were prepared in this work to evaluate their adsorption characteristics of human immunoglobulin G (hIgG). The results showed that the binding capacities to hIgG of the five resins showed a strong pH-dependency, which was high at pH 7.0–9.0, and extremely low at pH 5.0–6.0, providing a benefit choice to separate hIgG. The dynamic binding capacities (DBC) at 10% breakthrough of five resins could reach up to 28–37 mg/mLgel, and Ac-FYRH-4FF resin showed the best adsorption performance. The eluti...

High throughput process development workflow with advanced decision-support for antibody purification

Chromatography remains the workhorse in antibody purification; however process development and characterisation still require significant resources. The high number of operating parameters involved requires extensive experimentation, traditionally performed at small- and pilot-scale, leading to demands in terms of materials and time that can be a challenge. The main objective of this research was the establishment of a novel High Throughput Process Development (HTPD) workflow combining scale-down chromatography experimentation with advanced decision-support techniques in order to minimise the consumption of resources and accelerate the development timeframe. Additionally, the HTPD workflow provides a framework to rapidly manipulate large datasets in an automated fashion. The central component of the HTPD workflow is the systematic integration of a microscale chromatography experimentation strategy with an advanced chromatogram evaluation method, design of experiments (DoE) and multivariate data analysis. The outputs of this are leveraged into the screening and optimisation components of the workflow. For the screening component, a decision-support tool was developed combining different multi-criteria decision-making techniques to enable a fair comparison of a number of CEX resin candidates and determine those that demonstrate superior purification performance. This provided a rational methodology for screening chromatography resins and process parameters. For the optimisation component, the workflow leverages insights provided through screening experimentation to guide subsequent DoE experiments so as to tune significant process parameters for the selected resin. The resulting empirical correlations are linked to a stochastic modelling technique so as to predict the optimal and most robust chromatographic process parameters to achieve the desired performance criteria.

Development of a hybrid biomimetic ligand with high selectivity and mild elution for antibody purification

A hybrid biomimetic ligand composed of three amino acid residues and a hydrophobic charge-induction group was developed and its performance of immunoglobulin G (IgG) purification from human serum was evaluated. The sequence of peptides of the hybrid ligand (Phenylalanine-Tyrosine-Glutamate-“5-amino-benzimidazole”, FYE-ABI) was designed and evaluated via molecular simulation, and the ligand was synthesized and coupled onto agarose beads. Adsorption isotherm results showed that the optimum IgG adsorption was found at pH 7.0 with saturated adsorption capacity of 91 mg/mL resin and dissociation constant of 1.6 µM. The hybrid biomimetic resin showed high separation purity of 99.0% and recovery of 91.5% when used in IgG separation from human serum albumin (HSA) containing solutions. Moreover, study on IgG separation from human serum showed that the hybrid biomimetic ligand could provide comparable IgG purity (93.9%) and recovery (88.9%), but much milder elution condition (pH 4.5) as those of Protein A affinity chromatography. The combination of biomimetic and hydrophobic charge-induction chromatography presented high selectivity and mild elution for antibody purification.

Continuous integrated antibody precipitation with two-stage tangential flow microfiltration enables constant mass flow.

Continuous precipitation is a new unit operation for the continuous capture of antibodies. The capture step is based on continuous precipitation with PEG6000 and Zn++ in a tubular reactor integrated with a two‐stage continuous tangential flow filtration unit. The precipitate cannot be separated with centrifugation, because a highly compressed sediment results in poor resolubilization. We developed a new two‐stage tangential flow microfiltration method, where part of the concentrated retentate of the first stage was directly fed to the second stage, together with the wash buffer. Thus, the precipitate was concentrated and washed in a continuous process. We obtained 97% antibody purity, a 95% process yield during continuous operation, and a fivefold reduction in pre‐existing high‐molecular‐weight impurities. For other unit operations, surge tanks are often required, due to interruptions in the product mass flow out of the unit operation (e.g., the bind/elute mode in periodic counter‐current chromatography). Our setup required no surge tanks; thus, it provided a truly continuous antibody capture operation with uninterrupted product mass flow. Continuous virus inactivation and other flow‐through unit operations can be readily integrated downstream of the capture step to create truly continuous, integrated, downstream antibody processing without the need for hold tanks.

Improvement of protein binding capacity of acrylic-acid-grafted fibers by polymer root-to-brush shift

A weak acid cation exchange fiber used for large-scale antibody purification was prepared by radiation-induced graft polymerization of acrylic acid (AA) onto a commercially available nylon-6 fiber. The AA-grafted fiber was post-treated by conditioning in NaOH aqueous solution at 298 K followed by immersion in water at 353 K to increase the protein binding capacity of the cation exchange fiber. A 2 g/L lysozyme solution buffered at pH 6.0 flowed through a column charged with a treated or untreated AA-grafted fiber at a space velocity of 20 h−1. The 10% dynamic binding capacity of the column charged with the former fiber of 200 mg/mL-column was 5.9-fold that of the column charged with the latter fiber, and 2.3-fold that (88 mg/mL-column) of a column charged with CM Sepharose™ Fast Flow (GE Healthcare).

Optimization of Sequence, Display, and Mode of Operation of IgG-Binding Peptide Ligands to Develop Robust, High-Capacity Affinity Adsorbents That Afford High IgG Product Quality.

This work presents the use of peptide ligand HWRGWV and its cognate sequences to develop affinity adsorbents that compete with Protein A in terms of binding capacity and quality of the eluted product. First, the peptide ligand was conjugated to crosslinked agarose resins (WorkBeads) at different densities and using different spacer arms. The optimization of ligand density and display resulted in values of static and dynamic binding capacity of 85 mg/mL and 65 mg/mL, respectively. A selected peptide-WorkBeads adsorbent was utilized for purifying Mabs from Chinese Hamster Ovary (CHO) cell culture supernatants. The peptide-WorkBeads adsorbent was found able to withstand sanitization with strong alkaline solutions (0.5 M NaOH). The purity of the eluted product was consistently higher than 95%, with logarithmic removal value (LRV) of 1.5 for host cell proteins (HCPs) and 4.0 for DNA. HCP clearance was significantly improved by adding a post-load washing step with either 0.1 M Tris HCl pH 9 or 1 M NaCl. The cognate peptide of HWRGWV, constructed by replacing arginine (R) with citrulline, further increased the HCP LRV to 2.15. The peptide-based adsorbent also showed a remarkable performance in terms of removal of Mab aggregates; unlike Protein A, in fact, HWRGWV was found to bind only monomeric IgG. Collectively, these results demonstrate the potential of peptide-based adsorbents as alternative to Protein A for the purification of therapeutic antibodies.

Purification of Antibodies: Diethylaminoethyl (DEAE) Chromatography.

Because IgG from most species (other than rodents) tends to have an isoelectric point around neutral, two approaches can be used when separating IgG using diethylaminoethyl (DEAE) resins. When serum containing antibodies is applied to DEAE at a slightly acidic pH, the IgG flows through the column while most other serum proteins bind to the DEAE. This method is best performed using a batch method. The DEAE beads can be kept in a disposable syringe containing a polypropylene frit, a glass reactor containing a coarse-sintered glass frit, or other suitable vessel. If the antibody solution is adjusted to a basic pH of 8-8.5, then IgG binds to the DEAE resin. After washing the column, the antibody is eluted by adding a buffer of increasing ionic strength to the column. Prepacked columns of many sizes are available for the isolation of antibodies by DEAE chromatography. Alternatively, DEAE medium can be swelled or prepped according to the manufacturer's instructions and a column can be poured when needed.

Protein A and Protein G Purification of Antibodies.

Protein A and Protein G are immunoglobulin-binding proteins expressed in Staphylococcus aureus and Streptococcus sp., respectively, that have been adapted for use in purifying large amounts of IgG. They are available covalently attached to affinity resins such as 4% cross-linked agarose, making them suitable for low-pressure antibody isolation. Protein A is not recommended for the isolation of mouse mAbs because it lacks affinity for mouse IgG1, or for the isolation of antibodies from sheep, goat, chicken, hamster, or rat. IgGs from most species bind to Protein G at near physiological pH and ionic strength with a higher affinity than IgG binding to Protein A. Therefore, the pH required to dissociate bound IgG is lower, resulting in the loss of activity for some antibodies. If this is observed, Protein A may be an alternative if the IgG from the species being isolated can be purified using Protein A. Neither Protein A nor Protein G can be used for the isolation of chicken antibodies.

Immunohistochemical Localization of Proteins in the Phloem: Problems and Solutions.

Immunolocalization of proteins in differentiated phloem cells is a challenging task given their special anatomy, organellar infrastructure, and the phloem tissue's heterogeneity. Incorporation of specific wall components in the thickened cell walls of phloem cells is often the source of unspecific labeling, leading to erroneous localization. Therefore, special care is required regarding generation and purification of specific antibodies. In addition, tissue preservation of phloem cells, which contain a high osmotic pressure in their functional state, is a very challenging task prone to various pitfalls. This chapter provides practical advice for cautious tissue preparation and antibody purification. Furthermore, methods that can be used to verify immunohistochemical localization data, such as promoter-reporter studies or activity tests, are discussed. Such confirmation experiments are essential for unambiguous determination of protein location in cells of the phloem.

Optimization of sample preparation condition for sodium dodecyl sulfate capillary electrophoresis of non-reduced monoclonal antibodies

Sodium dodecyl sulfate capillary electrophoresis has become the mainstream method for purity analysis of monoclonal antibodies because of its advantages of fast and high resolution. Sample preparation has a significant impact on the purity detection of non-reduced monoclonal antibodies. In order to optimize sample preparation, the purity of monoclonal antibodies of different types and batches in sample buffers with iodoacetamide and N-ethyl maleimide as sulfhydryl sealants and at pH 6.0-9.0 was investigated. It was found that in the two types of sulfhydryl sealants, the high pH sample buffer could affect the sealing effect of the sulfhydryl sealant and produce more antibody fragments. Conversely, under the low pH condition, the antibody fragments were fewer and the purities of monoclonal antibodies were higher. Therefore, the sample buffer with pH 6.0 is the optimal preparation condition for the purity detection of non-reduced monoclonal antibodies.

Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in <i> Escherichia coli </i>

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody.

Purification of Human Monoclonal Antibodies and Their Fragments.

This chapter summarizes the most common chromatographic mAb and mAb fragment purification methods, starting by elucidating the relevant properties of the compounds and introducing the various chromatography modes that are available and useful for this application. A focus is put on the capture step affinity and ion-exchange chromatography. Aspects of scalability play an important role in judging the suitability of the methods. The chapter introduces also analytical chromatographic methods that can be utilized for quantification and purity control of the product. In the case of mAbs, for most purposes the purity obtained using an affinity capture step is sufficient. Polishing steps are required if material of particularly high purity needs to be generated. For mAb fragments, affinity chromatography is not yet fully established, and the capture step potentially may not provide material of high purity. Therefore, the available polishing techniques are touched upon briefly. In the case of mAb isoform and bispecific antibody purification, countercurrent chromatography techniques have proven to be very useful and a part of this chapter has been dedicated to them, paying tribute to the rising interest in these antibody formats in research and industry.

Fully Automated 89Zr Labeling and Purification of Antibodies.

Dozens of monoclonal antibodies (mAbs) have been approved for clinical use, and hundreds more are under development. To support these developments and facilitate a personalized medicine approach, PET imaging and quantification of mAbs, after chelation with desferrioxamine B (DFO) and radiolabeling with 89Zr, has become attractive. Also, the use of 89Zr-mAbs in preclinical and clinical studies is expanding rapidly. Despite these rapid developments, 89Zr radiolabeling is still performed manually. Therefore, we aimed to develop a simple, fully automated, good-manufacturing-practice (GMP)-compliant production procedure for the 89Zr labeling of mAbs. Such procedures should increase the robustness and capacity of 89Zr-mAb production while minimizing the radiation dose to the operator. Here, the procedures for fully automated 89Zr-mAb production are described and applied to produce batches of 89Zr-DFO-N-suc-cetuximab and 89Zr-DFO-N-suc-rituximab suitable for clinical use. Both products had to meet the GMP-compliant quality standards with respect to yield, radiochemical purity, protein integrity, antigen binding, sterility, and endotoxin levels. Methods: Automated 89Zr labeling of mAbs was developed on a Scintomics GRP 2V module and comprised the following steps: reagent transfer to the 89Zr-containing reaction vial, mixing of the reagents followed by a 60-min reaction at room temperature to obtain optimal radiolabeling yields, and product purification using a PD-10 desalting column. Results: Radiochemical yields of 89Zr-DFO-N-suc-cetuximab and 89Zr-DFO-N-suc-rituximab were all more than 90% according to instant thin-layer chromatography. Isolated yields were 74.6% ± 2.0% and 62.6% ± 3.0% for 89Zr-DFO-N-suc-cetuximab and 89Zr-DFO-N-suc-rituximab, respectively, which are similar to isolated yields obtained using GMP protocols for manual 89Zr labeling of mAbs. To meet the GMP-compliant quality standards, only the radiochemically pure fractions were collected from PD-10, resulting in a lower isolated yield than the radiochemical yield according to instant thin-layer chromatography. The radiochemical purity and protein integrity were more than 95% for both products, and the antigen binding was 95.6% ± 0.6% and 87.1% ± 2.2% for 89Zr-DFO-N-suc-cetuximab and 89Zr-DFO-N-suc-rituximab, respectively. The products were sterile, and the endotoxin levels were within acceptable limits, allowing future clinical production using this procedure. Conclusion: Procedures for fully automated GMP-compliant production of 89Zr-mAbs were developed on a commercially available synthesis module, which also allows the GMP production of other radiolabeled mAbs.