Purification Of Immunoglobulin G Research Articles

Comparative Analysis of Laboratory-Scale Immunoglobulin G Purification Methods from Human Serum.

Immunoglobulin G (IgG) purification is a critical process for evaluating its role in autoimmune diseases, which are defined by the occurrence of autoantibodies. Affinity chromatography with protein G is widely considered to be the optimal technique for laboratory-scale purification. However, this technique has some limitations, including the exposure of IgG to low pH, which can compromise the quality of the purified IgG. Here, we show that alternative methods for IgG purification are possible while maintaining the quality of IgG. Different techniques for IgG purification from serum were evaluated and compared with protein G-based approaches: Melon Gel, caprylic acid-ammonium sulfate (CAAS) precipitation, anion-exchange chromatography with diethylamino ethyl (DEAE) following ammonium sulfate (AS) precipitation, and AS precipitation alone. The results demonstrated that the purification yield of these techniques surpassed that of protein G. However, differences in the purity of IgG were observed using GeLC-MS/MS. The avidity of purified IgG against selected targets (SARS-CoV-2 and topoisomerase-I) was similar between purified IgG obtained using all techniques and unpurified sera. Our work provides valuable insights for future studies of IgG function by recommending alternative purification methods that offer advantages in terms of yield, time efficiency, cost-effectiveness, and milder pH conditions than protein G.

A Thioether‐Bridging Surface Modification of Polymeric Microspheres Offers Nonbiological Protein A‐Mimetic Affinity for IgG

AbstractSurface modification of polymeric materials to control their interaction with proteins has been studied extensively, leading to widespread bio‐applications. However, the development of nonbiological, smart polymer surfaces, mimicking the recognition ability of biomolecules, remains a challenge. The present study presents a thioether‐bridging surface modification of polymeric microspheres as a new approach for mimicking protein A affinity for immunoglobulin G (IgG). The bridge‐modified surface is created through an epoxide linking reaction of porous polymeric microspheres with potassium thioacetate in a 2:1 molar ratio, acting as a protein A ligand, which is essential for industrial IgG purification. This surface exhibits buffer responsiveness and selective high‐affinity binding to the IgG Fc region. Remarkably, a comparison among the binding behaviors of a series of thioether‐modified microspheres indicates that bridging structures composed of β,β′‐dihydroxysulfide play a predominant role in IgG recognition. This straightforward approach can lead to the development of economical and practical nonbiological alternatives to protein A‐conjugated materials, providing solutions for various applications such as biosensors and site‐specific reagents utilizing the affinity of the IgG Fc region. The improved understanding of protein interactions at bridged/non‐bridged interfaces can be valuable in various applications such as implant materials and biomaterials.

Recombinant streptococcal protein G-modified metal-organic framework ZIF-8 for the highly selective purification of immunoglobulin G from human serum

A novel solid phase extractant His-rSPG@ZIF-8 was prepared by covalently coupling recombinant streptococcal protein G (His-rSPG) with ZIF-8. The His-rSPG@ZIF-8 composite was characterized by Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy (Raman), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Due to the specific binding between the immunoglobulin binding region of His-rSPG and the Fc region of immunoglobulin G (IgG), the His-rSPG@ZIF-8 composite demonstrated exceptional selectivity in adsorbing IgG. In Britton-Robinson buffer (BR buffer) with a salt concentration of 500 mmol L−1 (0.04 mol L−1, pH 8.0), the His-rSPG@ZIF-8 composite exhibited a remarkable adsorption efficiency of 99.8 % for 0.05 mg of the composite on 200 μL of IgG solution (100 μg mL−1). The adsorption behavior of the His-rSPG@ZIF-8 composite aligns with the Langmuir adsorption model, and the theoretical maximum adsorption capacity is 1428.6 mg g−1. The adsorbed IgG molecules were successfully eluted using a SDS solution (0.5 %, m/m), resulting in a recovery rate of 91.2 %. Indeed, the His-rSPG@ZIF-8 composite was successfully utilized for the isolation and purification of IgG from human serum samples. The obtained IgG exhibited high purity, as confirmed by SDS-PAGE analysis. Additionally, LC-MS/MS analysis was employed to identify the human serum proteins following the adsorption and elution process using the His-rSPG@ZIF-8 composite material. The results revealed that the recovered solution contained an impressive content of immunoglobulin, accounting for 62.4 % of the total protein content. Furthermore, this process also led to the significant enrichment of low abundance proteins such as Serpin B4 and Cofilin-1. Consequently, the His-rSPG@ZIF-8 composite holds great promise for applications such as IgG purification and immunoassays. At the same time, it expands the application of metal-organic frameworks in the field of proteomics.

Development of an impedance-based biosensor for determination of IgG galactosylation levels

The glycan profile of immunoglobulin G (IgG) molecule and its changes are associated with a number of different diseases. Galactosylation of IgG was recently suggested as a potential biomarker for rheumatoid arthritis, inflammatory bowel disease and many cancers. In this paper, we propose a portable impedance-based biosensor that utilizes lectin array technology to detect glycans in IgG. Biotinylated Griffonia simplicifolia (GSL II) and Ricinus communis agglutinin I (RCA I) lectins were used in our biosensor design for determination of the ratio of N-acetyl glucosamine (GlcNAc) to galactose (Gal) respectively, which is termed agalactosylation factor (AF). Streptavidin gold nanoparticles (GNP) were conjugated to biotinylated lectin bonded to the carbohydrate in the glycoprotein to magnify the change in impedance signal and enhance detection sensitivity. The method was successfully applied to differentiation of the galactosylation levels in human and rat IgG. In addition, we present proof of concept use of our biosensor for differentiation of COVID-19 positive patient samples from negative patients. Consequently, the sensor can be useful in future applications to distinguish between glycan profiles of IgG from healthy and patient samples in disease studies. Our biosensor permits analysis of human serum without conventional time-consuming IgG purification steps or pretreatment using enzyme digestion to cut the sugars from the glycoprotein molecule. The results suggest that the proposed point of care (POC) biosensor can be used for evaluating disease progression and treatment efficacy via monitoring changes in the galactosylation profiles of IgG in patients.

Efficient separation of IgG from IgM antibodies via conjugated surfactant micelles

Immunoglobulin-G (IgG) (∼150 kDa) antibodies confer longer term immunity against bacterial or viral infections than the heavier IgM’s (∼900 kDa), which are generally detectable in blood circulation in response to more recently acquired infections. There may be, however, a time overlap, which is problematic for diagnostic purposes, in the interests of which it is essential to separate IgM's from IgG’s. We describe a purification platform, functioning at pH 6.5, containing Tween-20, or Brij-O20, non-ionic detergent micelles, mixed with the sugar-rich detergent dodecyl maltoside (DDM), amino acid monomer tyrosine (Tyr), and conjugated by the amphiphilic complex [(bathophenanthroline)3: Fe2+]. Using conjugated Brij-O20 micelles, with input molar ratio IgG: IgM 9:1, IgG is recovered at 10 °C with 85–90% yield, (by SDS-PAGE densitometry) and ≥95% purity (also by SDS-PAGE), while IgM's are recovered at lower yields (28–34%) and contain small amounts of co-extracted IgG's. Addition of E. coli lysate as an artificial contamination background does not reduce the yield or purity of the recovered IgG. Tween-20/DDM/Tyr micelles lead to IgG purity ≥95% similar to that of Brij-O20, but with lower process yields (64–70%, by densitometry). Chromatographic separation with Protein A or Protein G resins leads to yields comparable to those obtained with Brij-O20 micelles, but with lower purity.

Influence of Pore Size in Protein G'-Grafted Mesoporous Silica Nanoparticles as a Serum Pretreatment System for In Vitro Allergy Diagnosis.

Particles with the capacity to bind to immunoglobulin G (IgG) can be used for the purification of IgG or to process clinical samples for diagnostic purposes. For in vitro allergy diagnosis, the high IgG levels in serum can interfere with the detection of allergen-specific IgE, the main diagnostic biomarker. Although commercially available, current materials present a low IgG capture capacity at large IgG concentrations or require complex protocols, preventing their use in the clinic. In this work, mesoporous silica nanoparticles are prepared with different pore sizes, to which IgG-binding protein G' is grafted. It is found that for one particular optimal pore size, the IgG capture capacity of the material is greatly enhanced. The capacity of this material to efficiently capture human IgG in a selective way (compared to IgE) is demonstrated in both solutions of known IgG concentrations as well as in complex samples, like serum, from healthy controls and allergic patients using a simple and fast incubation protocol. Interestingly, IgG removal using the best-performing material enhances in vitro IgE detection in sera from patients allergic to amoxicillin. These results highlight the great translation potential of this strategy to the clinic in the context of in vitro allergy diagnosis.

Development of biomimetic triazine-based affinity ligands for efficient immunoglobulin G purification from human and rabbit plasma

Immunoglobulin purification from different biological fluids is considered one of the most critical steps in antibody production for diagnostic, therapeutic, and research purposes. The current study aimed to elucidate the role of the different aryl substituents in triazine-based affinity ligands on the performance of an affinity chromatography purification media to separate immunoglobulin G (IgG). The biomimetic triazine-based affinity ligand was chosen as a varied containing fix spacer and support. The sepharose beads were activated by epichlorohydrin, and five types of aryl substituents were replaced in the triazine ring and covalently immobilized to the resin surface by 1, 4-diaminobutane spacer. All affinity resins with various ligands were characterized and validated using FTIR, SEM, EDX, and microscopic images. The findings revealed that using R1=3-aminophenol and R2=3-aminophenol substituents in the triazine ring, as affinity ligands attached to the sepharose surface with a 10-atom linker CAES-6B-Cl@R1= MAF, R2= MAF (No. 4), leads to better purification of IgG from human and rabbit plasma with 22.8 mg/mL resin binding capacity in 73±5% yield and 95% of purity. All results confirmed that the designed triazine-based affinity ligands could effectively purify IgG compatible with a fast and low-cost approach.

Preparation of Molecularly Imprinted Poly(N-Isopropylacrylamide) Thermosensitive Based Cryogels.

Cryogels are defined as polymeric gel matrices with interconnected macropores providing an advantage to be efficient carriers enabling unhindered diffusion of interested molecule. Cryogels could be easily prepared with the combination of molecular imprinting. Molecular imprinting technology provides selective and sensitive recognition for biomolecules. Immunoglobulin G (IgG) is a main effector component in human response. It is recently well recognized that the purification strategies for IgG have intensively gained attention to treat immune defects. Several methods including affinity chromatography have been applied for the purification of IgG from complex media. The purification of IgG plays a crucial role in medical applications using several materials. Among these, cryogels have been widely applied for the purification of several biomolecules. They offer to create low-cost affinity systems with high chemical and physical stability. Above all, temperature sensitive polymers enable a reversible phase transition against small temperature changes, by the way, reversible swelling and shrinking manner is observed.In this chapter, immunoglobulin G imprinted thermosensitive poly(N-isopropylacrylamide-N methacryloyl-(l)-histidine) [p(NIPA-MAH)/IgG-MIP] monolithic cryogel is explained. The preparation and characterization of cryogels are summarized. In addition, IgG binding studies with different parameters are briefly described. Herein, an effective design principle is presented to create imprinted temperature-sensitive cryogels for IgG purification. A p(NIPA-MAH)/IgG-MIP monolithic cryogel was synthesized for IgG purification. Afterward, IgG binding capacity of p(NIPA-MAH)/IgG-MIP cryogels was examined in different experimental conditions. Apart from these, selectivity of the p(NIPA-MAH)/IgG-MIP cryogel was shown by comparing IgG binding capacity of nonimprinted [p(NIPA-MAH)/NIP] one. Finally, the IgG purification ability of the p(NIPA-MAH)/IgG-MIP cryogel from human plasma was demonstrated proving its application in affinity chromatography using real sample.

Production of an in-House Rabbit Anti-Camel Immunoglobulin G (IgG) Conjugated With Horseradish Peroxidase (HRP) For Use in Immunoblots

The present study was aimed to develop anti-camel immunoglobulin G (IgG) conjugate. Camel serum was obtained from healthy camels and the IgG was extracted from this serum using the commercially available IgG purification kits. The electrophoretic profile of camel IgG showed four bands of molecular weight 66, 50, 44 and 33 kDa. Two rabbits were immunised with the purified camel IgG for production of rabbit anti-camel IgG. IgG fraction from serum collected from the immunised rabbits at the end of the immunisation protocol was purified using the commercially available IgG purification kits. The purified IgG tested for reactivity using ELISA against camel serum and then coupled to horseradish peroxidase. The produced conjugate then tested for reactivity using western immunoblotting against serum from camels infected with Haemonchus longistipes and normal camel serum. The conjugate was able to react with camel serum and was able to detect three components of H. longistipes of molecular weight 126, 76 and 18 kDa.

Shear-induced structural changes and denaturation of bovine immunoglobulin G and serum albumin at different temperatures

Shearing can modify native properties of proteins alone or in combination with heating. Effects of shearing on secondary structure, extent of denaturation and interactions of immunoglobulin G (IgG) and bovine serum albumin (BSA) were studied at temperatures where these changes start to take place. Pure IgG (0.8 mL mL−1) or BSA (0.4 mg mL−1) and their binary mixture were subjected to selected temperature and time combinations (70, 72, 75 or 85 °C for 0–2 min or 20 °C for 20 min) without or with a constant shear at 1000 s−1. Shearing of pure IgG or BSA for 20 min at 20 °C resulted in protein denaturation (≥4%) and loss of secondary structure, mainly β-sheets in IgG and α-helices and β-turns in BSA. The shearing appeared to preserve native proteins to a varying degree when pure IgG and BSA were sheared at 72 °C and 75 °C for 0–2 min, respectively, in comparison to their non-sheared counterparts, possibly through a countereffect generated by shear against thermal effect. The shearing mostly induced denaturation of IgG and BSA under other conditions tested. The changes in the secondary structure largely coincided with protein denaturation or preservation results. The shearing had no apparent impact on aggregation of protein molecules due to covalent interactions in both, pure and binary, dispersions at 75 or 85 °C for 0–2 min. Heating at 85 °C for 2 min resulted in formation of covalently bound intra- and intermolecular aggregates in the pure and binary mixture, respectively, regardless of shearing.

Performance of phospho-L-tyrosine immobilized onto alginate/polyacrylamide-based cryogels: Effect of ligand coupling on human IgG adsorption and Fab fragments separation

The ortho-phospho-tyrosine (P-Tyr) pseudoaffinity ligand was immobilized via ether linkage onto polyacrylamide-alginate (PAAm-Alg)-epoxy cryogels prepared according to two different approaches in order to explore their performance in the immunoglobulin G (IgG) purification from human serum. In the first approach, the P-Tyr was attached to cryogel prepared by cryocopolymerization of acrylamide and alginate with allyl glycidyl ether (AGE) as functional comonomer, and methylenebisacrylamide and Ca(II) as crosslinkers, obtaining the PAAm-Alg-AGE-P-Tyr. In the second approach, the PAAm-Alg was synthesized under the same conditions, but without AGE, and the P-Tyr was coupled to epichlorohydrin (ECH)-activated cryogel, obtaining the PAAm-Alg-ECH-P-Tyr. Both pseudoaffinity cryogels were characterized by scanning electron microscopy, swelling tests, porosity, ligand density, and flow dynamics. The human IgG differently interacted with the PAAm-Alg-ECH-P-Tyr and PAAm-Alg-AGE-P-Tyr cryogels, depending on the pH and adsorption buffer system used. The selectivity analyzed by electrophoretic profiles was similar for both cryogels, but PAAm-Alg-ECH-P-Tyr achieved higher IgG adsorption capacity (dynamic capacity of 12.62 mg of IgG/mL of cryogel). The IgG purity assayed by ELISA was 95%. The maximum IgG adsorption capacity and dissociation constant of the PAAm-Alg-ECH-P-Tyr, determined by Langmuir isotherm, were found to be 91.75 mg IgG/g of dry cryogel and 4.60 × 10−6 mol/L at pH 6.0 from aqueous solutions. The PAAm-Alg-AGE-P-Tyr showed potential to purify the Fab fragments from papain-digested human IgG solution at pH 7.0. Fab fragments were separated from Fc fragments (but with uncleaved IgG) in eluted fractions (analyzed by the Western blot technique), with yield of 82% and purity of 95% (determined by radial immunodiffusion).

Rapid identification of tailor-made aqueous two-phase systems for the extractive purification of high-value biomolecules

The industrial production of high-value biomolecules, such as monoclonal antibodies or industrially applicable enzymes (used as biocatalysts), has grown significantly within the last decades. Aqueous two-phase extraction (ATPE) using an aqueous two-phase system (ATPS) has long been shown to be a promising alternative to cost-intensive chromatography for purification of these potent biomolecules. However, state-of-the-art ATPS design is still based on “trial-and-error” screening approaches identifying un-optimized ATPS that often perform below requirements.Within this work, an optimized and enhanced method for the identification of a tailor-made ATPS for the ATPE of high-value biomolecules is presented. Through thermodynamic modeling using the ePC-SAFT equation-of-state, a first choice in phase-former candidates can be identified based on their molecular interactions in solution, leading to optimal water conditions for the biomolecule. Simultaneously, the conformational stability in presence of the phase-former candidates is investigated by measuring the unfolding temperature of the biomolecule to refine this selection. The aggregation propensity is estimated by determining the second osmotic virial coefficient B22, thus the colloidal stability of the biomolecule is taken into account. Based on these results, the partition coefficient of the biomolecule as well as a process window for the ATPE can be estimated. This method was applied to immunoglobulin G (IgG) and allows for a selection of phase-formers offering optimal working conditions regarding stability and minimized aggregation propensity. Moreover, a process window concerning temperature and phase-former concentration for the ATPE was defined allowing for a selective IgG purification.

Recovery of immunoglobulin G from rabbit serum using κ-carrageenan-modified hybrid magnetic nanoparticles

Immunoglobulin G (IgG) has been used in the treatment of cancer, autoimmune diseases and neurological disorders, however, the current technologies to purify and recover IgG from biological media are of high-cost and time-consuming, resulting in high-cost products. In this sense, the search for cost-effective technologies to obtain highly pure and active IgG is highly required. The present work proposes a simple and efficient method for the purification and recovery of IgG from rabbit serum using magnetic iron oxide nanoparticles (magnetite, Fe3O4) coated with hybrid shells of a siliceous material modified with the anionic polysaccharide κ-carrageenan. Experimental parameters such as pH, contact time between the hybrid magnetic nanoparticles (HMNPs) and rabbit serum, and total protein concentration or dilution factor of serum were evaluated. The best results were achieved at pH 5.0, with a contact time of 60 min and using a rabbit serum with a total protein concentration of 4.8 mg·mL−1. Under these conditions, it was obtained an IgG purification factor and adsorption yield onto the HMNPs of 3.0 and 90%, respectively. The desorption of IgG from the HMNPs was evaluated using two strategies: a KCl aqueous solution and buffered aqueous solutions. Comparing to the initial rabbit serum, an IgG purification factor of 2.7 with a recovery yield of 74% were obtained using a buffered aqueous solution at pH 7.0. After desorption, the secondary structure of IgG and other proteins was evaluated by circular dichroism and no changes in the secondary structure were observed, meaning that the IgG integrity is kept after the adsorption and desorption steps. In summary, the application of HMNPs in the purification of IgG from serum samples has a high potential as a new downstream platform.

Antibody Screening by Microarray Technology-Direct Identification of Selective High-Affinity Clones.

The primary screening of hybridoma cells is a time-critical and laborious step during the development of monoclonal antibodies. Often, critical errors occur in this phase, which supports the notion that the generation of monoclonal antibodies with hybridoma technology is difficult to control and hence, a risky venture. We think that it is crucial to improve the screening process to eliminate most of the critical deficits of the conventional approach. With this new microarray-based procedure, several advances could be achieved: Selectivity for excellent binders, high-throughput, reproducible signals, avoidance of misleading avidity (multivalency) effects, and performance of simultaneous competition experiments. The latter can also be used to select clones of desired cross-reactivity properties. In this paper, a model system with two excellent clones against carbamazepine, two weak clones, and blank supernatant containing fetal bovine serum was designed to examine the effectiveness of the new system. The excellent clones could be detected largely independent of the immunoglobulin G (IgG) concentration, which is usually unknown during the clone screening since the determination and subsequent adjustment of the antibody concentration are not feasible in most cases. Furthermore, in this approach, the enrichment, isolation, and purification of IgG for characterization is not necessary. Raw cell culture supernatant can be used directly, even when fetal calf serum (FCS) or other complex media is used. In addition, an improved method for the oriented antibody-immobilization on epoxy-silanized slides is presented. Based on the results of this model system with simulated hybridoma supernatants, we conclude that this approach should be preferable to most other protocols leading to many false positives, causing expensive and lengthy elimination steps to weed out the poor clones.

Polyacrylamide-alginate (PAAm-Alg) and phospho-L-tyrosine-linked PAAm-Alg monolithic cryogels: Purification of IgG from human serum

In recent decades cryogels as monolithic materials have gained interest as stationary phase in chromatography for purification of biomolecules. In this study, polyacrylamide-alginate (PAAm-Alg) monolithic cryogels were prepared by cryo-copolymerization of acrylamide and alginate monomers and methylene-bisacrylamide as crosslinker to be used as a matrix in affinity chromatography for purification of proteins. Ortho-phospho-L-tyrosine (P-Tyr) was covalently attached onto PAAm-Alg cryogels via bisoxirane-activation (PAAm-Alg-Bix-P-Tyr) and both derivatized and non-derivatized cryogels were utilized for the purification of immunoglobulin G (IgG) from human serum. Cryogels were characterized by scanning electron microscopy, swelling tests, elemental analysis, FTIR, and flow dynamics. The effects of buffer systems, conductivity, and pH on IgG adsorption were studied. Through breakthrough curve analysis a dynamic capacity of 9.2 mg IgG/mL with an IgG purity of 94% was obtained (based on ELISA analysis of IgG and albumin) for PAAm-Alg-Bix-P-Tyr cryogel when human serum was diluted in 10 mmol/L NaP buffer at pH 6.0. The adsorption isotherm data were well described by the Langmuir model with value of maximum adsorption capacity of 36.12 ± 3.63 mg of IgG/g for PAAm-Alg-Bix-P-Tyr. The PAAm-Alg-Bix-P-Tyr cryogel provides an attractive alternative for adsorption of IgG from human serum.

Use of Ionic Liquids as Cosurfactants in Mixed Aqueous Micellar Two-Phase Systems to Improve the Simultaneous Separation of Immunoglobulin G and Human Serum Albumin from Expired Human Plasma

A large fraction of unused clinical transfusion plasma is nowadays discarded. Nevertheless, some proteins, including serum albumin, immunoglobulins, and clot factors, are still viable for use in diagnosis or research. However, lasma is a complex matrix, requiring multiple steps and time-consuming methods for the isolation of proteins. In this work, aqueous micellar two-phase systems (AMTPS) composed of nonionic surfactants (Triton X-114 or Tergitol 15-S-7) were investigated to simultaneously separate immunoglobulin G (IgG) and human serum albumin (HSA) from human expired plasma. A factorial planning was applied to the nonionic surfactant and plasma concentrations to enhance the systems separation performance. After identifying the best conditions with AMTPS formed by nonionic surfactants, mixed AMTPS formed by Tergitol 15-S-7 and various surface-active ionic liquids (SAILs) acting as cosurfactants were additionally investigated to tailor the proteins partition between the two phases. The mixed AMTPS composed of Tergitol 15-S-7 as the nonionic surfactant and tributyltetradecylphosphonium chloride as the cosurfactant at pH 8.0 improved the simultaneous separation of both proteins to the opposite phases. IgG purification of 1.14-fold in the surfactant-poor phase and HSA purification of 1.36-fold in the surfactant-rich phase were obtained. Method reproducibility was investigated using additional samples of human serum and plasma and serum mixtures. Although improvements in the purification factor of each protein are still a future goal, mixed AMTPS comprising nonionic surfactants and SAILs as cosurfactants may be considered as novel platforms to be used in the simultaneous separation of value-added compounds from complex and natural matrices.

A minimalist peptide ligand for IgG by minimizing the binding domain of protein A

Large scale application of antibody related drug has been promoting the development of short ligand with low cost and high stability for affinity chromatographic purification of immunoglobulin G (IgG). This study presents a strategy to design a short peptide ligand by minimizing the IgG binding portion from a commercial ligand of Protein A. A short hexapeptide (FYEILH) was thus screened out by the real screening technique of STD-NMR. It presented a comparable binding activity to Protein A, with the dissociation constant (Kd) of 1.8 × 10−6 mol/L, the static binding capacity of 49.7 mg/mL, and the purity of 94% for hIgG from chromatographic purification of serum feedstock in a single step. Epitope mapping analysis by STD-NMR indicated that the carboxy groups of the charged residue E (glutamic acid) in FYEILH was the most adjacent part in binding with hIgG molecule and thus the electronic interactions among them was the dominating binding mechanism. Such mechanism was a little different from Protein A and consequently leaded to its much milder chromatographic conditions of loading at pH 6.0 and eluting at 0.5 mol/L NaCl. The study provided a strategy of reducing larger binding domains of huge Protein A molecule to smaller functional peptide ligand, and offered a potential alternative ligand with low synthesis cost for IgG purification.

Alginate Encapsulation of Bioengineered Protein‐Coated Polyhydroxybutyrate Particles: A New Platform for Multifunctional Composite Materials

AbstractImmobilization and display of proteins is extensively used to enhance stability and performance of proteins for technical uses such as in biotechnology. Here, self‐assembled nonporous polyhydroxybutyrate (PHB) particles bioengineered to display proteins of interest are subjected to alginate encapsulation processes. The novel composite spheres are fabricated using ionotropic gelation methods. The immunoglobulin G (IgG) binding domain Z and organophosphate hydrolase (OpdA) are attached to PHB particles, and are examples for bioseparation and bioremediation applications, respectively. Alginate microspheres entrapping Z domain coated PHB particles enable flow‐through purification of IgG. Microsphere porosity is pH tunable and at acidic pH IgG is released from Z domains but retained within microspheres. OpdA‐PHB particles are functionally entrapped in alginate microspheres enabling flow‐through substrate conversion. Attachment of functional proteins to PHB particles enhances retention within the alginate microspheres. The hydrophobic PHB particle core within alginate beads provides payload for lipophilic substances, which adsorption kinetics are aligned with a pseudo‐second‐order kinetic model in agreement with the Freundlich isotherm model. This study describes the development of a multifunctional composite material platform based on alginate spheres encapsulating PHB particles that provide payload for lipophilic substances and can be engineered to display protein functions of interest.

One-step orientated immobilization of nanobodies and its application for immunoglobulin purification

Affinity chromatography technologies play an important role in the purification of antibodies. To prepare affinity materials, prior isolation and purification of affinity ligands are required before coupling onto solid supports, which is quite expensive and laborious in large-scale applications. In this study, a one-step approach which circumvents the ligand purification procedures was developed to fabricate affinity gel for purifying immunoglobulin G (IgG). A self-labeling tag, haloalkane dehalogenase, was fused to the C-terminal of an anti-Fc variable domain of the heavy chain of the heavy-chain antibody (AFV) which was isolated in previous work. The AFV binds to various sources of IgG and is highly thermal stable. The fusion protein, namely HAFV, was expressed in Escherichia coli as a soluble protein. The binding affinity of HAFV to the Fc region of IgG was characterized and compared with the untagged anti-Fc nanobody. Next, the HAFV was immobilized directly from the crude cell lysate of isopropylthio-β-D-galactoside (IPTG) induced E. coli. The effects of NaCl concentrations and pH on the capacity of the HAFV resin were investigated. In addition, the one-step coupled HAFV resin was compared with the AFV resin and commercial resins (Protein A and Protein G) by evaluating the static capacity and stability. Though the Protein A (8.34 ± 0.37 mg/ml) and Protein G (9.19 ± 0.28 mg/ml) showed higher static capacity, the static capacity of HAFV resin (8.21 ± 0.30 mg/ml) was better than that of the untagged AFV gel (6.48 ± 0.56 mg/ml). The recovery results calculated for the reusability and stability show that there is no significant difference between the results obtained for the HAFV gel with those of the untagged AFV gel and commercial Protein A and G. After stored at 37 ℃ for 7 days and recycled 10 times, the static capacity of HAFV gel remains above 78%. Our strategy is site-specific, cost-effective, reproducible, and has the potential to dramatically cut down the costs of affinity materials for IgG purification.

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.