Using atomistic solution scattering modelling to elucidate the role of the Fc glycans in human IgG4.

Human immunoglobulin G (IgG) exists as four subclasses IgG1-4, each of which has two Fab subunits joined by two hinges to a Fc subunit. IgG4 has the shortest hinge with 12 residues. The Fc subunit has two glycan chains, but the importance of glycosylation is not fully understood in IgG4. Here, to evaluate the stability and structure of non-glycosylated IgG4, we performed a multidisciplinary structural study of glycosylated and deglycosylated human IgG4 A33 for comparison with our similar study of human IgG1 A33. After deglycosylation, IgG4 was found to be monomeric by analytical ultracentrifugation; its sedimentation coefficient of 6.52 S was reduced by 0.27 S in reflection of its lower mass. X-ray and neutron solution scattering showed that the overall Guinier radius of gyration RG and its cross-sectional values after deglycosylation were almost unchanged. In the P(r) distance distribution curves, the two M1 and M2 peaks that monitor the two most common distances within IgG4 were unchanged following deglycosylation. Further insight from Monte Carlo simulations for glycosylated and deglycosylated IgG4 came from 111,382 and 117,135 possible structures respectively. Their comparison to the X-ray and neutron scattering curves identified several hundred best-fit models for both forms of IgG4. Principal component analyses showed that glycosylated and deglycosylated IgG4 exhibited different conformations from each other. Within the constraint of unchanged RG and M1-M2 values, the glycosylated IgG4 models showed more restricted Fc conformations compared to deglycosylated IgG4, but no other changes. Kratky plots supported this interpretation of greater disorder upon deglycosylation, also observed in IgG1. Overall, these more variable Fc conformations may demonstrate a generalisable impact of deglycosylation on Fc structures, but with no large conformational changes in IgG4 unlike those seen in IgG1.

Site-Specific Conjugation of Native Antibody: Transglutaminase-Mediated Modification of a Conserved Glutamine While Maintaining the Primary Sequence and Core Fc Glycan via Trimming with an Endoglycosidase.

A versatile chemo-enzymatic tool to site-specifically modify native (nonengineered) antibodies is using transglutaminase (TGase, E.C. 2.3.2.13). With various amines as cosubstrates, this enzyme converts the unsubstituted side chain amide of glutamine (Gln or Q) in peptides and proteins into substituted amides (i.e., conjugates). A pleasant surprise is that only a single conserved glutamine (Gln295) in the Fc region of IgG is modified by microbial TGase (mTGase, EC 2.3.2.13), thereby providing a highly specific and generally applicable conjugation method. However, prior to the transamidation (access to the glutamine residue by mTGase), the steric hindrance from the nearby conserved N-glycan (Asn297 in IgG1) must be reduced. In previous approaches, amidase (PNGase F, EC 3.5.1.52) was used to completely remove the N-glycan. However, PNGase F also converts a net neutral asparagine (Asn297) to a negatively charged aspartic acid (Asp297). This charge alteration may markedly change the structure, function, and immunogenicity of an IgG antibody. In contrast, in our new method presented herein, the N-glycan is trimmed by an endoglycosidase (EndoS2, EC 3.2.1.96), hence retaining both the core N-acetylglucosamine (GlcNAc) moiety and the neutral asparaginyl amide. The trimmed glycan also reduces or abolishes Fc receptor-mediated functions, which results in better imaging agents by decreasing nonspecific binding to other cells (e.g., immune cells). Moreover, the remaining core glycan allows further derivatization such as glycan remodeling and dual conjugation. Practical and robust, our method generates conjugates in near quantitative yields, and both enzymes are commercially available.

Identification and quantification of chain-pairing variants or mispaired species of asymmetric monovalent bispecific IgG1 monoclonal antibody format using reverse-phase polyphenyl chromatography coupled electrospray ionization mass spectrometry.

Developing a knob-into-hole asymmetric bispecific IgG1 monoclonal antibody (mAb) poses manufacturing challenges due to the expression of chain pairing variants, also called mispaired species, in the desired product. The incorrect pairing of light and heavy chains could result in heterogeneous mispaired species of homodimers, heterodimers, light chain swapping, and low molecular weight species (LMWS). Standard chromatography, capillary electrophoretic, or spectroscopic methods poorly resolve these from the main variants. Here, we report a highly sensitive reverse-phase polyphenyl ultra-high-performance liquid chromatography (RP-UHPLC) method to accurately measure mispaired species of Duet mAb format, an asymmetric IgG1 bispecific mAb, for both process development and quality control analytical tests. Coupled with electrospray ionization mass spectrometry (ESI-MS), it enabled direct online characterization of mispaired species. This single direct assay detected diverse mispaired IgG-like species and LMWS. The method resolved eight disulfide bonds dissociated LMWS and three mispaired LMWS. It also resolved three different types of IgG-like mispaired species, including two homodimers and one heterodimer. The characterization and quantification simultaneously enabled the cell line selection that produces a lesser heterogeneity and lower levels of mispaired species with the desired correctly paired product. The biological activity assessment of samples with increased levels of these species quantified by the method exhibited a linear decline in potency with increasing levels of mispaired species in the desired product. We also demonstrated the utility of the technique for testing in-process intermediate materials to determine and assess downstream purification process capability in removing diverse mispaired IgG-like species and LMWS to a certain level during the downstream purification process. Our investigation demonstrates that adopting this method was vital in developing asymmetric bispecific mAb from the initial stage of cell line development to manufacturing process development. Therefore, this tool could be used in the control strategy to monitor and control mispaired species during manufacturing, thus improving the quality control of the final product.

Reduction of monoclonal antibody viscosity using interpretable machine learning.

Early identification of antibody candidates with drug-like properties is essential for simplifying the development of safe and effective antibody therapeutics. For subcutaneous administration, it is important to identify candidates with low self-association to enable their formulation at high concentration while maintaining low viscosity, opalescence, and aggregation. Here, we report an interpretable machine learning model for predicting antibody (IgG1) variants with low viscosity using only the sequences of their variable (Fv) regions. Our model was trained on antibody viscosity data (>100 mg/mL mAb concentration) obtained at a common formulation pH (pH 5.2), and it identifies three key Fv features of antibodies linked to viscosity, namely their isoelectric points, hydrophobic patch sizes, and numbers of negatively charged patches. Of the three features, most predicted antibodies at risk for high viscosity, including antibodies with diverse antibody germlines in our study (79 mAbs) as well as clinical-stage IgG1s (94 mAbs), are those with low Fv isoelectric points (Fv pIs < 6.3). Our model identifies viscous antibodies with relatively high accuracy not only in our training and test sets, but also for previously reported data. Importantly, we show that the interpretable nature of the model enables the design of mutations that significantly reduce antibody viscosity, which we confirmed experimentally. We expect that this approach can be readily integrated into the drug development process to reduce the need for experimental viscosity screening and improve the identification of antibody candidates with drug-like properties.

Study of Monoclonal Antibody Aggregation at the Air-Liquid Interface under Flow by ATR-FTIR Spectroscopic Imaging.

Throughout bioprocessing, transportation, and storage, therapeutic monoclonal antibodies (mAbs) experience stress conditions that may cause protein unfolding and/or chemical modifications. Such structural changes may lead to the formation of aggregates, which reduce mAb potency and may cause harmful immunogenic responses in patients. Therefore, aggregates need to be detected and removed or ideally prevented from forming. Air-liquid interfaces, which arise during various stages of bioprocessing, are one of the stress factors causing mAb aggregation. In this study, the behavior of an immunoglobulin G (IgG) at the air-liquid interface was investigated under flow using macro attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic imaging. This chemically specific imaging technique allows observation of adsorption of IgG to the air-liquid interface and detection of associated secondary structural changes. Chemical images revealed that IgG rapidly accumulated around an injected air bubble under flow at 45 °C; however, no such increase was observed at 25 °C. Analysis of the second derivative spectra of IgG at the air-liquid interface revealed changes in the protein secondary structure associated with increased intermolecular ß-sheet content, indicative of aggregated IgG. The addition of 0.01% w/v polysorbate 80 (PS80) reduced the amount of IgG at the air-liquid interface in a static setup at 30 °C; however, this protective effect was lost at 45 °C. These results suggest that the presence of air-liquid interfaces under flow may be detrimental to mAb stability at elevated temperatures and demonstrate the power of ATR-FTIR spectroscopic imaging for studying the structural integrity of mAbs under bioprocessing conditions.

Design of inhibitor peptide sequences based on the interfacial knowledge of the protein G-IgG crystallographic complex and their binding studies with IgG.

Protein-protein interactions (PPI) have emerged as valuable targets in medicinal chemistry due to their key roles in important biological processes. The modulation of PPI by small peptides offers an excellent opportunity to develop drugs against human diseases. Here, we exploited the knowledge of the binding interface of the IgG-protein G complex (PDB:1FCC) for designing peptides that can inhibit these complexes. Herein, we have designed several closely related peptides, and the comparison of results from experiments and computational studies indicated that all the peptides bind close to the expected binding site on IgG and the complexes are stable. A minimal sequence consisting of 11 amino acids (P5) with binding constants in the range of 100 nM was identified. We propose that the main affinity differences across the series of peptides arose from the presence of polar amino acid residues. Further, the molecular dynamic studies helped to understand the dynamic properties of complexes in terms of flexibility of residues and structural stability at the interface. The ability of P5 to compete with the protein G in recognizing IgG can help in the detection and purification of antibodies. Further, it can serve as a versatile tool for a better understanding of protein-protein interactions.

Evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometry.

Aggregates are recognized as one of the most critical product-related impurities in monoclonal antibody (mAb)-based therapeutics due to their negative impact on the stability and safety of the drugs. So far, investigational efforts have primarily focused on understanding the causes and effects of mAb self-aggregation, including both internal and external factors. In this study, we focused on understanding mAb stability in the presence of its monovalent fragment, formed through hinge cleavage and loss of one Fab unit (referred to as "Fab/c"), a commonly observed impurity during manufacturing and stability. The Fab/c fragments were generated using a limited IgdE digestion that specifically cleaves above the IgG1 mAb hinge region, followed by hydrophobic interaction chromatographic (HIC) enrichment. Two IgG1 mAbs containing different levels of Fab/c fragments were incubated under thermally accelerated conditions. A method based on size exclusion chromatography coupled with native mass spectrometry (SEC-UV-native MS) was developed and used to characterize the stability samples and identified the formation of heterogeneous dimers, including intact dimer, mAb-Fab/c dimer, Fab/c-Fab/c dimer, and mAb-Fab dimer. Quantitative analyses on the aggregation kinetics suggested that the impact of Fab/c fragment on the aggregation rate of individual dimer differs between a glycosylated mAb (mAb1) and a non-glycosylated mAb (mAb2). An additional study of deglycosylated mAb1 under 25°C accelerated stability conditions suggests no significant impact of the N-glycan on mAb1 total aggregation rate. This study also highlighted the power of SEC-UV-native MS method in the characterization of mAb samples with regard to separating, identifying, and quantifying mAb aggregates and fragments.

An integrated methodology for quality assessment of therapeutic antibodies with potential long circulation half-life in harvested cell culture fluid using FcRn immobilized hydrophilic magnetic graphene.

Designing modified therapeutic antibodies with enhanced FcRn-binding affinity holds promise in the extension of circulation half-lives and potential refinement of pharmacokinetics. During the development of these new-generation therapeutic antibodies, FcRn binding affinity of IgGs is emphasized and monitored as a critical quality attribute (CQA), alongside other critical assessments including titer and aggregation level. However, the traditional workflow for assessing the overall quality of expressed IgGs in harvested cell culture fluid (HCCF) is blamed to be cumbersome and time-consuming. This study presents an integrated methodology for the rapid quality assessment of IgGs in HCCF by selectively extracting IgGs with favorable high FcRn affinity for subsequent analysis using size exclusion chromatography (SEC). The approach utilizes innovative adsorbents known as FcRn immobilized hydrophilic magnetic graphene (MG@PDA@PAMAM-FcRn) in a magnetic solid-phase extraction (MSPE) process. To simulate the in vivo binding dynamics, MSPE binding and dissociation was performed at pH 6.0 and 7.4, respectively. The composite have demonstrated enhanced extraction efficiency and impurity removal ability in comparison to commercially available magnetic beads. The SEC monomer peak area value provides the output of this method, the ranking of which enabled the facile identification of superior HCCF samples with high overall quality of IgG. Optimization of MSPE parameters was performed, and the method was validated for specificity, precision, sensitivity, and accuracy. The proposed method exhibited an analytical time of 0.6 h, which is 7-22 times shortened in comparison to the conventional workflow.

IgG glycopeptide enrichment using hydrophilic interaction chromatography-based solid-phase extraction on an aminopropyl column.

The sample preparation step is pivotal in glycoproteomic analysis. An effective approach in glycoprotein sample preparation involves enriching glycopeptides by solid-phase extraction (SPE) using polar stationary phases in hydrophilic interaction liquid chromatography (HILIC) mode. The aim of this work is to show how different experimental conditions influence the enrichment efficiency of glycopeptides from human immunoglobulin G (IgG) on an aminopropyl-modified SPE column. Different compositions of the elution solvent (acetonitrile, methanol, and isopropanol), along with varying concentrations of elution solvent acidifiers (formic and acetic acid), and different concentrations of acetonitrile for the conditioning and washing solvents (65%, 75%, and 85% acetonitrile) were tested to observe their effects on the glycopeptide enrichment process. Isopropanol proved less effective in enriching glycopeptides, while acetonitrile was the most efficient, with methanol in between. Higher formic acid concentrations in the elution solvent weakened the ionic interactions, particularly with sialylated glycopeptides. Substituting formic acid with acetic acid led to earlier elution of more glycopeptides. The acetonitrile concentration in conditioning and washing solutions played a key role; at 65% acetonitrile, glycopeptides were not retained on the SPE column and were detected in the flow-through fraction. Ultimately, it was proven that the enrichment method was applicable to human plasma samples, resulting in a significant decrease in the abundances of non-glycosylated peptides. To the best of our knowledge, this study represents the first systematic investigation into the impact of the mobile phase on glycopeptide enrichment using an aminopropyl-modified SPE column in HILIC mode. This study demonstrates the substantial impact of even minor variations in experimental conditions, which have not yet been considered in the literature, on SPE-HILIC glycopeptide enrichment. Consequently, meticulous optimization of these conditions is imperative to enhance the specificity and selectivity of glycoproteomic analysis, ensuring accurate and reliable quantification.

In-line buffer exchange in the coupling of Protein A chromatography with weak cation exchange chromatography for the determination of charge variants of immunoglobulin G derived from chinese hamster ovary cell cultures.

Immunoglobulin G (IgG) is the most common monoclonal antibody (mAb) grown for therapeutic applications. While IgG is often selectively isolated from cell lines using protein A (ProA) chromatography, this is only a stepping stone for complete characterization. Further classification can be obtained from weak cation exchange chromatography (WCX) to determine IgG charge variant distributions. The charge variants of monoclonal antibodies can influence the stability and efficacy in vivo, and deviations in charge heterogeneity are often cell-specific and sensitive to upstream process variability. Current methods to characterize IgG charge variants are often performed off-line, meaning that the IgG eluate from the ProA separation is collected, diluted to adjust the pH, and then transferred to the WCX separation, adding time, complexity, and potential contamination to the sample analysis process. More recently, reports have appeared to streamline this separation using in-line two-dimensional liquid chromatography (2D-LC). Presented here is a novel, 2D-LC coupling of ProA in the first dimension (1D) and WCX in the second dimension (2D) chromatography. As anticipated, the initial direct column coupling proved to be challenging due to the pH incompatibility between the mobile phases for the two stages. To solve the solvent compatibility issue, a size exclusion column was placed in the switching valve loop of the 2D-LC instrument to act as a means for the on-line solvent exchange. The efficacy of the methodology presented was confirmed through a charge variant determination using the NIST monoclonal antibody standard (NIST mAb), yielding correct acidic, main, and basic variant compositions. The methodology was employed to determine the charge variant profile of IgG from an in-house cultured Chinese hamster ovary (CHO) cell supernatant. It is believed that this methodology can be easily implemented to provide higher-throughput assessment of IgG charge variants for process monitoring and cell line development.

Poly(glutamic acid)-Based Viscosity Reducers for Concentrated Formulations of a Monoclonal IgG Antibody.

Above a concentration threshold, the viscosity of solutions of proteins increases abruptly, which hampers the injectability of therapeutic formulations. Concentrations above 200 g/L are an ideal goal for subcutaneous application of antibodies. Molecular additives, such as amino acids (e.g., arginine) help decrease the viscosity, but they are used at concentrations as high as about 200 mmol/L. We addressed the question of whether poly(amino acids) could be more efficient than small molecular additives. We observed marked fluidification of a model therapeutic monoclonal antibody (mAb) solution by poly(d,l-glutamic acid) and poly(l-glutamic acid) derivatives added at concentrations of <6.5 g/L (i.e., a mAb/polymer chain molar ratio between 4:1 and 1:1 mol/mol). The bare poly(glutamate) parent chains were compared with polyethylene glycol-grafted chains as PEGylation is a common way to enhance stability. Viscosity could be decreased to ∼20 mPa s as compared to values of ∼100 mPa s in the absence of polymers at 200 g/L mAb. Formation of complexes between the mAb and the polyglutamates was characterized by capillary electrophoresis analysis in dilute solutions (1 g/L mAb) and by observation of phase separation at higher concentrations, suggesting tight association at about 2:1 mol/mol mAb/polymer. Altogether, these results show that polyglutamate derivatives hold an untapped potential as an excipient for fluidification of concentrated protein solutions.

Insight into the Mechanism Underlying the Reduction of Digestibility and IgG/IgE Binding Ability in Ovalbumin during Different High-Temperature Conduction Modes-Induced Glycation.

Effects of different high-temperature conduction modes [high-temperature air conduction (HAC), high-temperature contact conduction (HCC), high-temperature steam conduction (HSC)]-induced glycation on the digestibility and IgG/IgE-binding ability of ovalbumin (OVA) were studied and the mechanisms were investigated. The conformation in OVA-HSC showed minimal structural changes based on circular dichroism, fluorescence, and ultraviolet spectroscopy. The degree of hydrolysis analysis indicated that glycated OVA was more resistant to digestive enzymes. Liquid chromatography-Orbitrap mass spectrometry identified 11, 14, and 15 glycation sites in OVA-HAC, OVA-HCC, and OVA-HSC, respectively. The IgG/IgE-binding ability of OVA was reduced during glycation and digestion, and the interactions among glycation, allergenicity, and digestibility were further investigated. Glycation sites masked the IgG/IgE epitopes resulting in a reduction in allergenicity. Digestion enzymes destroyed the IgG/IgE epitopes thus reducing allergenicity. Meanwhile, the glycation site in proximity to the digestion site of pepsin was observed to cause a reduction in digestibility.

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.

Kinetics of Trisulfide-to-Disulfide Conversion of Therapeutic IgG1 Monoclonal Antibodies Under Physiological Conditions: A Case Study of Casirivimab And Imdevimab.

The percentage of trisulfide variants is a product quality metric that is monitored during the manufacture of monoclonal antibody (mAb)-based therapeutics. Results from earlier preclinical studies revealed that trisulfide linkages in mAbs are rapidly converted to disulfides in circulation. In this study, casirivimab and imdevimab, which are both IgG1 subclass mAbs that target the non-overlapping epitopes in SARS-CoV2 Spike protein, are used as models to study the kinetics of trisulfide-to-disulfide conversion in vivo in human circulation. To determine the percentage of trisulfide variants in systemic circulation immediately after intravenous injection, both mAbs were immunoprecipitated from serum samples collected from COVID-19 patients that received this cocktail antibody treatment as part of a first-in-human study. The immunoprecipitated mAbs were then digested under non-reducing conditions and evaluated by liquid-chromatography-mass spectrometry (LC-MS). Significant reductions in the percentages of trisulfide variants were observed in serum samples as early as 1 hr after completion of the intravenous infusion. A flow-through dialysis model designed to mimic the redox potential of blood revealed a plausible chemical mechanism for the rapid trisulfide-to-disulfide conversion of IgG1 subclass mAbs under physiological conditions.

Effects of unfolding treatment assisted glycation on the IgE/IgG binding capacity and antioxidant activity of ovomucoid.

Ovomucoid is the immune-dominant allergen in the egg white of hens. Due to its structure based on nine disulfide bonds as well as its resistance to heat and enzymatic hydrolysis, the allergenicity of this food protein is difficult to decrease by technological processes. We sought to reduce its allergenicity through the Maillard reaction. The unfolding of ovomucoid with L-cysteine-mediated reduction was used to increase accessibility to conformational and linear epitopes by modifying the secondary and tertiary structures of the allergen. Glycation with different saccharides revealed the beneficial effect of maltose glycation on the IgG-binding capacity reduction. By determining the better glycation conditions of unfolded ovomucoid, we produced ovomucoid with reduced IgE binding capacity due to the glycation sites (K17, K112, K129, and K164) on epitopes. Moreover, after simulated infant and adult gastrointestinal digestion, the unfolded plus glycated ovomucoid showed higher ABTS˙+ scavenging activity, O2˙- scavenging activity, ˙OH scavenging activity, Fe2+ chelating activity, and a FRAP value; in particular, for ˙OH scavenging activity, there was a sharp increase of more than 100%.

Production and characterization of an Fv-clasp of rheumatoid factor, a low-affinity human autoantibody.

Rheumatoid factor (RF) is an autoantibody against IgG that affects autoimmune diseases and inhibits the effectiveness of pharmaceuticals and diagnostic agents. Although RFs derived from various germline genes have been identified, little is known about their molecular recognition mechanisms. In this study, the Fv-clasp format was used to prepare YES8c, an RF. We developed an Escherichia coli secretion expression system capable of producing milligram-scale of YES8c Fv-clasp per 1 L of culture. Although YES8c is an autoantibody with very low affinity, the produced Fv-clasp maintained specific binding to IgG. Interestingly, the molecules prepared by E. coli secretion had a higher affinity than those prepared by refolding. In the structure of the YES8c-Fc complex, the N-terminus of the light chain is close to Fc; therefore, it is suggested that the addition of the N-terminal methionine may cause collisions with Fc, resulting in reduced affinity. Our findings suggest that the Fv-clasp, which provides sufficient stability and a high bacterial yield, is a useful format for studying RFs with very low affinity. Furthermore, the Fv-clasp produced from a secretion expression system, which can properly process the N-terminus, would be suitable for analysis of RFs in which the N-terminus may be involved in interactions.

Enhancing thermal stability in the CH2 domain to suppress aggregation through the introduction of simultaneous disulfide bonds in Pichia pastoris.

Protein aggregations decrease production yields and impair the efficacy of therapeutics. The CH2 domain is a crucial part of the constant region of human IgG. But, it is also the least stable domain in IgG, which can result in antibody instability and aggregation problems. We created a novel mutant of the CH2 domain (T250C/L314C, mut10) by introducing a disulfide bond and expressed it using Pichia pastoris. The mut10 variant exhibited enhanced thermal stability, resistance to enzymatic degradation, and reduced aggregation in comparison to the original CH2 domain. However, it was less stable than mut20 (L242C/K334C), which is the variant prepared in a previous study (Gong et al., J. Biol. Chem., 2009). A more advanced mutant, mut25, was created by combining mut10 and mut20. Mut25 artificially contains two disulfide bonds. The new mutant, mut25, showed enhanced thermal stability, increased resistance to enzymatic digestion, and reduced aggregation in comparison to mut20. According to our knowledge, mut25 achieves an unprecedented level of stability among the humanized whole CH2 domains that have been reported so far. Mut25 has the potential to serve as a new platform for antibody therapeutics due to its ability to reduce immunogenicity by decreasing aggregation.

Phase-Diagram Observation of Liquid-Liquid Phase Separation in the Poly(l-lysine)/ATP System and a Proposal for Diagram-Based Application Strategy.

Liquid-liquid phase separation (LLPS) is essential to understanding the biomacromolecule compartmentalization in living cells and to developing soft-matter structures for chemical reactions and drug delivery systems. However, the importance of detailed experimental phase diagrams of modern LLPS systems tends to be overlooked in recent times. Even for the poly(l-lysine) (PLL)/ATP system, which is one of the most widely used LLPS models, any detailed phase diagram of LLPS has not been reported. Herein, we report the first phase diagram of the PLL/ATP system and demonstrate the feasibility of phase-diagram-based research design for understanding the physical properties of LLPS systems and realizing biophysical and medical applications. We established an experimentally handy model for the droplet formation-disappearance process by generating a concentration gradient in a chamber for extracting a suitable condition on the phase diagram, including the two-phase droplet region. As a proof of concept of pharmaceutical application, we added a human immunoglobulin G (IgG) solution to the PLL/ATP system. Using the knowledge from the phase diagram, we realized the formation of IgG/PLL droplets in a pharmaceutically required IgG concentration of ca. 10 mg/mL. Thus, this study provides guidance for using the phase diagram to analyze and utilize LLPS.

Non-enzymatic glycation and aggregation of camel immunoglobulins induce breast cancer cell proliferation.

Glycation of biomolecules results in the formation of advanced glycation end products (AGEs). Immunoglobulin G (IgG) has been implicated in the progression of various diseases, including diabetes and cancer. This study purified three IgG subclasses (IgG1, IgG2, and IgG3) from Camelus dromedarius colostrum using ammonium sulfate fractionation and chromatographic procedures. SDS-PAGE was performed to confirm the purity and molecular weight of the IgG subclasses. Several biochemical and biophysical techniques were employed to study the effect of glycation on camel IgG using methylglyoxal (MGO), a dicarbonyl sugar. Early glycation measurement showed an increase in the fructosamine content by ~four-fold in IgG2, ~two-fold in IgG3, and a slight rise in IgG1. AGEs were observed in all classes of IgGs with maximum hyperchromicity (96.6%) in IgG2. Furthermore, glycation-induced oxidation of IgGs led to an increase in carbonyl content and loss of -SH groups. Among subclass, IgG2 showed the highest (39.7%) increase in carbonyl content accompanied by 82.5% decrease in -SH groups. Far UV-CD analysis illustrated perturbation of ß-sheet structure during glycation reaction with MGO. Moreover, glycation of IgG proceeds to various conformational states like aggregation and increased hydrophobicity. In addition, the cytotoxicity assay (MTT) illustrated the proliferation of breast cancer cells (MCF-7) with IgG2 treatment.

A comprehensive evaluation of arginine and its derivatives as protein formulation stabilizers.

Arginine and its derivatives (such as arginine ethyl ester and acetyl arginine) have varying degrees of protein aggregation suppressor effect across different protein solutions. To understand this performance ambiguity, we evaluated the activity of arginine, acetyl arginine, and arginine ethyl ester for aggregation suppressor effect against human intravenous immunoglobulin G (IgG) solution at pH 4.8. Both arginine and its cationic derivative arginine ethyl ester in their hydrochloride salt forms significantly reduced the colloidal and conformational stability (reduced kd and Tm) of IgG. Consequently, the monomer content was decreased with an increase in subvisible particulates after agitation or thermal stress. Furthermore, compared to arginine, arginine ethyl ester with one more cationic charge and hydrochloride salt form readily precipitated IgG at temperatures higher than 25 °C. On the contrary, acetyl arginine, which mostly exists in a neutral state at pH 4.8, efficiently suppressed the formation of subvisible particles retaining a high amount of monomer owing to its higher colloidal and conformational stability. Concisely, the charged state of additives significantly impacts protein stability. This study demonstrated that contrary to popular belief, arginine and its derivatives may either enhance or suppress protein aggregation depending on their net charge and concentration.