IgG Solution Research Articles

Reconsidering freeze-induced protein aggregation: Air bubbles as the root cause of ice-water interface stress

Freeze-induced stress causing aggregation of proteins has typically been primarily attributed to the ice-water interface. However, we hypothesize that the underlying observed and perceived detrimental effect of ice is, to some extent, attributed to air bubbles expelled from ice crystal lattices or to nanobubbles existing prior to freezing. The reduction of dissolved air was achieved via a deaeration process by placing samples in a reduced pressure chamber, while the reduction of nanobubbles was achieved by filtering samples via a syringe filter. The results showed that the reduction of both dissolved air molecules and stable colloidal nanobubbles in a bovine IgG solution prior to freezing led to a significant decrease in aggregation after thawing compared to untreated samples (∼6,000 vs. ∼ 40,000 particles/mL at a freezing rate of 100 K/s, respectively). The deaeration-filtration treatment works additively with cryoprotectants such as trehalose, further reducing the freeze-induced aggregation of IgG. The results also demonstrated that air–water interfacial aggregation of IgG in bulk liquid samples is a time-dependent process. The number of IgG subvisible particles increased with time and temperature, suggesting that random collisions of denatured molecules promoted the formation of aggregates with spherical morphology. In contrast, the IgG subvisible count after freeze-thawing had already reached its nominal value, suggesting a time-independent process where denatured protein molecules were compressed between ice crystals into filament-like aggregates. In summary, the findings shift the current paradigm from ice crystals being the main destabilizing factor during freezing to air bubbles, although the two are intertwined. From a translational aspect, this study underscores the value of deaeration-filtration as an essential supplemental process that can be applied in addition to formulation approaches such as the use of cryoprotectants to further reduce freezing stress on proteins and increase their stability.

Quantification of SARS-CoV-2 monoclonal IgG mass fraction by isotope dilution mass spectrometry.

The availability of serology assays to measure antibodies against the SARS coronavirus 2 (SARS-CoV-2) expanded rapidly during the Covid-19 pandemic. The interchangeable use of such assays to monitor disease progression and immune protection requires their standardization, for which suitably characterized monoclonal antibody materials can be useful. The methods, based on isotope dilution mass spectrometry, to value assign the mass fraction of such a material in solution within the context of an international interlaboratory comparison study (CCQM-P216) are described. The mass fraction in solution of a humanized IgG monoclonal antibody (mAb) against the SARS-CoV-2 Spike glycoprotein in the study sample has been value assigned through a combination of liquid chromatography, isotope dilution mass spectrometry (LC-ID-MS) methods and size exclusion chromatography with UV detection (SEC-UV). The former were developed for the quantification of amino acids and proteotypic peptides as surrogate analytes of the mAb while the latter was applied for the determination of the relative monomeric mass fraction. High-resolution mass spectrometry (hrMS) allowed the molecular weight evaluation and ruled out the presence of significant impurities. Method trueness was assessed using a subclass homologous IgG1 material value assigned by amino acid analysis. The assigned mass fraction of monomeric SARS-CoV-2 IgG in solution was 390 ± 16mg/g. The associated expanded uncertainty originated mainly from acid hydrolysis variability and Trypsin/Lys-C digestion variability and efficiency.

The immunomodulating activity of trimodulin (polyvalent IgM, IgA, IgG solution): a post hoc analysis of the phase II CIGMA trial

BackgroundThe phase II CIGMA trial performed in 160 patients with severe community-acquired pneumonia (sCAP) found treatment with trimodulin (human polyvalent immunoglobulin [Ig]: ~ 23% IgM, ~ 21% IgA, ~ 56% IgG) was associated with a lower mortality in those patients with elevated baseline serum levels of C-reactive protein (CRP) and/or subnormal IgM.MethodsIn this post hoc analysis, the pharmacodynamic effects of trimodulin treatment (182.6 mg/kg/day for 5 days) were investigated on Ig replenishment, cellular markers of inflammation (absolute neutrophil [ANC] and lymphocyte [ALC] count, neutrophil-to-lymphocyte ratio [NLR]), and soluble markers of inflammation (procalcitonin [PCT] and CRP). The impact of these pharmacodynamic effects on mortality was also evaluated.ResultsCompared with healthy subjects, baseline serum levels of IgM, IgG, and ALC were significantly lower, and ANC, NLR, PCT and CRP significantly higher in sCAP patients (p < 0.0001). Low Ig concentrations increased with trimodulin. Normalization of ANC (analysis of variance [ANOVA] p = 0.016) and PCT (ANOVA p = 0.027) was more rapid with trimodulin compared with placebo. These and other effects were more evident in patients with low baseline IgM levels. Normalization of PCT and CRP levels was both steadier and faster with trimodulin treatment. In patients with low baseline ALC, trimodulin was associated with a lower 28-day all-cause mortality rate (14.5% vs 32.1% in placebo, p = 0.043) and more ventilator-free days ([VFD]; median VFD: 3.5 vs 11 in placebo, p = 0.043). These numerical differences were greater if baseline IgM was also low (low ALC, low IgM: 8.1% mortality vs 34.1% placebo, p = 0.006; 3 VFD vs 15 VFD, p = 0.009, respectively). Results were consistent in patients with high baseline CRP (low ALC, high CRP: 10.9% mortality vs 34.1% placebo, p = 0.011).ConclusionsThis post hoc pharmacodynamic analysis of a blinded phase II trial suggests that trimodulin compensates for, and more rapidly modifies, the dysregulated inflammatory response seen in sCAP patients. Trimodulin was associated with significantly lower mortality and more VFD in subgroups with high CRP and low ALC. This effect was particularly marked in patients who also had low baseline IgM values. These findings require confirmation in prospective trials.

Biochemical Characterization of a New 10% IVIG Preparation [IgG Next Generation (BT595)/Yimmugo®] Obtained from a Manufacturing Process Preserving IgA/IgM Potential of Human Plasma.

Human plasma is used for the generation of several life-saving drugs and contains valuable antibodies from the immunoglobulin classes IgG, IgM and IgA. Purified intravenous IgG solutions (IVIGs) form the majority of plasma-derived medicine to treat patients with various forms of immunodeficiencies. In conventional IVIG manufacturing processes, immunoglobulin classes IgM and IgA are often discarded as contaminants, but these antibody classes have been proven to be effective for the treatment of acute bacterial infections. Considering the increase in demand for human plasma-derived products and the ethical value of the raw material, a more resource-saving usage of human plasma is needed. Intensive research over the last decades showed that adverse reactions to IVIGs depend on the presence of thrombogenic factors, partially unfolded proteins, non-specific activation of the complement system, and blood group specific antibodies. Therefore, new IVIG preparations with reduced risks of adverse reactions are desirable. A new manufacturing process that yields two biologics was established and quality attributes of the new IVIG solution (Yimmugo®) obtained from this process are presented. Here, we provide a biochemical characterization of Yimmugo®, a new 10%IVIG preparation. It is derived from human blood plasma by a combined manufacturing process, where IgM and IgA are retained for the production of a new biologic (trimodulin, currently under investigation in phase III clinical trials). Several improvements have been implemented in the manufacturing of Yimmugo® to reduce the risk of adverse reactions. Gentle and efficient mixing by vibration (called "vibromixing") during a process step where proteins are at risk to aggregate was implemented to potentially minimize protein damage. In addition, a dedicated process step for the removal of the complement system activator properdin was implemented, which resulted in very low anticomplementary activity levels. The absence of measurable thrombogenic activity in combination with a very high degree of functional monomeric antibodies predict excellent efficacy and tolerability. Yimmugo® constitutes a new high quality IVIG preparation derived from a novel manufacturing process that takes advantage of the full therapeutic immunoglobulin potential of human plasma.

Extended Work Function Shift of Large‐Area Biofunctionalized Surfaces Triggered by a Few Single‐Molecule Affinity Binding Events

AbstractFew binding events are here shown to elicit an extended work function change in a large‐area Au‐surface biofunctionalized with ≈108 capturing antibodies. This is demonstrated by Kelvin probe force microscopy (KPFM), imaging a ≈105 µm2 wide Au‐electrodes covered by a dense layer (≈104 µm−2) of physisorbed anti‐immunoglobulin‐M (anti‐IgM). A 10 min incubation in 100 µL phosphate buffer saline solution encompassing ≈10 IgM antigens (10−19 mole L−1  102 × 10−21 m) produces a work function shift ΔW ≈ –60 meV. KPFM images prove that this shift involves the whole inspected area. Notably, no work function change occurs upon incubation in highly concentrated (3 × 10−15 m) nonbinding IgG solutions. The ΔW measured by KPFM is in quantitative agreement with the threshold voltage shift of an electrolyte‐gated single‐molecule large‐area transistor (SiMoT). The findings provide direct experimental evidence for the SiMoT ultrahigh sensitivity, by imaging the extensive shift of the gate work function, likely arising from collective surface phenomena, elicited by single‐molecule binding events.

Supported ionic liquids as customizable materials to purify immunoglobulin G

Over the past few years, antibodies such as immunoglobulin G, IgG, have increased their market share as alternative therapeutics. However, their production at high purity levels is still costly due to the absence of a cost-effective platform for their recovery and purification from the complex biological media in which they are produced. This work describes, for the first time, that materials modified with ionic liquids (ILs) can be designed for the effective capture and purification of antibodies from complex matrices, allowing both the selective adsorption of IgG or the selective adsorption of other proteins present in the media. The best results correspond to IgG with 59 % of yield and 84 % of purity in the aqueous solution, and IgG with 76 % of yield and 100 % of purity on the surface of one SIL due to the selective adsorption of IgG from human serum. The best conditions and materials were then applied to other IgG-containing matrices, namely rabbit serum and Chinese hamster ovary (CHO) cell culture supernatants, proving the robustness of the developed strategy. Furthermore, it is demonstrated that the secondary structure of IgG is preserved during the purification process and that these antibodies remain biologically active. In summary, it is shown that by only changing the IL chemical structure at the material surface it is possible to selectively adsorb IgG or to adsorb other proteins leaving IgG in solution. These findings prove that SILs are customizable materials with future potential to act in the flow-through or bind-and-elute modes. Therefore, SILs can be envisioned as potential chromatographic columns capable of substituting the high-cost commercial chromatographic columns based on biological ligands currently used to purify IgG.

Development of Selective Cortisol Biosensors in the Control of Fatigue and Related Diseases

The study of fatigue becomes complex due to several subjective factors related to its causality, making the relationship between the level of fatigue and the concentration of cortisol in an individual characteristic. However, cortisol is identified as the main element released in response to fatigue, and as such, it can be used as a biomarker for its determination and other associated diseases. Inadequate amounts of cortisol are related to non-specific diseases such as diabetes and osteoporosis. The body's cortisol levels fluctuate throughout the day, with the greatest levels in the morning and the lowest in the evening. To correlate these various fluctuations to the degree of fatigue, is required a real-time determination of cortisol levels with an effective sensory device, which overcomes the demand of costly and time-consuming laboratory analysis. Cortisol, as expressed in sweat, has a wide dynamic concentration range that can be used to monitor fatigue during different activities, being useful in preventing diseases and accidents. Currently, cortisol evaluation may be critical as one of the prospective biomarkers for Covid-19-induced significant inflammatory consequences. Some studies show possible severity between the production of pro-inflammatory cytokines related to viral infection by the new Sars-Cov-2 in people with above-average serum cortisol levels. In this regard, the establishment of a simple and low-cost biosensor with rapid diagnosis and in loco application would be beneficial due to the analytical center's independence and the simplicity with patients positive for the Sars-Cov-2 virus or others may be followed and treated. For the cortisol biosensor development, electrochemically exfoliated graphene (EEG) was used as a current collector nanomaterial in combination with chitosan (CS), a biomaterial derived from discarded crustacean shells. The carbon DropSens were modified with nanocomposite containing CS and EEG prepared using an ultrasonication. 21µL of CS_EEG dispersion by drop casting and exposed for 4 hours by white light, in order to dry the nanocomposite and recover all the working electrode. The material not bounded to electrode was then removed after 3 washes of 50µL of Milli-Q water. 10 µL of glutaraldehyde 1 % solution (GA) was dripped on the electrodes and allowed to react for 15 minutes to guarantee greater interaction of the nanocomposite with the antibody. The wet surface of electrodes was then evaluated for the incorporation of monoclonal antibody solution by IgG type specific for cortisol at varying concentrations. A total of 7.5 µL of diluted IgG solution (3.33 ng mL-1) and pure IgG solution were added to the biosensor immune complex (100 ng mL-1) and allowed to act for 30 minutes. Sensory measurements are performed in standard cortisol dilutions in PBS 7.4 in the following concentrations: 1 ng mL-1, 10ng mL -1, 100ng mL-1, 200ng mL-1, 500ng mL-1 and 1 µg. Then, the electrodes with and without antibody were characterized, via electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), FEG microscopy, Goniometry and Zeta Potential. Goniometry assays showed an increase of hydrophilic profile of the nanocomposites after antibody modification conferring a suit surface for biological elements deposition, Fig 1. FTIR and FEG Microscopy provided the interaction of antibody-nanocomposite and antibody- antigen complex, Fig 2 and 3. Infrared analysis showed bands assigned to methyl groups disappeared after antibody adhesion and this condition may relates to the recovery of heavy chains of IgG on the nanocomposite. The increase in capacitance value after each sensory electrochemical measurement provided a cortisol curve calibration, Fig 4. The zeta potential of the immune complex formation showed an increase of values indicating a high interaction of immune system, mainly for GA condition, Fig 5. The cortisol calibration curve was improved by GA intercalation and the detections were enhanced in concentrated antibody condition, Fig 6. Figure 1

All Fiber-Optic Immunosensors Based on Elliptical Core Helical Intermediate-Period Fiber Grating with Low-Sensitivity to Environmental Disturbances.

An all fiber-optic immunosensor based on elliptical core helical intermediate-period fiber grating (E-HIPFG) is proposed for the specific detection of human immunoglobulin G (human IgG). E-HIPFGs are all-fiber transducers that do not include any additional coating materials or fiber architectures, simplifying the fabrication process and promising the stability of the E-HIPFG biosensor. For human IgG recognition, the surface of an E-HIPFG is functionalized by goat anti-human IgG. The functionalized E-HIPFG is tested by human IgG solutions with a concentration range of 10–100 μg/mL and shows a high sensitivity of 0.018 nm/(μg/mL) and a limit of detection (LOD) of 4.7 μg/mL. Notably, the functionalized E-HIPFG biosensor is found to be insensitive to environmental disturbances, with a temperature sensitivity of 2.6 pm/°C, a strain sensitivity of 1.2 pm/με, and a torsion sensitivity of −23.566 nm/(rad/mm). The results demonstrate the considerable properties of the immunosensor, with high resistance to environmental perturbations, indicating significant potential for applications in mobile biosensors and compact devices.

Protein microbeadification to achieve highly concentrated protein formulation with reversible properties and in vivo pharmacokinetics after reconstitution

A protein precipitation technique was optimized to produce biophysically stable ‘protein microbeads’, applicable to highly concentrated protein formulation. Initially, production of BSA microbeads was performed using rapid dehydration by vortexing in organic solvents followed by cold ethanol treatment and a vacuum drying. Out of four solvents, n-octanol produced the most reversible microbeads upon reconstitution. A Shirasu porous glass (SPG) membrane emulsification technique was utilized to enhance the size distribution and manufacturing process of the protein microbeads with a marketized human IgG solution. Process variants such as dehydration time, temperature, excipients, drying conditions, and initial protein concentration were evaluated in terms of the quality of IgG microbeads and their reversibility. The hydrophobized SPG membrane produced a narrow size distribution of the microbeads, which were further enhanced by shorter dehydration time, low temperature, minimized the residual solvents, lower initial protein concentration, and addition of trehalose to the IgG solution. Final reversibility of the IgG microbeads with trehalose was over 99% at both low and high protein concentrations. Moreover, the formulation was highly stable under repeated mechanical shocks and at an elevated temperature compared to its liquid state. Its in vivo pharmacokinetic profiles in rats were consistent before and after the ‘microbeadification’.

Hyper-Enriched Anti-RSV Immunoglobulins Nasally Administered: A Promising Approach for Respiratory Syncytial Virus Prophylaxis.

Respiratory syncytial virus (RSV) is a public health concern that causes acute lower respiratory tract infection. So far, no vaccine candidate under development has reached the market and the only licensed product to prevent RSV infection in at-risk infants and young children is a monoclonal antibody (Synagis®). Polyclonal human anti-RSV hyper-immune immunoglobulins (Igs) have also been used but were superseded by Synagis® owing to their low titer and large infused volume. Here we report a new drug class of immunoglobulins, derived from human non hyper-immune plasma that was generated by an innovative bioprocess, called Ig cracking, combining expertises in plasma-derived products and affinity chromatography. By using the RSV fusion protein (F protein) as ligand, the Ig cracking process provided a purified and concentrated product, designated hyper-enriched anti-RSV IgG, composed of at least 15-20% target-specific-antibodies from normal plasma. These anti-RSV Ig displayed a strong in vitro neutralization effect on RSV replication. Moreover, we described a novel prophylactic strategy based on local nasal administration of this unique hyper-enriched anti-RSV IgG solution using a mouse model of infection with bioluminescent RSV. Our results demonstrated that very low doses of hyper-enriched anti-RSV IgG can be administered locally to ensure rapid and efficient inhibition of virus infection. Thus, the general hyper-enriched Ig concept appeared a promising approach and might provide solutions to prevent and treat other infectious diseases.ImportanceRespiratory Syncytial Virus (RSV) is the major cause of acute lower respiratory infections in children, and is also recognized as a cause of morbidity in the elderly. There are still no vaccines and no efficient antiviral therapy against this virus. Here, we described an approach of passive immunization with a new class of hyper-enriched anti-RSV immunoglobulins (Ig) manufactured from human normal plasma. This new class of immunoglobulin plasma derived product is generated by an innovative bioprocess, called Ig cracking, which requires a combination of expertise in both plasma derived products and affinity chromatography. The strong efficacy in a small volume of these hyper-enriched anti-RSV IgG to inhibit the viral infection was demonstrated using a mouse model. This new class of immunoglobulin plasma-derived products could be applied to other pathogens to address specific therapeutic needs in the field of infectious diseases or even pandemics, such as COVID-19.

Optimization of High-Density Fe-Au Nano-Arrays for Surface-Enhanced Raman Spectroscopy of Biological Samples.

The method of realizing nanostructures using porous alumina templates has attracted interest due to the precise geometry and cheap cost of nanofabrication. In this work, nanoporous alumina membranes were utilized to realize a forest of nanowires, providing a bottom-up nanofabrication method suitable for surface-enhanced Raman spectroscopy (SERS). Gold and iron were electroplated through the straight channels of the membrane. The resulting nanowires are, indeed, made of an active element for plasmonic resonance and SERS as the hexagonal distribution of the nanowires and the extreme high density of the nanowires allows to excite the plasmon and detect the Raman signal. The method to reduce the distance between pores and, consequently, the distance of the nanowires after electrodeposition is optimized here. Indeed, it has been predicted that the light intensity enhancement factor is up to 1012 when the gap is small than 10 nm. Measurements of Raman signal of thiol groups drying on the gold nanowires show that the performance of the device is improved. As the thiol group can be linked to proteins, the device has the potential of a biosensor for the detection of a few biomolecules. To assess the performance of the device and demonstrate its ability to analyze biological solutions, we used it as SERS substrates to examine solutions of IgG in low abundance ranges. The results of the test indicate that the sensor can convincingly detect biomolecules in physiologically relevant ranges.

Multiphysics Modeling and Simulation of Subcutaneous Injection and Absorption of Biotherapeutics: Model Development.

Many monoclonal antibodies (mAbs) are administered via subcutaneous (SC) injection. Local transport and absorption kinetics and mechanisms, however, remain poorly understood. A multiphysics computational model was developed to simulate the injection and absorption processes of a protein solution in the SC tissue. Quantitative relationships among tissue properties and transport behaviors of an injected solution were described by respective physical laws. SC tissue was treated as a 3-dimensional homogenous, poroelastic medium, in which vasculatures and lymphatic vessels were implicitly treated. Tissue deformation was considered, and interstitial fluid flow was modeled by Darcy's law. Transport of the drug mass was described based on diffusion and advection, which was integrated with tissue mechanics and interstitial fluid dynamics. Injection and absorption of albumin and IgG solutions were simulated. Upon injection, a sharp rise in tissue pressure, porosity, and fluid velocity could be observed at the injection tip. Largest tissue deformation appeared at the model surface. Transport of drug mass out of the injection zone was minimal. Absorption by local lymphatics was found to last several weeks. A bottom-up method was developed to simulate drug transport and absorption of protein solutions in skin tissue base on physical principles. The results appear to match experimental observations.

Control of viscosity in biopharmaceutical protein formulations

Controlling the viscosity of concentrated protein solutions – usually reducing – is an open challenge, with major recent relevance in protein formulations for biopharmaceutical, medical, food, and other applications. The addition of viscosity-reducing additives generally not only changes the viscosity of the protein solutions but also the actual secondary/tertiary structure of the proteins, which is usually highly undesirable, and can be even toxic in systems, such as for biopharmaceutical applications. Therefore, it is of major importance to be able to establish control over the combination of viscosity-affecting additives and adequate protein stability, usually at high protein concentrations. Here, we demonstrate the control and manipulation of the viscosity profile of a selected protein solution (monoclonal antibody of immunoglobulin gamma type IgG) of direct biopharmaceutical relevance, by identifying elementary viscosity contributions via selected additives that target different protein–protein interactions. Specifically, a combined study of viscosity control and protein aggregation is performed with viscosity characterized by microfluidic measurements and protein aggregation by size-exclusion chromatography, where aggregation data is further supplemented with conformational stability measurements via thermal and chemical protein denaturation. A dissection of contributions to total viscosity – steric, electrostatic, hydrophobic, van der Waals – is performed. A novel mechanism of the impact of electrostatic interactions on the viscosity of IgG solutions is proposed based on interacting charged protein patches subjected to orientational alignment under flow birefringence. More generally, we show a control over the interplay of viscosity, potency and stability in a distinct protein system, as a general contribution to understanding the viscosity in different colloidal, biological, and soft materials.

Carbamylation reduces the capacity of IgG for hexamerization and complement activation.

SummaryCarbamylation is a post‐translational modification that can be detected on a range of proteins, including immunoglobulin (Ig)G, in several clinical conditions. Carbamylated IgG (ca‐IgG) was reported to lose its capacity to trigger complement activation, but the mechanism remains unclear. Because C1q binds with high affinity to hexameric IgG, we analyzed whether carbamylation of IgG affects binding of C1q, hexamerization and complement‐dependent cytotoxicity (CDC). Synovial tissues of rheumatoid arthritis (RA) patients were analyzed for the presence of ca‐IgG in vivo. Synovial tissues from RA patients were analyzed for the presence of ca‐IgG using mass spectrometry (MS). Monomeric or hexameric antibodies were carbamylated in vitro and quality in solution was controlled. The capacity of ca‐IgG to activate complement was analyzed in enzyme‐linked immunosorbent (ELISAs) and cellular CDC assays. Using MS, we identified ca‐IgG to be present in the joints of RA patients. Using in vitro carbamylated antibodies, we observed that ca‐IgG lost its capacity to activate complement in both solid‐phase and CDC assays. Mixing ca‐IgG with non‐modified IgG did not result in effective inhibition of complement activation by ca‐IgG. Carbamylation of both monomeric IgG and preformed hexameric IgG greatly impaired the capacity to trigger complement activation. Furthermore, upon carbamylation, the preformed hexameric IgG dissociated into monomeric IgG in solution, indicating that carbamylation influences both hexamerization and C1q binding. In conclusion, ca‐IgG can be detected in vivo and has a strongly reduced capacity to activate complement which is, in part, mediated through a reduced ability to form hexamers.

Immunoglobulin G structure and rheumatoid factor epitopes.

Antibodies are important for immunity and exist in several classes (IgM, IgD, IgA, IgG, IgE). They are composed of symmetric dimeric molecules with two antigen binding regions (Fab) and a constant part (Fc), usually depicted as Y-shaped molecules. Rheumatoid factors found in patients with rheumatoid arthritis are autoantibodies binding to IgG and paradoxically appear to circulate in blood alongside with their antigen (IgG) without reacting with it. Here, it is shown that rheumatoid factors do not react with native IgG in solution, and that their epitopes only become accessible upon certain physico-chemical treatments (e.g. heat treatment at 57 °C), by physical adsorption on a hydrophobic surface or by antigen binding. Moreover, chemical cross-linking in combination with mass spectrometry showed that the native state of IgG is a compact (closed) form and that the Fab parts of IgG shield the Fc region and thereby control access of rheumatoid factors and presumably also some effector functions. It can be inferred that antibody binding to pathogen surfaces induces a conformational change, which exposes the Fc part with its effector sites and rheumatoid factor epitopes. This has strong implications for understanding antibody structure and physiology and necessitates a conceptual reformulation of IgG models.

Freeze/thaw of IGG solutions

In this communication, the effect of mannitol and trehalose crystallization on the unfolding of IgG1, a monoclonal antibody, in the frozen state with repeat freeze/thaw under different pH conditions was explored. Formulations were annealed at −20 °C for 20 h five times (interrupted by freeze/thaw). This was done to induce excipient crystallization. Characterization of the frozen-thawed samples was performed by circular dichroism, particle analysis, and size exclusion chromatography. At a pH of 3, formation of insoluble and soluble aggregates was observed however, these could be reduced by the use of a surfactant. Cryoprotectant free formulations showed higher monomer content after freeze/thaw. At pH5, a single freeze/thaw cycle did not result in a significant increase in particle numbers. At pH range of 4–7 however, aggregate formation in the size range of 1–25 µm was observed after 5freeze/thaw cycles.

Surface Area Enhancement of Nanomechanical Disk Resonators Using MWCNT for Mass-Sensing Applications.

This work presents fabrication of thermal-piezoresistive nanoelectromechanical silicon disk resonators and their characterization as highly sensitive mass sensors. Forest of multiwall carbon nanotubes (MWCNTs) has been grown on the top surface of the fabricated devices, increasing the resonator effective surface area, which, in turn, increases the adsorption capacity and, therefore, the frequency shift of the sensor in molecular or particulate detection applications. To investigate the effect of the enhanced surface area on frequency shift, devices with and without MWCNTs were exposed to an aqueous solution of manganese sulfate for different deposition times and the resonance frequency shift was recorded accordingly. The measured frequency shift for the devices covered with MWCNTs was 14× higher than similar bare silicon devices. Furthermore, mass loading experiments were performed using gold nanoparticles as the loading mass. A novel way to attach gold nanoparticles on the carbon nanotubes (CNTs) wall was developed here. Oxygen plasma treatment introduced dangling bonds on the MWCNTs walls to facilitate the bonding between them and trimethoxysilane aldehyde molecules, forming the self-assembled monolayer (SAM). After functionalization of the device with SAM and antiinfluenza H1N1 viruses (AB), the device was exposed to a solution of Antimouse IgG (whole molecule)-gold antibody produced in goat products. The results showed more than three times response enhancement for the resonators with MWCNTs.

Holographic Characterization of Protein Aggregates in the Presence of Silicone Oil and Surfactants

Characterizing protein aggregates in the presence of silicone oil is a long standing challenge for the pharmaceutical industry. Silicone oil is often used as a lubricant in devices that deliver and store therapeutic protein products and has been linked to protein aggregation, which can compromise a drug’s effectiveness or cause autoimmune responses in patients. Most traditional technologies cannot quantitatively distinguish protein aggregates and silicone oil in their native formulations for sizes less than 5 μm. We use holographic video microscopy to study protein aggregation to demonstrate its capability to quantitatively distinguish protein aggregates and silicone oil in the presence of varying amounts of the surfactants SDS and polysorbate 80 in the size range of 0.5-10 μm without the need for dilution or special sample preparation. We show that SDS denatures proteins and stabilizes silicone oil. We also show that polysorbate 80 may limit protein aggregate formation if it is added to an IgG solution before introducing silicone oil.

Suppression of Aggregation of Therapeutic Monoclonal Antibodies during Storage by Removal of Aggregation Precursors Using a Specific Adsorbent of Non-Native IgG Conformers.

The quality of preparations of therapeutic IgG molecules, widely used for the treatment of various diseases, should be maintained during storage and administration. Nevertheless, recent studies demonstrate that IgG aggregation is one of the most critical immunogenicity risk factors that compromises safety and efficacy of therapeutic IgG molecules in the clinical setting. During the IgG manufacturing process, 0.22-μm membrane filters are commonly used to remove aggregates. However, particles with a diameter below 0.22 μm (small aggregates) are not removed from the final product. The residual species may grow into large aggregates during the storage period. In the current study, we devised a strategy to suppress IgG aggregate growth by removing aggregation precursors using the artificial protein AF.2A1. This protein efficiently binds the Fc region of non-native IgG conformers generated under chemical and physical stresses. Magnetic beads conjugated with AF.2A1 were used to remove non-native monomers and aggregates from solutions of native IgG and from native IgG solutions spiked with stressed IgG. The time-dependent growth of aggregates after the removal treatment was monitored. The removal of aggregation precursors, i.e., non-native monomers and nanometer aggregates (<100 nm), suppressed the aggregate growth. The presented findings demonstrate that a removal treatment with a specific adsorbent of non-native IgG conformers enables long-term stable storage of therapeutic IgG molecules and will facilitate mitigation of the immunogenicity of IgG preparations.

Arginine suppresses opalescence and liquid–liquid phase separation in IgG solutions

Antibody formulation often necessitates the protein concentration to be increased above 100 mg/ml, because of the large therapeutic doses of antibodies required and the volume limitations of subcutaneous injections. However, high concentrations of antibody lead to opalescent states in solution, resulting in safety and application problems. In this study, we investigated the effect of additives on opalescence in IgG solutions. Arginine (Arg) was observed to most effectively suppress opalescence in IgG solutions among the additives tested, which included guanidine hydrochloride, NaCl, and other amino acids. Moreover, Arg also suppressed liquid–liquid phase separation (LLPS) of highly concentrated IgG solutions during incubation at low temperature. Comparative analysis showed that the effects of Arg on opalescence and LLPS in IgG solutions result from its unique structure, which comprises an amino acid main chain, a guanidinium group, and a counter ion. These results indicate that Arg has high potency as an excipient in antibody drug formulations for the suppression of opalescence and LLPS as well as protein aggregation.