IgG Molecules Research Articles

Superparamagnetic MoS2@Fe3O4 nanoflowers for rapid resonance-Raman scattering biodetection.

Sensors for rapid and reliable detection of biomolecules are crucial for clinical medical diagnoses. Here, a rapid, ultra-sensitive, magnetic-assisted biosensor based on resonance Raman scattering at MoS2@Fe3O4 composite nanoflowers is presented. Raman shifts and X-ray photoelectron spectra indicated that the composite was formed via Fe–S covalent bonds. Convenient magnetic separations could be performed because of the superparamagnetic Fe3O4 nanoparticles. MoS2 E12g and A1g Raman peaks were used as probe signals for anti-interference immunoassays. The probe unit of the immunoassay also included goat anti-human IgG molecules that were used as the target analyte. Au substrates coupled with the goat anti-human IgG were used as capture units to form sandwich biosensors. Because of the magnetic enrichment, the detection limit was improved by three orders-of-magnitude and the detection time was reduced from 1.5 h to 1 min. Sandwich biosensors using MoS2@Fe3O4 nanoflowers as Raman probes could be very promising sensors for proteins, antigens, and other immunogenic biopolymers, as well as for corpuscular viruses and cells.

A TNFR2-Specific TNF Fusion Protein With Improved In Vivo Activity.

Tumor necrosis factor (TNF) receptor-2 (TNFR2) has attracted considerable interest as a target for immunotherapy. Indeed, using oligomeric fusion proteins of single chain-encoded TNFR2-specific TNF mutants (scTNF80), expansion of regulatory T cells and therapeutic activity could be demonstrated in various autoinflammatory diseases, including graft-versus-host disease (GvHD), experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA). With the aim to improve the in vivo availability of TNFR2-specific TNF fusion proteins, we used here the neonatal Fc receptor (FcRn)-interacting IgG1 molecule as an oligomerizing building block and generated a new TNFR2 agonist with improved serum retention and superior in vivo activity.MethodsSingle-chain encoded murine TNF80 trimers (sc(mu)TNF80) were fused to the C-terminus of an in mice irrelevant IgG1 molecule carrying the N297A mutation which avoids/minimizes interaction with Fcγ-receptors (FcγRs). The fusion protein obtained (irrIgG1(N297A)-sc(mu)TNF80), termed NewSTAR2 (New selective TNF-based agonist of TNF receptor 2), was analyzed with respect to activity, productivity, serum retention and in vitro and in vivo activity. STAR2 (TNC-sc(mu)TNF80 or selective TNF-based agonist of TNF receptor 2), a well-established highly active nonameric TNFR2-specific variant, served as benchmark. NewSTAR2 was assessed in various in vitro and in vivo systems.ResultsSTAR2 (TNC-sc(mu)TNF80) and NewSTAR2 (irrIgG1(N297A)-sc(mu)TNF80) revealed comparable in vitro activity. The novel domain architecture of NewSTAR2 significantly improved serum retention compared to STAR2, which correlated with efficient binding to FcRn. A single injection of NewSTAR2 enhanced regulatory T cell (Treg) suppressive activity and increased Treg numbers by > 300% in vivo 5 days after treatment. Treg numbers remained as high as 200% for about 10 days. Furthermore, a single in vivo treatment with NewSTAR2 upregulated the adenosine-regulating ectoenzyme CD39 and other activation markers on Tregs. TNFR2-stimulated Tregs proved to be more suppressive than unstimulated Tregs, reducing conventional T cell (Tcon) proliferation and expression of activation markers in vitro. Finally, singular preemptive NewSTAR2 administration five days before allogeneic hematopoietic cell transplantation (allo-HCT) protected mice from acute GvHD.ConclusionsNewSTAR2 represents a next generation ligand-based TNFR2 agonist, which is efficiently produced, exhibits improved pharmacokinetic properties and high serum retention with superior in vivo activity exerting powerful protective effects against acute GvHD.

Biophysical Characterization of the Contribution of the Fab Region to the IgG-FcγRIIIa Interaction.

The cell-surface receptor FcγRIIIa is crucial to the efficacy of therapeutic antibodies as well as the immune response. The interaction of the Fc region of IgG molecules with FcγRIIIa has been characterized, but until recently, it was thought that the Fab regions were not involved in the interaction. To evaluate the influence of the Fab regions in a biophysical context, we carried out surface plasmon resonance analyses using recombinant FcγRIIIa ligands. A van't Hoff analysis revealed that compared to the interaction of the papain-digested Fc fragment with FcγRIIIa, the interaction of commercially available, full-length rituximab with FcγRIIIa had a more favorable binding enthalpy, a less favorable binding entropy, and a slower off rate. Similar results were obtained from analyses of IgG1 molecules and an IgG1-Fc fragment produced by Expi293 cells. For further validation, we also prepared a maltose-binding protein-linked IgG1-Fc fragment (MBP-Fc). The binding enthalpy of MBP-Fc was nearly equal to that of the IgG1-Fc fragment for the interaction with FcγRIIIa, indicating that such alternatives to the Fab domains as MBP do not positively contribute to the IgG-FcγRIIIa interactions. Our investigation strongly suggests that the Fab region directly interacts with FcγRIIIa, resulting in an increase in the binding enthalpy and a decrease in the dissociation rate, at the expense of favorable binding entropy.

Efficient and Sustainable Platform for Preparation of a High-Quality Immunoglobulin G as an Urgent Treatment Option During Emerging Virus Outbreaks.

During the pre-vaccine era of the COVID-19 pandemic convalescent plasma has once again emerged as a major potential therapeutic form of passive immunization that in specific cases still represents irreplaceable treatment option. There is a growing concern that variable concentration of neutralizing antibodies, present in convalescent plasma which originates from different donors, apparently affects its effectiveness. The drawback can be overcome through the downstream process of immunoglobulin fraction purification into a standardized product of improved safety and efficacy. All modern procedures are quite lengthy processes. They are also based on fractionation of large plasma quantities whose collection is not attainable during an epidemic. When outbreaks of infectious diseases are occurring more frequently, there is a great need for a more sustainable production approach that would be goal-oriented towards assuring easily and readily available immunoglobulin of therapeutic relevance. We propose a refinement strategy for the IgG preparation achieved through simplification and reduction of the processing steps. It was designed as a small but scalable process to offer an immediately available treatment option that would simultaneously be harmonized with an increased availability of convalescent plasma over the viral outbreak time-course. Concerning the ongoing pandemic status of the COVID-19, the proof of concept was demonstrated on anti-SARS-CoV-2 convalescent plasma but is likely applicable to any other type depending on the current needs. It was guided by the idea of persistent keeping of IgG molecules in the solution, so that protection of their native structure could be assured. Our manufacturing procedure provided a high-quality IgG product of above the average recovery whose composition profile was analyzed by mass spectrometry as quality control check. It was proved free from IgA and IgM as mediators of adverse transfusion reactions, as well as of any other residual impurities, since only IgG fragments were identified. The proportion of S protein-specific IgGs remained unchanged relative to the convalescent plasma. Undisturbed IgG subclass composition was accomplished as well. However, the fractionation principle affected the final product’s capacity to neutralize wild-type SARS-CoV-2 infectivity, reducing it by half. Decrease in neutralization potency significantly correlated with the amount of IgM in the starting material.

Potential for a Plant-Made SARS-CoV-2 Neutralizing Monoclonal Antibody as a Synergetic Cocktail Component.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a public health crisis over the last two years. Monoclonal antibody (mAb)-based therapeutics against the spike (S) protein have been shown to be effective treatments for SARS-CoV-2 infection, especially the original viral strain. However, the current mAbs produced in mammalian cells are expensive and might be unaffordable for many. Furthermore, the emergence of variants of concern demands the development of strategies to prevent mutant escape from mAb treatment. Using a cocktail of mAbs that bind to complementary neutralizing epitopes is one such strategy. In this study, we use Nicotiana benthamiana plants in an effort to expedite the development of efficacious and affordable antibody cocktails against SARS-CoV-2. We show that two mAbs can be highly expressed in plants and are correctly assembled into IgG molecules. Moreover, they retain target epitope recognition and, more importantly, neutralize multiple SARS-CoV-2 variants. We also show that one plant-made mAb has neutralizing synergy with other mAbs that we developed in hybridomas. This is the first report of a plant-made mAb to be assessed as a potential component of a SARS-CoV-2 neutralizing cocktail. This work may offer a strategy for using plants to quickly develop mAb cocktail-based therapeutics against emerging viral diseases with high efficacy and low costs.

Two-peak Elution Profile of a Bispecific VHH-IgG Fusion Protein in Ion Exchange Purification Process caused by Atypical N-Glycosylation

In-depth analysis and thorough understanding of Nglycosylation is critical for therapeutic proteins. For IgG or IgG-like molecules, the conserved N-glycosylation at Asn297 has critical safety and efficacy implications and therefore is under close scrutiny. Any additional N-glycosylation that increases complexity and heterogeneity of the molecule, should be avoided at the early stage of drug discovery. However, unexpected glycosylation at atypical sites brings unforeseen challenges for antibody drug development process. Here, we observed a two-peak elution profile when developing ion exchange purification process for a novel bispecific VHH-IgG fusion protein. The charge and size properties of the two peaks were characterized by a variety of analytical methods. Two atypical N-glycosylation sites in the VHH domain were identified by means of mass spectrometry and confirmed by site-specific mutagenesis. Further characterization indicated that G0F was the predominant glycan species for both sites with varying occupancy. Interestingly, the addition of neutral glycans changed the charge behaviour of the fusion protein, leading to the unexpected ion exchange profile. These findings suggest that glycosylation may occur at non-canonical sites and may have significant effect on the process development and drug developability.

Development of a miniature bioreactor model to study the impact of pH and DOT fluctuations on CHO cell culture performance as a tool to understanding heterogeneity effects at large-scale.

Understanding the impact of spatial heterogeneities that are known to occur in large‐scale cell culture bioreactors remains a significant challenge. This work presents a novel methodology for mimicking the effects of pH and dissolved oxygen heterogeneities on Chinese hamster ovary (CHO) cell culture performance and antibody quality characteristics, using an automated miniature bioreactor system. Cultures of 4 different cell lines, expressing 3 IgG molecules and one fusion protein, were exposed to repeated pH and dissolved oxygen tension (DOT) fluctuations between pH 7.0–7.5 and DOT 10%–30%, respectively, for durations of 15, 30, and 60 min. Fluctuations in pH had a minimal impact on growth, productivity, and product quality although some changes in lactate metabolism were observed. DOT fluctuations were found to have a more significant impact; a 35% decrease in cell growth and product titre was observed in the fastest growing cell line tested, while all cell lines exhibited a significant increase in lactate accumulation. Product quality analysis yielded varied results; two cell lines showed an increase in the G0F glycan and decrease in G1F, G2F, and Man5; however, another line showed the opposite trend. The study suggests that the response of CHO cells to the effects of fluctuating culture conditions is cell line specific and that higher growing cell lines are most impacted. The miniature bioreactor system described in this work therefore provides a platform for use during early stage cell culture process development to identify cell lines that may be adversely impacted by the pH and DOT heterogeneities encountered on scale‐up. This experimental data can be combined with computational modeling approaches to predict overall cell culture performance in large‐scale bioreactors.

Increased Capillary Permeability in Heart Induces Diastolic Dysfunction Independently of Inflammation, Fibrosis, or Cardiomyocyte Dysfunction.

While endothelial dysfunction is suggested to contribute to heart failure with preserved ejection fraction pathophysiology, understanding the importance of the endothelium alone, in the pathogenesis of diastolic abnormalities has not yet been fully elucidated. Here, we investigated the consequences of specific endothelial dysfunction on cardiac function, independently of any comorbidity or risk factor (diabetes or obesity) and their potential effect on cardiomyocyte. The ubiquitine ligase Pdzrn3, expressed in endothelial cells (ECs), was shown to destabilize tight junction. A genetic mouse model in which Pdzrn3 is overexpressed in EC (iEC-Pdzrn3) in adults was developed. EC-specific Pdzrn3 expression increased cardiac leakage of IgG and fibrinogen blood-born molecules. The induced edema demonstrated features of diastolic dysfunction, with increased end-diastolic pressure, alteration of dP/dt min, increased natriuretic peptides, in addition to limited exercise capacity, without major signs of cardiac fibrosis and inflammation. Electron microscopic images showed edema with disrupted EC-cardiomyocyte interactions. RNA sequencing analysis of gene expression in cardiac EC demonstrated a decrease in genes coding for endothelial extracellular matrix proteins, which could be related to the fragile blood vessel phenotype. Irregularly shaped capillaries with hemorrhages were found in heart sections of iEC-Pdzrn3 mice. We also found that a high-fat diet was not sufficient to provoke diastolic dysfunction; high-fat diet aggravated cardiac inflammation, associated with an altered cardiac metabolic signature in EC-Pdzrn3 mice, reminiscent of heart failure with preserved ejection fraction features. An increase of endothelial permeability is responsible for mediating diastolic dysfunction pathophysiology and for aggravating detrimental effects of a high-fat diet on cardiac inflammation and metabolism.

Hexamerization of Anti-SARS CoV IgG1 Antibodies Improves Neutralization Capacity.

The SARS-CoV-2 pandemic and particularly the emerging variants have deepened the need for widely available therapeutic options. We have demonstrated that hexamer-enhancing mutations in the Fc region of anti-SARS-CoV IgG antibodies lead to a noticeable improvement in IC50 in both pseudo and live virus neutralization assay compared to parental molecules. We also show that hexamer-enhancing mutants improve C1q binding to target surface. To our knowledge, this is the first time this format has been explored for application in viral neutralization and the studies provide proof-of-concept for the use of hexamer-enhanced IgG1 molecules as potential anti-viral therapeutics.

Development of an immunoassay for aglycosylated murine IgG1 in mouse serum via generation of a specific tool antibody.

Aim: To develop a method for the quantitation of effector functionless mouse surrogate IgG1 drug molecules in mouse matrices. Materials & methods: A panel of antibodies that bound specifically to N297G mutation-containing mouse IgG molecules was generated in rats. The panel was screened to identify an antibody that could be used as both the capture and detection reagent in an electrochemiluminescent immunoassay. Results & conclusion: The quantitative assay developed with the N297G-specific antibody passed acceptance criteria across multiple IgG1 fragment crystallizable (Fc)-containing protein formats and provides accurate quantitation of the total levels of mouse surrogate protein Fc present in in vivo mouse serum samples. These results are useful in understanding drug integrity and the development of precise pharmacokinetic/pharmacodynamic relationships.

BNT162b2 vaccine induces antibody release in saliva: a possible role for mucosal viral protection?

Vaccination against an airborne pathogen is very effective if it induces also the development of mucosal antibodies that can protect against infection. The mRNA‐based vaccine‐encoding SARS‐CoV‐2 full‐length spike protein (BNT162b2, Pfizer/BioNTech) protects also against infection despite being administered systemically. Here, we show that upon vaccination, cognate IgG molecules are also found in the saliva and are more abundant in SARS‐CoV‐2 previously exposed subjects, paralleling the development of plasma IgG. The antibodies titer declines at 3 months from vaccination. We identified a concentration of specific IgG in the plasma above which the relevant IgG can be detected in the saliva. Regarding IgA antibodies, we found only protease‐susceptible IgA1 antibodies in plasma while they were present at very low levels in the saliva over the course of vaccination of SARS‐CoV‐2‐naïve subjects. Thus, in response to BNT162b2 vaccine, plasma IgG can permeate into mucosal sites and participate in viral protection. It is not clear why IgA1 are detected in low amount, they may be proteolytically cleaved.

Coupling Anion Exchange Chromatography with Native Mass Spectrometry for Charge Heterogeneity Characterization of Monoclonal Antibodies.

Despite the recent success of coupling anion exchange chromatography with native mass spectrometry (AEX-MS) to study anionic proteins, the utility of AEX-MS methods in therapeutic monoclonal antibody (mAb) characterization has been limited. In this work, we developed and optimized a salt gradient-based AEX-MS method and explored its utility in charge variant analysis of therapeutic mAbs. We demonstrated that, although the developed AEX-MS method is less useful for IgG1 molecules that have higher isoelectric points (pIs), it is an attractive alternative for charge variant analysis of IgG4 molecules. By elevating the column temperature and lowering the mAb pI through PNGase F-mediated deglycosylation, the chromatographical resolution from AEX separation can be significantly improved. We also demonstrated that, after PNGase F and IdeS digestion, the AEX-MS method exhibited excellent resolving power for multiple attributes in the IgG4 Fc region, including unprocessed C-terminal Lys, N-glycosylation occupancy, and several conserved Fc deamidations, making it ideally suited for multiple attribute monitoring (MAM). Through fractionation and peptide mapping analysis, we also demonstrated that the developed AEX-MS method can provide site-specific and isoform-resolved separation of Fc deamidation products, allowing rapid and artifact-free quantitation of these modifications without performing bottom-up analysis.

Maternal HPV-antibodies and seroconversion to HPV in children during the first 3\xa0years of life

To assess the dynamics of human papillomavirus (HPV) serology, we analyzed HPV6-,11-,16-,18-, and 45 antibodies in infants during the first 36 months of their life. Serial serum samples of 276/327 mother–child pairs were collected at baseline (mothers) and at months 1, 2, 6, 12, 24 and 36 (offspring), and tested for HPVL1-antibodies using the GST-L1 assay. Concordance between maternal and infant HPV-antibody levels remained high until month-6 (p < = 0.001), indicating maternal antibody transfer. At 1 month, 40–62% of the infants tested seropositive to any of the 5 HPV-types. Between 1–3 years of age, 53% (58/109) of the children born to HPV-seronegative mothers tested HPV-seropositive. Times to positive seroconversion varied between13.4 and 18.7 months, and times to negative seroconversion (decay) between 8.5 and 9.9 months. Significant independent predictors of infants’ seroconversion to LR-HPV were hand warts and mother’s history of oral warts and seroconversion to LR-HPV. No predictors of seroconversion to HR-HPV were identified. Maternal HPV-IgG-antibodies are transferred to her offspring and remain detectable for 6 months, corroborating the IgG molecule’s half-life. Seroconversion to HPV-genotypes 6, 11, 16 and 18 was confirmed among children born to HPV-seronegative mothers, implicating an immune response to these HPV-genotypes during early infancy.

Functional Changes of Therapeutic Antibodies upon Exposure to Pro-Oxidative Agents.

Therapeutic monoclonal antibodies have exerted a transformative impact on clinical practice in last two decades. However, development of a therapeutic antibody remains a complex process. Various physiochemical and functional liabilities can compromise the production or the therapeutic efficacy of antibodies. One of these liabilities is the susceptibility to oxidation. In the present study, we portrayed an oxidation-dependent vulnerability of immunoglobulins that can be of concern for therapeutic antibodies. By using a library of 119 monoclonal IgG1 molecules, containing variable domain matching clinical-stage antibodies, we demonstrated that a substantial number of these molecules acquired antigen-binding polyreactivity upon exposure to ferrous ions. Statistical analyses revealed that the potential for induction of polyreactivity by the redox-active metal ions correlated with a higher number of somatic mutations in V genes encoding variable domains of heavy and light immunoglobulin chains. Moreover, the sensitive antibodies used with biased frequencies particular V gene families encoding variable domains of their light chains. Besides the exposure to ferrous ions the induction of polyreactivity of therapeutic antibodies occurred after contact with an unrelated pro-oxidative substance—hypochlorite ions. Our data also revealed that induction of polyreactivity by pro-oxidative agents did not impact the binding of antibodies to their cognate antigens. The results from this study may contribute for better selection of antibody therapeutics with suitable developability profiles.

P074 SZN-1326 promotes colonic mucosal healing in an acute injury model of IBD by first accelerating epithelial regeneration and secondarily reducing inflammation

Abstract Background Wnt signaling plays critical roles in regulating intestinal stem cell maintenance and differentiation. The inflammatory bowel disease, ulcerative colitis (UC), is characterized by epithelial lesions, and there is a need for therapies that promote mucosal healing in addition to limiting inflammation. The Surrozen proprietary Wnt mimetic, SZN-1326, is a FZD5,8 and LRP6-specific, full-length, effectorless, bi-specific IgG1 molecule that is well-suited for impacting the intestinal epithelium where FZD5 is highly expressed. Previously, we have shown that in an acute dextran sodium sulfate (DSS) epithelial injury model, where 4% DSS was provided in the drinking water for seven days followed by 1% DSS until termination at day 10, a single dose of SZN-1326 at 1 mg/kg or higher on Day 4 robustly induced intestinal epithelial healing and reduced inflammation, and we wanted to understand more thoroughly the mechanism underlying this activity. Methods Here, we report on additional studies in the same DSS model including scRNA-seq and tissue analysis timepoints at days 3, 4, 5, 6, 7, and 10 (6-days post treatment). Results DSS impacted all tissue layers by day 3. The direct effect of SZN-1326 on the epithelium occurred as early as 24-hours after treatment (day 5 of the experiment). SZN-1326 induced and/or increased Wnt target and cell cycle gene expression in the epithelium leading to progenitor cell expansion, and following this wave of enhanced progenitor proliferation, the cells transited toward colonocyte differentiation more quickly than the progenitor cells in control treated samples. Even in the presence of 1% DSS, relative to the anti-GFP treatment samples, the absorptive cell lineage in the SZN-1326-treated epithelium was more similar at a transcriptomic level to the uninjured condition. In addition, when comparing SZN-1326 treatment to anti-GFP at 24-hours, there were no differentially expressed genes in either the stroma or immune cells. However, analysis of the later timepoints revealed a robust reduction in cytokine/inflammatory signaling in the stromal and immune cells and a drastic reduction of infiltrating monocytes and neutrophils, which we validated in the tissue by immunohistochemistry. We detected this reduction by 72-hours after treatment, and it was more pronounced by day 10, 6-days after treatment, implicating a secondary effect. Conclusion In this acute damage context, SZN-1326 promoted mucosal healing by transiently inducing epithelial progenitor cell expansion and accelerating epithelial repair. Secondary to its initial impact on the epithelium, SZN-1326 reduced stromal and immune cytokine signaling and immune cell infiltration.

A new flow cytometry assay identifies recipient IgG subtype antibodies binding donor cells: increasing donor availability for highly sensitised patients

ObjectivesThere are four immunoglobulin (IgG) subtypes that have varying complement‐activating ability: strong (IgG3 and IgG1) and weak (IgG2 and IgG4). The standard flow cytometric crossmatch (FCM) assay does not distinguish between the various subtypes of the IgG molecule. This study outlines the development and use of a novel cell‐based IgG subtype‐specific FCM assay that is able to detect the presence of and quantitate the IgG subtypes bound to donor cells.MethodsA six‐colour lyophilised reagent was designed that specifically detects the four IgG subtypes, as well as distinguishes between T cells and B cells in the lymphocyte population. To test the efficacy of this reagent, a retrospective evaluation of a group of highly sensitised patients awaiting heart and kidney transplant was carried out, who, because of positive standard FCM results, had been deemed incompatible with numerous prior potential donors.ResultsObservations in this study demonstrate that the positive standard FCM results were mainly because of the presence of noncomplement‐activating IgG2 or IgG4 antibodies. The results were supported by the absence of C3d‐binding donor‐specific antibodies (DSA) and a negative complement‐dependent cytotoxicity crossmatch (CDC).ConclusionPreliminary data presented in this study demonstrate the reliability of the novel IgG subtype assay to detect the presence of pretransplant, complement‐activating antibodies bound to donor cells. The knowledge gained from the IgG subtype assay and the C3d‐binding specificities of DSAs provides improved identification of donor suitability in pretransplant patients, potentially increasing the number of transplants.

Sample displacement chromatography of monoclonal antibody charge variants and aggregates.

The rise of biosimilar monoclonal antibodies has renewed the interest in monoclonal antibody (mAb) charge variants composition and separation. The sample displacement chromatography (SDC) has the potential to overcome the low separation efficiency and productivity associated with bind-elute separation of mAb charge variants. SDC in combination with weak cation exchanging macroporous monolithic chromatographic column was successfully implemented for a separation of charge variants and aggregates of monoclonal IgG under overloading conditions. The charge variants composition was at-line monitored by a newly developed, simple and fast analytical method, based on weak cation exchange chromatography. It was proven that basic charge variants acted as displacers of IgG molecules with lower pI, when the loading was performed 1 to 1.5pH unit below the pI of acidic charge variants. The efficiency of the SDC process is flow rate independent due to a convection-based mass transfer on the macroporous monolith. The productivity of the process at optimal conditions is 35mg of purified IgG fraction per milliliters of monolithic support with 75-80% recovery. As such, an SDC approach surpasses the standard bind-elute separation in the productivity for a factor of 3, when performed on the same column. The applicability of the SDC approach was confirmed for porous particle-based column as well, but with 1.5 lower productivity compared to the monoliths.

Прогностическое значение исследования экспонирования гликозильных детерминант на молекулах IgG в сыворотке крови больных рассеянным склерозом в зависимости от проводимой медикаментозной терапии

Актуальность. Большое значение в аутоиммунных процессах при рассеянном склерозе имеет определение антител к собственным антигенам организма. Функции иммуноглобулинов (Ig) определяются составом биомолекул гликановых остатков, присоединенных к N-гликозирующему домену в кристаллизирующем фрагменте (Fc-области) тяжелых цепей Ig. Считают, что изменения в структуре гликанов способны влиять на функционирование Ig. Так, наличие или отсутствие определенных остатков углеводов в составе антител связано с их про- или противовоспалительными свойствами. В свою очередь, это модифицирует протекание воспалительных реакций при аутоиммунных заболеваниях и может определять активность рассеянного склероза. Поэтому исследования структурных изменений Ig сыворотки крови больных рассеянным склерозом имеет важное клиническое значение. Материалы и методы. Было проведено исследование сывороток 6 когорт больных рассеянным склерозом в зависимости от группы препарата болезнемодифицирующей терапии, которую пациенты принимают, а именно: 17 пациентов до момента забора крови для указанных исследований никогда не получали никакой терапии; 15 больных рассеянным склерозом, получавших болезнемодифицирующую терапию препаратами из группы интерферонов β-1b; 12 человек после пульс-терапии метилпреднизолоном в дозе 1000 мг в течение 5–7 дней; 12 больных рассеянным склерозом разного пола и возраста, получавших терапию препаратами из группы моноклональных антител, а именно окрелизумаб в дозе 600 мг, частотой 1 раз в 6 месяцев; 9 человек — на препарате глатирамера ацетат дозой 40 мг трижды в неделю; 10 больных рассеянным склерозом, получавших лечение препаратом терифлуномид в дозе 14 мг при приеме один раз в сутки; а также 23 практически здоровых лиц. Результаты. В результате проведенных исследований выявлено, что различные виды терапии влияют на изменение гликозильных детерминант иммуноглобулинов. Выводы. Гликановые маркеры можно использовать как высокочувствительные и специфичные биомаркеры для диагностики рассеянного склероза и прогнозирования его течения.

Development of an E. coli strain for cell-free ADC manufacturing.

Recent advances in cell‐free protein synthesis have enabled the folding and assembly of full‐length antibodies at high titers with extracts from prokaryotic cells. Coupled with the facile engineering of the Escherichia coli translation machinery, E. coli based in vitro protein synthesis reactions have emerged as a leading source of IgG molecules with nonnatural amino acids incorporated at specific locations for producing homogeneous antibody–drug conjugates (ADCs). While this has been demonstrated with extract produced in batch fermentation mode, continuous extract fermentation would facilitate supplying material for large‐scale manufacturing of protein therapeutics. To accomplish this, the IgG‐folding chaperones DsbC and FkpA, and orthogonal tRNA for nonnatural amino acid production were integrated onto the chromosome with high strength constitutive promoters. This enabled co‐expression of all three factors at a consistently high level in the extract strain for the duration of a 5‐day continuous fermentation. Cell‐free protein synthesis reactions with extract produced from cells grown continuously yielded titers of IgG containing nonnatural amino acids above those from extract produced in batch fermentations. In addition, the quality of the synthesized IgGs and the potency of ADC produced with continuously fermented extract were indistinguishable from those produced with the batch extract. These experiments demonstrate that continuous fermentation of E. coli to produce extract for cell‐free protein synthesis is feasible and helps unlock the potential for cell‐free protein synthesis as a platform for biopharmaceutical production.

A Sensitivity-Enhanced Electrolyte-Gated Graphene Field-Effect Transistor Biosensor by Acoustic Tweezers.

Low-abundance biomolecule detection is very crucial in many biological and medical applications. In this paper, we present a novel electrolyte-gated graphene field-effect transistor (EGFET) biosensor consisting of acoustic tweezers to increase the sensitivity. The acoustic tweezers are based on a high-frequency bulk acoustic resonator with thousands of MHz, which has excellent ability to concentrate nanoparticles. The operating principle of the acoustic tweezers to concentrate biomolecules is analyzed and verified by experiments. After the actuation of acoustic tweezers for 10 min, the IgG molecules are accumulated onto the graphene. The sensitivities of the EGFET biosensor with accumulation and without accumulation are compared. As a result, the sensitivity of the graphene-based biosensor is remarkably increased using SMR as the biomolecule concentrator. Since the device has advantages such as miniaturized size, low reagent consumption, high sensitivity, and rapid detection, we expect it to be readily applied to many biological and medical applications.