Size-exclusion High-performance Liquid Chromatography Research Articles

A Novel Tetravalent CD95/Fas Fusion Protein With Superior CD95L/FasL Antagonism.

Inhibition of CD95/Fas activation is currently under clinical investigation as a therapy for glioblastoma multiforme and preclinical studies suggest that disruption of the CD95-CD95L interaction could also be a strategy to treat inflammatory and neurodegenerative disorders. Besides neutralizing anti-CD95L/FasL antibodies, mainly CD95ed-Fc, a dimeric Fc fusion protein of the extracellular domain of CD95 (CD95ed), is used to prevent CD95 activation. In view of the fact that full CD95 activation requires CD95L-induced CD95 trimerization and clustering of the resulting liganded CD95 trimers, we investigated whether fusion proteins of the extracellular domain of CD95 with a higher valency than CD95ed-Fc have an improved CD95L-neutralization capacity. We evaluated an IgG1(N297A)-based tetravalent CD95ed fusion protein which was obtained by replacing the variable domains of IgG1(N297A) with CD95ed (CD95ed-IgG1(N297A)) and a hexavalent variant obtained by fusion of CD95ed with a TNC-Fc(DANA) scaffold (CD95ed-TNC-Fc(DANA)) promoting hexamerization. The established N297A and DANA mutations were used to minimize FcγR binding of the constructs under maintenance of neonatal Fc receptor (FcRn) binding. Size exclusion high-performance liquid chromatography indicated effective assembly of CD95ed-IgG1(N297A). More important, CD95ed-IgG1(N297A) was much more efficient than CD95ed-Fc in protecting cells from cell death induction by human and murine CD95L. Surprisingly, despite its hexavalent structure, CD95ed-TNC-Fc(DANA) displayed an at best minor improvement of the capacity to neutralize CD95L suggesting that besides valency, other factors, such as spatial organization and agility of the CD95ed domains, play also a role in neutralization of CD95L trimers by CD95ed fusion proteins. More studies are now required to evaluate the superior CD95L-neutralizing capacity of CD95ed-IgG1(N297A) in vivo.

Aqueous ozone effects on wheat gluten: Yield, structure, and rheology.

This study investigates the impact of aqueous ozone (AO) on the yield, molecular structure, and rheological properties of wheat gluten separated using the batter procedure. Employing strong gluten flour (SGF) and weak gluten flour (WGF), we demonstrate that AO pretreatment significantly enhances the yield and purity of separated starch and gluten. Surface hydrophobicity, free sulfhydryl groups, Fourier transform infrared spectroscopy (FTIR), Raman, and size exclusion-high-performance liquid chromatography (SE-HPLC) analyses were used to evaluate the effects of AO on the molecular structure of gluten. Our analysis reveals that low concentrations of AO induce specific modifications in gluten proteins. AO treatment increases cross-linking in glutenin macropolymer (GMP), reduces surface hydrophobicity, and stabilizes secondary and tertiary structures. These changes include an increase in β-sheet content by approximately 9% and a corresponding decrease in β-turn structures, leading to enhanced viscoelastic properties of the gluten. The research highlights AO's potential as a sustainable and efficient agent in wheat flour processing, offering advancements in both product quality and eco-friendly processing techniques. Future research should optimize AO treatment parameters and explore its effects on different cereal types further to enhance its applicability and benefits in food processing. PRACTICAL APPLICATION: Our work substantially advances the existing knowledge on wheat flour processing by demonstrating the multifaceted benefits of AO pretreatment. We unveil significant improvements in the yield and purity of starch and gluten when compared to conventional separation methods. Moreover, our in-depth analysis of molecular changes induced by AO, including increased cross-linking, alterations in surface hydrophobicity, and modifications in glutenin macropolymer content, provides new insights into how AO affects the viscoelastic properties of gluten. This contribution is pivotal for the development of more efficient, sustainable, and eco-friendly wheat flour processing technologies.

Novel identification of mAbs by Raman spectroscopy

AbstractThe identification of the critical quality attributes (CQAs) of monoclonal antibodies (mAbs) is a key component of quality by design. Traditional detection methods for the identification of mAbs, such as peptide mapping and weak cation‐exchange chromatography (WCX‐HPLC), require sophisticated equipment, experienced staff, and a considerable amount of time. In this study, the novel identification of mAbs was performed using Raman spectroscopy, combined with the evaluation of other CQAs, such as the appearance and purity of size‐exclusion high‐performance liquid chromatography(SEC‐HPLC), charge heterogeneity of WCX‐HPLC, and bioassay. Raman spectroscopy achieved comparable results to the conventional, but complex, approach to identifying anti‐vascular endothelial growth factor antibodies. Raman spectroscopy was used to identify and distinguish between different antibody types. Additionally, the Raman technique with principal component analysis of multivariate algorithms is rapid, efficient, and accurate for mAb identification; this technique has great potential to support biopharmaceutical development and counterfeiters.

Enhancing monoclonal antibody stability during protein a chromatography using 2-methyl imidazolium dihydrogen phosphate

This study investigates the impact of 2-methyl imidazolium dihydrogen phosphate (2-MIDHP) on monoclonal antibody (mAb) aggregation during the Protein A purification stage, at a low pH (pH 3.0), and the viral inactivation phase. Size-exclusion high-performance liquid chromatography (SE-HPLC) and dynamic light scattering (DLS) were used to assess the mAb aggregation. Additionally, the influence of 2-MIDHP on mAb recovery, host cell protein (HCP) clearance, and Protein A leaching was investigated. Thermal stability of mAb, eluted in buffers containing 5 % to 25 % 2-MIDHP was analysed, using differential scanning calorimetry (DSC). Structural insights were obtained via circular dichroism (CD) and fluorescence spectroscopy. Our findings indicated that 2-MIDHP exerted a concentration-dependent protective effect against mAb aggregation, at the pH of 3.0. As the concentration of 2-MIDHP was increased from 0 % to 25 %, the aggregation was significantly reduced from 3.8 ± 0.01 % to 0.56 ± 0.002 %, as analysed by SE-HPLC. Addition of 2-MIDHP did not significantly impact the mAb recovery, HCP clearance, or Protein A leaching. DSC data supported these results, with higher 2-MIDHP concentrations leading to increased melting temperatures of mAb. CD and fluorescence spectroscopy revealed no significant changes in the secondary structure or aromatic residue environment in 2-MIDHP-treated samples, despite the observed reduction in aggregation. The results suggested that 2-MIDHP mitigated mAb aggregation during Protein A purification, possibly by stabilizing the protein structure under acidic stress conditions. These findings offer valuable insights for improving the robustness of mAb purification processes, enhancing product quality and yield.

Monitoring of the disulfide scrambled species by mixed-mode SEC-HPLC during the purification of a bispecific antibody

Size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) is an analytical method routinely used for assessing aggregation content in protein samples. As SEC-HPLC separates analytes based on their hydrodynamic radius, it generally lacks the capability of differentiating species that are similar in size. Recently while purifying a bispecific antibody (bsAb), we noticed that SEC-HPLC can provide certain degree of resolution between the target bsAb and a disulfide scrambled form, although these two species were identical in molecular weight. In seeing the unexpected potential of SEC-HPLC at resolving species with similar size, we further tested Zenix SEC-300, a mixed-mode SEC-HPLC column from Sepax, which was reported to be capable of separating protein analytes based on other factors besides size. The Zenix column indeed provided resolution much better than the regular SEC-HPLC column. Upon further optimization, the Zenix column allowed close to baseline separation of the correctly folded and the disulfide scrambled species. The current study, as a complement to the previous reports, further demonstrates that mix-mode SEC-HPLC is capable of separating protein analytes that are close in size but are different in conformation and/or surface characteristics.

Method for quantifying free hemoglobin, distinct from the hemoglobin-haptoglobin complex, in human serum

The measurement of free hemoglobin (free Hb) in blood is crucial for assessing the risk of organ damage in patients with hemolytic diseases. However, the colorimetric method, commonly used in clinical practice, does not distinguish between free Hb and the hemoglobin-haptoglobin complex (Hb-Hp) in the blood, instead reflecting the total Hb level. Although size-exclusion high-performance liquid chromatography (SEC-HPLC) can specifically measure free Hb, its clinical use is limited by long assay times. Here, we developed a novel assay method for the rapid quantification of free Hb in serum, distinguishing it from Hb-Hp, using a latex agglutination immunoturbidimetric assay (LATIA). This method could be used to measure free Hb in sera in the range of 1–100 μg/mL in approximately 15 min using an automatic biochemistry analyzer. Using Hb-spiked serum samples from healthy adults, there was a high correlation with Hb levels determined using the newly developed method and SEC-HPLC, indicating a high specificity for free Hb. This novel assay can be used to monitor levels of free Hb in patients with various hemolytic diseases and to design therapeutic strategies based on measured values. However, further studies are required to assess its clinical performance.

Effect of carboxymethyl chitosan on the storage stability of rice dough during frozen storage

In this study, we aimed to determine the effect of carboxymethyl chitosan (CMCh) and carboxymethyl cellulose sodium (CMCNa) on the quality of frozen rice dough. We used a variety of methods to conduct a thorough investigation of frozen rice dough, including nuclear magnetic resonance (NMR), scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, size exclusion high-performance liquid chromatography (SE-HPLC), X-ray diffraction (X-RD), differential scanning calorimetry (DSC), and rapid visco analyzer (RVA). Our findings showed that frozen storage caused significant damage to the texture of rice dough, and this damage was reduced by the inclusion of CMCh, which led to a gradual change in the orderly structure of proteins. The degree of cross-linking between CMCh-B (DS:1; 0.5 %, 1 %, and 1.5 %) and the large protein polymer was significantly higher than that between CMCh-A (DS:0.8; 0.5 %, 1 %, and 1.5 %) and CMCNa (DS:1; 1 %), which decreased the ability of bound water to become free water. This resulted in the increase of tan δ, which effectively delayed the structural transformation of frozen rice dough. Furthermore, the introduction of CMCh delayed the immediate order of starch and crystal structure modifications, altering the thermal properties and pasting qualities of the frozen rice dough. Therefore, 1.5 % CMCh-B showed the best protective effect on frozen rice dough.

Extractability and chromatographic separation of proteins from potato (Solanum tuberosum L.) trimmings

By-products from the potato processing industry, like potato trimmings, are sustainable sources of proteins. Here, a size-exclusion high performance liquid chromatography (SE-HPLC) method was applied to simultaneously determine the extractability and aggregation state of proteins from three batches of potato trimmings of different cultivars. Obtained SE-HPLC profiles allowed distinguishing between the patatin and protease inhibitor fractions of potato proteins. Moreover, only 75% of the crude proteins could be extracted in phosphate buffer containing sodium dodecyl sulfate and a reducing agent, indicating the presence of physical extraction barriers. Ball milling for 5 min significantly increased protein extractability, but prolonged treatment resulted in aggregation of native patatin and a reduced protein extractability. Microwave-dried trimmings had a lower protein extractability than freeze-dried trimmings. In future research, the SE-HPLC method can be used to examine changes in potato protein (fractions) as a result of processing.

Purification, and characterization of a new pro-coagulant protein from Iranian Echis carinatus venom

This work aimed to purify the proteins that cause blood coagulation in the venom of the Iranian Echis carinatus snake species in a comprehensive manner. Gel filtration chromatography (GFC), Ion exchange chromatography (IEC), and Size Exclusion High-Performance Liquid Chromatography (SEC-HPLC) were utilized in the purification of the coagulation factors. The prothrombin clotting time (PRCT) and SDS-PAGE electrophoresis were performed to confirm the coagulative fractions. The fraction with the shortest coagulation time was selected. The components of this designated fraction were identified through matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF) following thorough purification. Circular dichroism (CD) was employed to determine the second structure of the coagulation factor. The crude venom (CV) was analyzed and had a total protein concentration of 97%.Furthermore, the PRCT of the crude venom solution at a concentration of 1 mg/ml was determined to be 24.19 ± 1.05 s. The dosage administered was found to be a factor in the venom's capacity to induce hemolysis. According to CD analysis, the protein under investigation had a helical structure of 16.7%, a beta structure of 41%, and a turn structure of 9.8%. CHNS proved that the purified coagulant protein had a Carbon content of 77.82%, 5.66% Hydrogen, 3.19% Nitrogen, and 0.49% Sulphur. In the present investigation, a particular type of snake venom metalloproteinase (SVMP) has undergone the process of purification and characterization and has been designated as EC-124. This purified fraction shows significant efficacy as a procoagulant. Our findings have shown that this compound has a function similar to factor X and most likely it can cause blood coagulation by activating factor II (FII).

Soy protein isolate-catechin complexes conjugated by pre-heating treatment for enhancing emulsifying properties: Molecular structures and binding mechanisms

This study aimed to investigate the impact of different pre-heating temperatures (ranging from 40 °C to 80 °C) on the interactions between soy protein isolate (SPI) and catechin to effectively control catechin encapsulation efficiency and optimize the emulsifying properties of soy protein isolate. Results showed that optimal heat treatment at 70 °C improved catechin encapsulation efficiency up to 93.71 ± 0.14 %, along with the highest solubility, enhanced emulsification activity index and improved thermal stability of the protein. Multiple spectroscopic techniques revealed that increasing pretreatment temperature (from 40 °C to 70 °C) altered the secondary structures of SPI, resulting in a more stable unfolded structure for the composite system with a significant increase in α-helical structures and a decrease in random coil and β-sheet structures. Moreover, optimal heat treatment also leads to an augmentation of free sulfhydryl groups within complex as well as exposure of more internal chromophore amino acids on molecular surface. Size-exclusion high-performance liquid chromatography and SDS-PAGE analysis indicated that the band intensity of newly formed high-molecular-weight soluble macromolecules (>180 kDa) increased as the pre-heating temperature rose. Furthermore, fluorescence spectroscopy and molecular docking analysis suggest that hydrophobic and covalent interactions were involved in complex formation, which intensified with increasing temperature.

Determining the Impact of Genotype × Environment on Oat Protein Isolate Composition Using HPLC and LC-MS Techniques.

The effect of genotype and environment on oat protein composition was analyzed through size exclusion-high-performance liquid chromatography (SE-HPLC) and liquid chromatography-mass spectrometry (LC-MS) to characterize oat protein isolate (OPI) extracted from three genotypes grown at three locations in the Canadian Prairies. SE-HPLC identified four fractions in OPI, including polymeric globulins, avenins, glutelins, and albumins, and smaller proteins. The protein composition was dependent on the environment, rather than the genotype. The proteins identified through LC-MS were grouped into eight categories, including globulins, prolamins/avenins, glutelins, enzymes/albumins, enzyme inhibitors, heat shock proteins, grain softness proteins, and allergenic proteins. Three main globulin protein types were also identified, including the P14812|SSG2-12S seed storage globulin, the Q6UJY8_TRITU-globulin, and the M7ZQM3_TRIUA-Globulin-1 S. Principal component analysis indicated that samples from Manitoba showed a positive association with the M7ZQM3_TRIUA-Globulin-1 S allele and Q6UJY8_TRITU-globulin, while samples from Alberta and Saskatchewan had a negative association with them. The results show that the influence of G × E on oat protein fractions and their relative composition is crucial to understanding genotypes' behavior in response to different environments.

Genetic and environmental variation in protein composition of Belgian soy determined with a novel size-exclusion chromatography method

To allow the determination of soy protein composition on protein subunit level in a quantitative manner, a novel method combining size-exclusion high-performance liquid chromatography (SE-HPLC) and peak deconvolution was developed. Peaks corresponding to the different soy protein (subunits) were identified based on the analysis of (i) marker proteins, (ii) 7S and 11S globulin-enriched fractions, and (iii) cross-validation with SDS-PAGE analysis. Given the recent introduction of soybean cultivation in Belgium, the optimized SE-HPLC method was here applied to study the impact of genotype, growing location, harvest year and inoculation strategy on the protein composition of Belgian soy. Especially genotype and harvest year were key to determining protein composition, while all factors influenced the chemical composition of soy flour. The developed SE-HPLC method can be used to further understand the impact of soybean cultivation practices in Belgium and elsewhere on protein composition, which is highly relevant for novel applications of soy. In addition, this SE-HPLC method can be used to determine how various soy processing methods may affect protein composition.

Changes in the molecular and structural of wheat gluten in the presence of zein during heating: Comparative studies on gluten, glutenin and gliadin

This study comparatively investigated the heat-triggered molecular and structural changes of wheat gluten in the presence of zein to clarify the gluten and zein interaction mechanism from the perspective of gluten, glutenin, and gliadin polymerization behavior during heating. Scanning electron microscopy (SEM) images showed that zein induced more uniform dispersion of the gluten network during heating. Size exclusion-high performance liquid chromatography (SE-HPLC) results demonstrated that zein addition increased the SDS-soluble protein content in the gluten, glutenin, and gliadin during heating. After heating at 95 °C, zein increased the free sulfhydryl content in gluten and glutenin by 23.15% and 38.97%, respectively. Zein primarily enhanced the non-covalent interaction with gliadin more than with gluten and glutenin. The addition of zein led to a significant decrease in β-sheets content in gluten, glutenin, and gliadin during heating. Zein improved the thermal stability of gluten and gliadin after heating, resulting in higher degradation temperatures and lower weight loss. The improvement in rheological properties of gluten, glutenin, and gliadin during the 95 °C heating phase could be mainly attributed to the prevention of excessive protein aggregation and the promotion of non-covalent interactions by zein.

Development of a Quantitative Determination Method for Polysorbate 80 in Biologicals by Size-Exclusion High-Performance Liquid Chromatography

Development of a Quantitative Determination Method for Polysorbate 80 in Biologicals by Size-Exclusion High-Performance Liquid Chromatography

Daboialipase, a phospholipase A2 from Vipera russelli russelli venom posesses anti-platelet, anti-thrombin and anti-cancer properties

Snake venoms are known to contain toxins capable of interfering with normal physiological processes of victims. Specificity of toxins from snake venoms give scope to identify new molecules with therapeutic action and/or help to understand different cellular mechanisms. Russell's viper venom (RVV) is a mixture of many bioactive molecules with enzymatic and non-enzymatic proteins. The present article describes Daboialipase (DLP), an enzymatic phospholipase A2 with molecular mass of 14.3 kDa isolated from RVV. DLP was obtained after cation exchange chromatography followed by size-exclusion high performance liquid chromatography (SE-HPLC). The isolated DLP presented strong inhibition of adenosine di-phosphate (ADP) and collagen induced platelet aggregation. It also showed anti-thrombin properties by significantly extending thrombin time in human blood samples. Trypan blue and resazurin cell viability assays confirmed time-dependent cytotoxic and cytostatic activities of DLP on MCF7 breast cancer cells, in vitro. DLP caused morphological changes and nuclear damage in MCF7 cells. However, DLP did not cause cytotoxic effects on non-cancer HaCaT cells. Peptide sequences of DLP obtained by O-HRLCMS analysis showed similarity with a previously reported PLA2 (Uniprot ID: PA2B_DABRR/PDB ID: 1VIP_A). An active Asp at 49th position, calcium ion binding site and anticoagulant activity sites were identified in 1 VIP_A. These findings are expected to contribute to designing new anti-platelet, anticoagulant and anti-cancer molecules.

Development of a [89Zr]Zr-labeled Human Antibody using a Novel Phage-displayed Human scFv Library.

Tax-interacting protein 1 (TIP1) is a cancer-specific radiation-inducible cell surface antigen that plays a role in cancer progression and resistance to therapy. This study aimed to develop a novel anti-TIP1 human antibody for noninvasive PET imaging in patients with cancer. A phage-displayed single-chain variable fragment (scFv) library was created from healthy donors' blood. High-affinity anti-TIP1 scFvs were selected from the library and engineered to human IgG1. Purified Abs were characterized by size exclusion chromatography high-performance liquid chromatography (SEC-HPLC), native mass spectrometry (native MS), ELISA, BIAcore, and flow cytometry. The labeling of positron emitter [89Zr]Zr to the lead Ab, L111, was optimized using deferoxamine (DFO) chelator. The stability of [89Zr]Zr-DFO-L111 was assessed in human serum. Small animal PET studies were performed in lung cancer tumor models (A549 and H460). We obtained 95% pure L111 by SEC-HPLC. Native MS confirmed the intact mass and glycosylation pattern of L111. Conjugation of three molar equivalents of DFO led to the optimal DFO-to-L111 ratio of 1.05. Radiochemical purity of 99.9% and specific activity of 0.37 MBq/μg was obtained for [89Zr]Zr-DFO-L111. [89Zr]Zr-DFO-L111 was stable in human serum over 7 days. The immunoreactive fraction in cell surface binding studies was 96%. In PET, preinjection with 4 mg/kg cold L111 before [89Zr]Zr-DFO-L111 (7.4 MBq; 20 μg) significantly (P < 0.01) enhanced the tumor-to-muscle standard uptake values (SUVmax) ratios on day 5 compared with day 2 postinjection. L111 Ab targets lung cancer cells in vitro and in vivo. [89Zr]Zr-DFO-L111 is a human antibody that will be evaluated in the first in-human study of safety and PET imaging.

Artificial intelligence-driven design of the assembled major cat allergen Fel d 1 to improve its spatial folding and IgE-reactivity

BackgroundCat-derived allergens are considered as one of the most common causes of allergic diseases worldwide. Fel d 1 is a major cat allergen and plays an important role in immunoglobulin E (IgE)-reaction diagnosis. However, the two separate chains of Fel d 1 exhibited lower IgE-reactivity than its complete molecule of an assembled form, which makes it difficult to efficiently prepare and limits the application of Fel d 1 in molecular diagnosis of cat allergy. MethodsWe first applied artificial intelligence (AI) based tool AlphaFold2 to build the 3-dimensional structures of Fel d 1 with different connection modes between two chains, which were evaluated by ERRAT program and were expressed in Escherichia coli. We then calculated the expression ratios of soluble form/inclusion bodies form of optimized Fel d 1. The Circular Dichroism (CD), High Performance Liquid Chromatography-Size Exclusion Chromatography (HPLC-SEC) and reducing/non-reducing SDS-PAGE were performed to characterize the folding status and dimerization of the optimized fusion Fel d 1. The improvement of specific-IgE reactivity to optimized fusion Fel d 1 was investigated by enzyme linked immunosorbent assay (ELISA). ResultsAmong several linkers, 2 × GGGGS got the highest scores, with an overall quality factor of 100. The error value of the residues around the junction of 2 × GGGGS was lower than others. It exhibited highest proportion of soluble protein than other Fel d 1 constructs with ERRAT (GGGGS, KK as well as direct fusion Fel d 1). The results of CD and HPLC-SEC showed the consistent folding and dimerization of two fused subunits between the optimized fusion Fel d 1 and previously well-defined direct fusion Fel d 1. The overall IgE-binding absorbance of optimized fusion Fel d 1 tested by ELISA was improved compared with that of the direct fusion Fel d 1. ConclusionWe firstly provided an AI-design strategy to optimize the Fel d 1, which could spontaneously fold into its native-like structure without additional refolding process or eukaryotic folding factors. The improved IgE-binding activity and simplified preparation method could greatly facilitate it to be a robust allergen material for molecular diagnosis of cat allergy.

SEC-HPLC analysis of column load and flow-through provides critical understanding of low Protein A step yield

For a certain number of mAbs, bispecific antibodies (bsAbs) and Fc-fusion proteins that we worked on, the Protein A capture step experienced low yield (i.e., ∼80%). A previous case study suggested that non-binding aggregate formed in cell culture was the root cause of low Protein A step yield. In the current work, we selected five projects with the low Protein A yield issue to further illustrate this phenomenon. In all cases, existence of non-binding aggregates was confirmed by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis of Protein A load and flow-through. In addition, we demonstrated that aggregates failed to bind to Protein A resin mainly due to their large sizes, which prevented them from entering the resin beads. As the data suggested, SEC-HPLC analysis of Protein A load and flow-through, although not a standard procedure, can provide information that is critical for understanding the unexpected performance of Protein A chromatography in cases like those being presented here. Thus, SEC-HPLC analysis of Protein A load and flow-through is highly recommended for antibodies/Fc-fusions suffering from low Protein A yield.

Alleviative effects of carboxymethyl chitosan on the quality deterioration of frozen rice dough during freeze thaw cycles

The objective of this study was to examine the impact of carboxymethyl chitosan (CMCh) on rice dough quality during freeze–thaw cycles (FTCs). We conducted a comprehensive analysis of frozen rice dough using various techniques, including scanning electron microscopy (SEM), low–field nuclear magnetic resonance (LF–NMR), confocal laser scanning microscopy (CLSM), Fourier transform infrared spectroscopy (FT–IR), size–exclusion high–performance liquid chromatography (SE–HPLC), small–angle X–ray scattering (SAXS), X–ray diffraction (X–RD), differential scanning calorimetry (DSC), and rapid visco–analysis (RVA). Our results revealed that adding carboxymethyl chitosan (CMCh) to frozen rice dough reduced damage to starch granules and gluten proteins during FTCs compared to dough without CMCh. This resulted in the deceleration of the modification of the ordered protein structure. Furthermore, the inclusion of CMCh resulted in increased cross–linking with the large protein polymer, impairing the capacity of bound water to transition into free water within the frozen rice dough. Consequently, this led to an elevation in the storage modulus (G′), effectively retarding the textural transformation of the frozen rice dough. Introducing CMCh to frozen rice dough subsequently decelerated the alterations in the short-range order and crystal structures of starch, consequently modifying the pasting and thermal properties of the frozen rice dough. Based on the results obtained from our experiments, CMCh has a decelerating effect on the impact of frozen rice dough on FTCs.

ZGGS15, a Novel IgG4 Bispecific Monoclonal Antibody Targeting Both LAG-3 and Tigit, Demonstrates Outstanding Anti-Tumor Activity and Can be Used As Monotherapy or in Combination with Anti-PD-1 Antibodies for Cancer Immunotherapy

Background This study aims to evaluate the anti-tumor efficacy of ZGGS15, a novel IgG4 bispecific monoclonal antibody (BsAb) targeting TIGIT and LAG-3, for cancer immunotherapy. Methods: ZGGS15 was generated in-house with the standard recombinant BsAb technology. ZGGS15 proteins were purified with protein A affinity column chromatography and evaluated for purity by size-exclusion high-performance liquid chromatography. The ZGGS15 affinity was assessed using a biolayer interferometry (BLI) assay. The EC50s of ZGGS15 binding to TIGIT and LAG-3 were determined using the ELISA technique. The EC50s of ZGGS15 binding to REH, Hela, MCF-7, and HCC1954 cells were measured by flow cytometry. Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities were also evaluated by flow cytometry. BALB/c-hPD-1/hLAG-3 and BALB/c-hPD-1/hTIGIT subcutaneously transplanted with CT26.WT tumor mouse models, and peripheral blood mononuclear cells (PBMC) -humanized NOG bearing A375 tumor mouse model were used for in vivo anti-tumor efficacy evaluation. Results ZGGS15 binds specifically to activated CD4 + and CD8 + T cells and human LAG-3 and TIGIT proteins expressed by CHO-K1 cells identically with a dose-dependent nanomolar affinity of 0.69 nM and 1.87 nM for human LAG-3 and TIGIT, respectively. In vitro kinetic analysis BLI results revealed ZGGS15 has high affinity for human LAG-3 and human TIGIT proteins, with KDs of 3.05 nM and 2.65 nM, respectively. ZGGS15 binds to human LAG-3 and human TIGIT simultaneously with similar affinity. Furthermore, ZGGS15 competitively inhibited the binding of LAG-3 to MHC-II (IC50 = 0.77 nM) and the binding of TIGIT to CD155 (IC50=0.24 nM). ZGGS15 induced better T cell activation than single anti-LAG3 or anti-TIGIT mAb agent or combined with different stimuli with or without cancer cells. When combined with nivolumab, ZGGS15 can further enhance the T cell activation in a dose-dependent manner. ADCC/CDC assay results showed that ZGGS15 binding to target cells does not induce ADCC or CDC. ZGGS15 interacts with the human neonatal Fc receptor in a pH-dependent manner, which can promote recirculation of ZGGS15 with a prolonged half-life in plasma. Besides, ZGGS15 interacts with human Fc-γ receptors and the human complement component, C1q, with low binding affinities. However, ZGGS15 does not induce obvious cytokine production from PBMCs, including IFN-γ, IL-2, IL-6, IL-10, and TNF-α. In vivo anti-tumor efficacy for ZGG15 in PBMC humanized NOG mice bearing A375 tumors showed that ZGGS15 had significant anti-tumor effects in the subcutaneous A375 xenograft model. ZGGS15 showed a better anti-tumor inhibition than single anti-LAG-3 or anti-TIGIT agent and a synergetic effect when combined with nivolumab. On day 18 post-treatment, the ZSAB015 plus nivolumab treatment demonstrated a significantly tumor growth inhibition (TGI) of 95.80% ( p =0.001), compared with the vehicle group. In vivo efficacy tests in BALB/c-hPD-1/hLAG-3 mice subcutaneously transplanted with CT26.WT tumor model showed that both high and low doses of ZGGS15 could inhibit the growth of CT26.WT colon cancer. The combination of high-dose ZGGS15 plus nivolumab has better efficacy than high-dose ZGGS15 and is slightly better than nivolumab alone. The tumor volume inhibition rate (TGITV) for ZGGS15 at 2 mg/kg was 69.70%, and for ZGGS15 at 5 mg/kg plus nivolumab at 1 mg/kg, it was 94.03% ( p &amp;lt; 0.001). During the experiment, the tumor-bearing mice tolerated it well, and no adverse reactions were observed. In vivo efficacy tests of ZGG15 in BALB/c-hPD-1/hTIGIT mice subcutaneously transplanted with CT26.WT tumor model showed that ZGGS15 at a low dose (1.5 mg/kg) demonstrated a significant tumor inhibitory effect, with the TGITV of 51.42% ( p &amp;lt; 0.01). The combination of ZGGS15 high-dose with nivolumab (5 mg/kg + 1 mg/kg) showed a better tumor inhibitory effect compared with ZGGS15 high-dose (5 mg/kg), with the TGITV of 81.15% and 41.54%, respectively ( p &amp;lt; 0.05). Conclusions ZGGS15 has excellent anti-tumor activity by inhibiting LAG-3 and TIGIT as a new IgG4 BsAb. ZGGS15 also exhibits potent anti-tumor efficacy in transgenic mouse models combined with nivolumab. Additionally, ZGGS15 does not elicit ADCC or CDC, therefore avoiding the cross-reaction of non-target binding. Finally, ZGGS15 does not induce cytokine production, mitigating any potential adverse effects of CRS.