Size Exclusion Chromatography Analysis Research Articles

Alternative Role of B/b Knob-Hole Interactions in the Fibrin Assembly.

The self-assembly of fibrin is a vital process in blood clotting, primarily facilitated by the interactions between knobs "A" and "B" in the central E region of one molecule and the corresponding holes "a" and "b" in the peripheral D regions of two other fibrin molecules. However, the precise function of the interactions between knob "B" and hole "b" during fibrin polymerization remains a subject of ongoing debate. The present study focuses on investigating intermolecular interactions between knob "B" and hole "b". We investigated the D-E-D interactions within the fibrin protofibril to accomplish this objective. Our investigation involved studying the formation of supramolecular complexes involving desAB fibrin with fibrin(ogen) fragments, specifically the D-dimer and D fragment. The research utilized analytical size-exclusion chromatography, SDS-PAGE and densitometry of SDS-PAGE images, dynamic light scattering measurements, turbidity studies, electron microscopy, and computer modeling. Our findings indicate that the interference of the D fragment into classical D-E-D interaction occurs through knob "B" of the fibrin molecule. Molecular dynamics simulations elucidate the binding of only one D region, attributed to the shift of the D-dimer toward the fibrin desAB molecule. The formation of such a complex can be considered evidence supporting the potential mechanism of the branching of protofibrils. According to this theoretical mechanism, the inclusion of the D region from an external fibrin molecule into D-E-D interactions is facilitated through "B/b" contacts.

Accurate Molar Mass Estimation of Polyolefin Blends Using High Temperature-size Exclusion Chromatography With Triple Detection: Insight Into Molecular Level Mixing of Polypropylene With Various Application Grades of Polyethylene.

In this study, a commercially available polypropylene homopolymer (H-PP) was blended with blow molding polyethylene (PE) grade via melt mixing using a compounding machine. The resulting blends were subjected to high-temperature size exclusion chromatography (SEC) analysis, coupled with infrared-5 (IR-5), viscometer (VISCO), and multi-angle laser light scattering (MALS) detectors. The molecular weight (MW) and MW distributions were investigated using SEC, and the exact blend compositions were evaluated using 13C nuclear magnetic resonance. The molecular-level mixing of the PP/PE blend composition was assessed by comparing the theoretical and measured weight-average MW (Mw) values obtained using different SEC detection modes. The measured average Mw values of different PP/PE blends obtained using VISCO and multi-angle light-scattering detection were found to be in close agreement with predicted blend ratios, compared to the conventional SEC technique (IR-5), with percentage errors ranging from -4.53 to -5.99 and 1.57 to 7.65, respectively. The linear relationship between the melt flow rate (MFR) and Mw was studied to assess the molecular-level mixing behavior of the prepared blends using different SEC detection modes. Furthermore, the SEC methodology was extended to other application grades of PE with varying MFRs to verify the blend mixing behavior with H-PP, and the obtained results are discussed.

An Underlying Cause and Solution to the Poor Size Exclusion Chromatography Performance of Antibody-Drug Conjugates.

Antibody-drug conjugate (ADC) size variants are frequently assessed by size exclusion chromatography (SEC). However, poor chromatography performance is often observed during SEC analysis. Existing studies have primarily focused on qualitatively describing non-specific interactions between ADCs and the column matrix. The purposes of the current study are to introduce an underlying cause from a novel perspective on the protein-protein interaction (PPI) mechanism, characterized by quantifying diffusion interaction parameter (kD) values, and to provide several strategies to reduce PPI and improve column performance during SEC analysis. Two kinds of ADCs with varying hydrophobicity properties and their corresponding monoclonal antibodies are used as models. The hydrophobicity of these products was verified using the relative calculated logarithm of the partition coefficient of a substance in n-octanol (oil) and water (rCLogP) and reversed-phase high performance liquid chromatography (RP-HPLC), and the size variants were analyzed using SEC. Finally, the PPI was characterized by kD values of these four products. The results of rCLogP and RP-HPLC indicated that ADC-1 is relatively hydrophobic, whereas ADC-2 is relatively hydrophilic. In the SEC analysis of the ADC-1, substituting sodium chloride with L-arginine hydrochloride or adding a specific concentration of acetonitrile as an organic solvent to the mobile phase resulted in reduced PPI and enhanced column performance. Conversely, the impact on ADC-2 was negligible. This study provides insights into improving the performance of SEC analysis for ADCs through strategies involving alterations in mobile phase composition. The changes in column performance can be quantitatively explained by the PPI mechanism.

Pirin does not bind to p65 or regulate NFκB-dependent gene expression but does modulate cellular quercetin levels.

Pirin is a non-heme iron binding protein with a variety of proposed functions including serving as a co-activator of p65 NFκB and quercetinase activity. We report here, failure to confirm pirin's primary proposed mechanism, binding of Fe(III)-pirin and p65. Analytical size exclusion chromatography (SEC) and fluorescence polarization (FP) studies did not detect an interaction. We also found no effects of pirin on TNFα-activated p65-regulated gene transcription using mouse embryonic fibroblasts (MEFs) from a pirin knockout mouse and a pirin knockdown NIH3T3 fibroblast cell line. TNFα - activated p65 response gene mRNA was neither increased nor decreased in cells with loss of pirin compared to wildtype cells. Furthermore, pirin immunofluorescence in NIH3T3 fibroblasts showed primarily a cytoplasmic localization, not nuclear as in most previous studies. This was confirmed by cell fractionation analysis. Pirin did show colocalization with the endoplasmic reticulum (ER) marker protein disulfide-isomerase (PDI) as well as cyotoplasmic labeling. We confirmed pirin's quercetinase activity in biochemical assays and demonstrated competitive inhibition by the pirin inhibitor CCG-257081. Cellular quercetin levels in cells exposed to quercetin in vitro were increased by knockdown of pirin or by treatment with pirin inhibitors. Since pirin is localized to ER and flavanols are protective of ER stress, we investigated whether pirin knockdown altered ER stress signaling but did not find any effect of pirin knockdown on ER stress response genes. Our results challenge the dominant model of pirin's function (NFκB regulation) but confirm its quercetinase activity with implications for the mechanisms of pirin binding small molecules.

Reassessing the Photochemical Upcycling of Polystyrene Using Acridinium Salts.

Polystyrene (PS) is a commodity plastic recalcitrant to chemical recycling or upcycling processes. Approaches aimed at deconstructing PS by photocatalytic means struggle to generate high-energy species capable of cleaving the robust C-H and C-C bonds of PS. We show that 9-mesityl-10-methylacridinium perchlorate (MA) is capable of upcycling various grades of PS substrates into up to 40 % benzoic acid (BAc), formic acid (FA) and small proportions of acetophenone (ACP), under visible light (456 nm) or through solar radiation. Time-resolved emission and absorption spectroscopy evidence that a reaction with oxygen is the primary photochemical step in oxygen-saturated solutions, accounting for 77 % of the photons absorbed vs. 1 % for the direct reaction with PS (0.303 M in repeating units). These results are in agreement with a mechanism in which MA-mediated photo upcycling of PS to BAc occurs through the abstraction of benzylic H atoms by reactive oxygen species generated by energy or electron transfer from the excited state of MA. Addition of triplet O2 to these radicals, followed by intra- or inter-molecular hydrogen atom transfer (HAT) generates C- or O-centered radicals then undergoing β-scission or hydroperoxide fragmentation. The formation of intermediate oligomers functionalized by terminal carbonyl groups is demonstrated by both infrared analysis and MALDI TOF mass spectrometry. These oligomers undergo further photoinduced conversion even in the absence of MA, as evidenced by size exclusion chromatography analysis of the irradiated samples.

Reassessing the Photochemical Upcycling of Polystyrene Using Acridinium Salts

AbstractPolystyrene (PS) is a commodity plastic recalcitrant to chemical recycling or upcycling processes. Approaches aimed at deconstructing PS by photocatalytic means struggle to generate high‐energy species capable of cleaving the robust C−H and C−C bonds of PS. We show that 9‐mesityl‐10‐methylacridinium perchlorate (MA) is capable of upcycling various grades of PS substrates into up to 40 % benzoic acid (BAc), formic acid (FA) and small proportions of acetophenone (ACP), under visible light (456 nm) or through solar radiation. Time‐resolved emission and absorption spectroscopy evidence that a reaction with oxygen is the primary photochemical step in oxygen‐saturated solutions, accounting for 77 % of the photons absorbed vs. 1 % for the direct reaction with PS (0.303 M in repeating units). These results are in agreement with a mechanism in which MA‐mediated photo upcycling of PS to BAc occurs through the abstraction of benzylic H atoms by reactive oxygen species generated by energy or electron transfer from the excited state of MA. Addition of triplet O2 to these radicals, followed by intra‐ or inter‐molecular hydrogen atom transfer (HAT) generates C‐ or O‐centered radicals then undergoing β‐scission or hydroperoxide fragmentation. The formation of intermediate oligomers functionalized by terminal carbonyl groups is demonstrated by both infrared analysis and MALDI TOF mass spectrometry. These oligomers undergo further photoinduced conversion even in the absence of MA, as evidenced by size exclusion chromatography analysis of the irradiated samples.

Elucidation of Mg2+ induced size and charge heterogeneity in monoclonal antibody therapeutics

Changes in charge variant profile are known to affect mAb stability and vice versa. This report elucidates the effects of magnesium metal (0.5 mM Mg2+) on trastuzumab (IgG1 antibody). Mg2+ is often used as an excipient (50–100 mM) and lubricant (5–10 % w/w) in biopharmaceutical formulations. Analytical size-exclusion chromatography (SEC) and cation-exchange chromatography (CEX) coupled with mass spectrometry (MS) were used to evaluate the size and charge heterogeneity in the thermal and metal stressed samples and compared to the control sample (room temperature). The present study unveils that presence of Mg2+ significantly increases the rate of aggregation with 9 % aggregation observed in Mg2+ stressed samples as compared to that from thermal stress (~2 %) or control sample (<1 %). Similarly, a 2-fold elevation in acidic variants was observed both in presence of Mg2+ and thermal stress, when contrasted with the control sample. Application of stress also led to the formation of 17 additional chemical modifications (7 due to thermal stress and 10 due to Mg2+ stress) which were not identified in control, predominantly involving deamidation, isomerization of aspartic acid, oxidation, and succinimide modifications. The results indicate the need for a detailed analysis of the impact of presence of metals in biotherapeutic formulations.

Evaluating the influence of the initial high molecular weight level on monoclonal antibody particle formation kinetics using a short-term chemical stress study

Protein formulations may form proteinaceous particles that vary in size from nanometers to millimeters. Monitoring the kinetics of protein particle formation, e.g., through accelerated degradation studies, is an attempt to understand and assess the rate and progression of particle populations. Little is known about whether the initial level of high molecular weight (HMW) species, or initial HMW level (IHL), of a protein solution influences the propagation of protein particle formation, and thus affects the storage stability of proteins.In this study, we have established a method to generate protein solutions of different IHLs by thermal stress. We have evaluated a 16-week thermal stability study at 40 °C of two monoclonal antibodies (mAb-A and mAb-B) at different IHLs using size exclusion chromatography (SEC) and sub-visible particle analysis. We have performed an isothermal stress study with guanidinium hydrochloride (GuaHCl) at room temperature for 300-min to evaluate the formation of HMWs analysed by SEC. The application of the Finke-Watzky (F-W) two-step nucleation model allowed us to mathematically describe the kinetics of HMW formation and to extract kinetic parameters of this process.For mAb-A, the IHLs had a marginal influence on the loss of monomer rate; instead, mAb-A exhibited fragmentation at 40 °C, which was independent of the IHL. Nevertheless, above a threshold of ≥ 7 % IHL, existing trimers/tetramers undergo conversion into higher-order oligomers at 40 °C, which is not observed at lower IHLs. In contrast, mAb-B exhibited an increased HMW formation rate above a threshold of ≥ 4 % IHL, which was reflected in the monomer decay rates at 40 °C and the F-W kinetic parameters of the chemical stress study.This case study shows that the initial level of HMWs exerts a differential influence on the progression of HMW formation. In one instance, there is a discernible acceleration in the formation of HMWs with rising IHLs. Conversely, in another example, the IHL exerts only a slight influence on HMW formation. Moreover, the results of our short-term chemical stress study are in accordance with those of a classical storage stability study conducted at 40 °C, which evaluated different IHLs. The analysis of HMW formation kinetics will enhance our understanding of the protein particle formation process and facilitate the formulation development of biotherapeutics.

High-yield production of recombinant human myelin oligodendrocyte glycoprotein in SHuffle bacteria without a refolding step

Experimental autoimmune encephalomyelitis (EAE) is a model for central nervous system (CNS) autoimmune demyelinating diseases such as multiple sclerosis (MS) and MOG antibody-associated disease (MOGAD). Immunization with the extracellular domain of recombinant human MOG (rhMOG), which contains pathogenic antibody and T cell epitopes, induces B cell-dependent EAE for studies in mice. However, these studies have been hampered by rhMOG availability due to its insolubility when overexpressed in bacterial cells, and the requirement for inefficient denaturation and refolding. Here, we describe a new protocol for the high-yield production of soluble rhMOG in SHuffle cells, a commercially available E. coli strain engineered to facilitate disulfide bond formation in the cytoplasm. SHuffle cells can produce a soluble fraction of rhMOG yielding >100 mg/L. Analytical size exclusion chromatography multi-angle light scattering (SEC-MALS) and differential scanning fluorimetry of purified rhMOG reveals a homogeneous monomer with a high melting temperature, indicative of a well-folded protein. An in vitro proliferation assay establishes that purified rhMOG can be processed and recognized by T cells expressing a T cell receptor (TCR) specific for the immunodominant MOG35–55 peptide epitope. Lastly, immunization of wild-type, but not B cell deficient, mice with rhMOG resulted in robust induction of EAE, indicating a B cell-dependent induction. Our SHuffle cell method greatly simplifies rhMOG production by combining the high yield and speed of bacterial cell expression with enhanced disulfide bond formation and folding, which will enable further investigation of B cell-dependent EAE and expand human research of MOG in CNS demyelinating diseases.

Efficient Synthesis of μ-A(BC)C Miktoarm Star Polymer Assemblies via Aqueous Photoinitiated Polymerization-Induced Self-Assembly.

In this study, green light-activated photoiniferter reversible addition-fragmentation chain transfer (RAFT) polymerization of glycerol methacrylate was performed using an ω,ω-heterodifunctional macro-RAFT agent. Because of the different RAFT controllability of two RAFT groups toward methacrylic monomers, only one RAFT group was activated under green light irradiation, leading to the formation of a diblock copolymer macro-RAFT agent with one RAFT group located at the chain end and the other RAFT group located between two blocks. The obtained diblock copolymer macro-RAFT agent was then used to mediate aqueous photoinitiated RAFT dispersion polymerization of diacetone acrylamide (DAAM), which formed μ-A(BC)C miktoarm star polymer assemblies with a diverse set of morphologies. Comparing with the ABC triblock copolymer, it was found that the architecture of the μ-A(BC)C miktoarm star polymer facilitated the formation of higher-order morphologies. Kinetic studies indicated that the aqueous photoinitiated RAFT dispersion polymerization exhibited ultrafast polymerization behavior, with quantitative monomer conversion being achieved within 5 min. Size exclusion chromatography analysis confirmed that good RAFT control was maintained during the polymerization. A morphological phase diagram for μ-A(BC)C miktoarm star polymer assemblies was constructed by varying the monomer concentration and the [DAAM]/[Macro-RAFT] ratio. We expect that this study not only develops an approach for the preparation of miktoarm star polymer assemblies but also provides mechanistic insights into the polymerization-induced self-assembly of nonlinear polymers.

MiR-182, miR-221 and miR-222 are potential urinary extracellular vesicle biomarkers for canine urothelial carcinoma

Current diagnostic methods for canine urothelial carcinoma (UC) are technically challenging or can lack specificity, hence there is a need for novel biomarkers of UC. To this end, we analysed the microRNA (miRNA) cargo of extracellular vesicles (EVs) from urine samples of dogs with UC to identify candidate miRNA biomarkers. Urine was fractionated using ultrafiltration combined with size-exclusion chromatography and small RNA sequencing analysis was performed on both the EV enriched and (EV free) protein fractions. A greater number of candidate miRNA biomarkers were detected in the EV fraction than the protein fraction, and further validation using droplet digital PCR (ddPCR) was performed on the EV enriched fraction of a second cohort of dogs with UC which indicated that miR-182, miR-221 and miR-222 were significantly overrepresented in dogs with UC when compared with healthy dogs and dogs with urinary tract infections. Pathway analysis confirmed that these three miRNAs are involved in cancer. In addition, their potential downstream gene targets were predicted and PIK3R1, a well-known oncogene is likely to be a shared target between miRNA-182 and miRNA-221/222. In summary, this study highlights the potential of urinary EV-associated miRNAs as a source of biomarkers for the diagnosis of canine UC.

Assessing Photostability of mAb Formulations In Situ Using Light-Coupled NMR Spectroscopy.

Biopharmaceuticals, such as monoclonal antibodies (mAbs), need to maintain their chemical and physical stability in formulations throughout their lifecycle. It is known that exposure of mAbs to light, particularly UV, triggers chemical and physical degradation, which can be exacerbated by trace amounts of photosensitizers in the formulation. Although routine assessments of degradation following defined UV dosages are performed, there is a fundamental lack of understanding regarding the intermediates, transient reactive species, and radicals formed during illumination, as well as their lifetimes and immediate impact post-illumination. In this study, we used light-coupled NMR spectroscopy to monitor in situ live spectral changes in sealed samples during and after UV-A illumination of different formulations of four mAbs without added photosensitizers. We observed a complex evolution of spectra, reflecting the appearance within minutes of transient radicals during illumination and persisting for minutes to tens of minutes after the light was switched off. Both mAb and excipient signals were strongly affected by illumination, with some exhibiting fast irreversible photodegradation and others exhibiting partial recovery in the dark. These effects varied depending on the mAb and the presence of excipients, such as polysorbate 80 (PS80) and methionine. Complementary ex situ high-performance size-exclusion chromatography analysis of the same formulations post-UV exposure in the chamber revealed significant loss of purity, confirming formulation-dependent degradation. Both approaches suggested the presence of degradation processes initiated by light but continuing in the dark. Further studies on photoreaction intermediates and transient reactive species may help mitigate the impact of light on biopharmaceutical degradation.

Gel‐forming properties of Bambara groundnut globulin and protein subfractions: structural and rheological characterisation

SummaryThe gel‐forming ability of the pulse proteins influences the formation of food products with desirable texture, structure, and stability. In this study, Bambara globulin was fractionated into legumin and vicilin for structural and physicochemical analyses, and heat‐induced gel rheological characterisation. Bambara globulin showed major vicilin (7S, Mw: 120 kDa) and minor legumin (11S, Mw: 410 kDa) components by size exclusion chromatography and gel electrophoresis analyses. Legumin had the lowest amount of all the charged amino acids. One basic subunit (22 kDa) was identified in the legumin fraction with predominant helical structures. The sol–gel transition temperatures increased in the order of globulin (40 °C) &lt; legumin (50 °C) &lt; vicilin (80 °C). The G′ and G″ of globulin showed relatively low dependency on heating time beyond the gel point compared to legumin and vicilin subfractions, suggesting a more rapid establishment of its gel network during gelation. Vicilin gel consisted of a microporous structure with a small lath sheet‐like structure compared to others. Bambara vicilin could be a prospective ingredient in the development of new products with defined textural characteristics.

Design of Artificial C-Peptides as Potential Anti-HIV-1 Inhibitors Based on 6-HB Formation Mechanism.

The six-helix bundle (6-HB) is a core structure formed during the membrane fusion process of viruses with the Class I envelope proteins. Peptide inhibitors, including the marketed Enfuvirtide, blocking the membrane fusion to exert inhibitory activity were designed based on the heptads repeat interactions in 6-HB. However, the drawbacks of Enfuvirtide, such as drug resistance and short half-life in vivo, have been confirmed in clinical applications. Therefore, novel design strategies are pivotal in the development of next-generation peptide-based fusion inhibitors. The de novo design of α-helical peptides against MERS-CoV and IAVs has successfully expedited the development of fusion inhibitors. The reported sequences were completely nonhomologous with natural peptides, which can provide some inspirations for the antiviral design against other pathogenic viruses with class I fusion proteins. Here, we design a series of artificial C-peptides based on the similar mechanism of 6-HB formation and general rules of heptads repeat interaction. The inhibitory activity of peptides against HIV-1 was assessed by HIV-1 Env-mediated cell-cell fusion assays. Interaction between artificial C-peptides and target peptides was evaluated by circular dichroism, polyacrylamide gel electrophoresis, size-exclusion chromatography, and sedimentation velocity analysis. Molecular docking studies were performed by using Schrödinger molecular modelling software. The best-performing artificial C-peptide, 1SR, was highly active against HIV-1 env-mediated cell-cell fusion. 1SR binds to the gp41 NHR region, assembling polymer to prevent endogenous 6-HB formation. We have found an artificial C-lipopeptide lead compound with inhibitory activity against HIV-1. Also, this paper enriched both N- and C-teminal heptads repeat interaction rules in 6-HB and provided an effective idea for next-generation peptide-based fusion inhibitors against HIV-1.

Structure–function characterization of two enzymes from novel subfamilies of manganese peroxidases secreted by the lignocellulose-degrading Agaricales fungi Agrocybe pediades and Cyathus striatus

BackgroundManganese peroxidases (MnPs) are, together with lignin peroxidases and versatile peroxidases, key elements of the enzymatic machineries secreted by white-rot fungi to degrade lignin, thus providing access to cellulose and hemicellulose in plant cell walls. A recent genomic analysis of 52 Agaricomycetes species revealed the existence of novel MnP subfamilies differing in the amino-acid residues that constitute the manganese oxidation site. Following this in silico analysis, a comprehensive structure–function study is needed to understand how these enzymes work and contribute to transform the lignin macromolecule.ResultsTwo MnPs belonging to the subfamilies recently classified as MnP-DGD and MnP-ESD—referred to as Ape-MnP1 and Cst-MnP1, respectively—were identified as the primary peroxidases secreted by the Agaricales species Agrocybe pediades and Cyathus striatus when growing on lignocellulosic substrates. Following heterologous expression and in vitro activation, their biochemical characterization confirmed that these enzymes are active MnPs. However, crystal structure and mutagenesis studies revealed manganese coordination spheres different from those expected after their initial classification. Specifically, a glutamine residue (Gln333) in the C-terminal tail of Ape-MnP1 was found to be involved in manganese binding, along with Asp35 and Asp177, while Cst-MnP1 counts only two amino acids (Glu36 and Asp176), instead of three, to function as a MnP. These findings led to the renaming of these subfamilies as MnP-DDQ and MnP-ED and to re-evaluate their evolutionary origin. Both enzymes were also able to directly oxidize lignin-derived phenolic compounds, as seen for other short MnPs. Importantly, size-exclusion chromatography analyses showed that both enzymes cause changes in polymeric lignin in the presence of manganese, suggesting their relevance in lignocellulose transformation.ConclusionsUnderstanding the mechanisms used by basidiomycetes to degrade lignin is of particular relevance to comprehend carbon cycle in nature and to design biotechnological tools for the industrial use of plant biomass. Here, we provide the first structure–function characterization of two novel MnP subfamilies present in Agaricales mushrooms, elucidating the main residues involved in catalysis and demonstrating their ability to modify the lignin macromolecule.

4-Vinyl Guaiacol: A Key Intermediate for Biobased Polymers.

In order to contribute to the shift from petro-based chemistry to biobased chemistry, necessary to minimize the environmental impacts of the chemical industry, 2-methoxy-4-vinylphenol (4-vinyl guaiacol, 4VG) was used to synthesize a platform of biobased monomers. Thus, nine biobased monomers were successfully prepared. The synthesis procedures were investigated through the green metrics calculations in order to quantify the sustainability of our approaches. Their radical homopolymerization in toluene solution initiated by 2,2'-azobis(2-methylpropionitrile) (AIBN) was studied and the effect of residual 4VG as a radical inhibitor on the kinetics of polymerization was also explored. The new homopolymers were characterized by proton nuclear magnetic resonance (1H-NMR) spectroscopy, size exclusion chromatography and thermal analyses (dynamical scanning calorimetry DSC, thermal gravimetric analysis TGA). By varying the length of the alkyl ester or ether group of the 4VG derivatives, homopolymers with Tg ranging from 117 °C down to 5 °C were obtained. These new biobased monomers could be implemented in radical copolymerization as substitutes to petro-based monomers to decrease the carbon footprint of the resulting copolymers for various applications.

Understanding the Effects of Site-Specific Light Chain Conjugation on Antibody Structure Using Hydrogen Exchange-Mass Spectrometry (HX-MS)

Antibody drug conjugates (ADCs) represent one of the fastest growing classes of cancer therapeutics. Drug incorporation through site-specific conjugation in ADCs leads to uniform drug load and distribution. These site-specific modifications may have an impact on ADC quality attributes including protein higher order structure (HOS), which might impact safety and efficacy. In this study, we conducted a side-by-side comparison between the conjugated and unconjugated mAb. In the ADC, the linker-pyrrolobenzodiazepine was site specifically conjugated to an engineered unpaired C215 residue within the Fab domain of the light chain. Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) indicated a decrease in thermal stability for the CH2 transition of the ADC. Size exclusion chromatography (SEC) analysis showed that conjugation of the mAb resulted in earlier aggregation onset and increased aggregation propensity after 4 weeks at 40 °C. Differential hydrogen-exchange mass spectrometry (HX-MS) indicated that upon conjugation, light chain residues 150-155 and 197-204, close to the conjugation site, showed significantly faster HX kinetics, suggesting an increase in backbone flexibility within this region, while heavy chain residues 32-44 exhibited significantly slower kinetics, suggesting distal stabilization of the mAb backbone.

Analytical evaluation of the performance of pyrolysis-gasification (Py-Gs) combination within hybrid thermochemical-biological biorefinery

Thermochemical treatments like pyrolysis and gasification, were proposed to circumvent hydrolysis bottleneck of conventional 2nd generation biorefineries. Within this big-picture, the target of this article was to establish the type and amount of bioavailable matter that can be obtained through intermediate pyrolysis of biomass followed by gasification of biochar. To establish the amount of “chemical energy” partitioned among different products’ chemical oxygen demand (COD), which is proportional to higher heating value (HHV) and often utilized for biological systems, was applied for the evaluation of thermochemical conversion of a lignocellulosic feedstock (fir wood). The most abundant product of intermediate pyrolysis was biochar, which retains from 33 % to 40 % of feedstock COD (with temperatures starting from 450 °C). The yield of water-soluble pyrolysis products (WS) slightly increases from 24 % at 450 °C to 27 % at 650 °C, whereas the yield of WI increases considerably with the same temperature. Pyrolysis temperature showed a minor effect on the composition of WS as revealed by GC-MS analysis of main compounds and size exclusion chromatography. Preliminary gasification experiments, performed at 850 °C under CO2 atmosphere, provided gasification rates for different biochars, which was equal to 0.004, 0.005, 0.006 min−1 for biochars obtained at 650, 550, and 450 °C respectively. Syngas obtained from gasification of 450 °C and 550 °C biochar was almost tar-free, whereas gasification of 650 °C biochar yielded a detectable amount of tars (0.4 %). Increasing the gasification temperature to 950 °C sharply increases the gasification rate of biochar obtained at 450 °C, allowing to obtain 55% conversion yield. Within the scope of hybrid thermochemical-biological (HTB) processes, the obtained results show that intermediate pyrolysis, when coupled with subsequent 950 °C CO2 gasification of biochar, can deliver 64% of chemical energy (by COD basis) of the lignocellulosic feedstock as bioavailable constituents, which are defined to syngas and WS based on recent biological studies. Whereas downstream fermentation can process syngas and WS materials in an effective way, such yields could surpass the holocellulose-targeted methods based on hydrolysis.

Investigating the Impact of Drone Transport on the Stability of Monoclonal Antibodies for Inter-Hospital Transportation

In the field of healthcare logistics, the reliance on conventional transport methods such as cars for the delivery of monoclonal antibodies (mAbs) is susceptible to challenges posed by traffic and infrastructure, leading to increased and unpredictable transport times. Recognizing the potential role of drones in mitigating these challenges, we aimed to investigate the impact of medical drone transport on the stability of mAbs. Compromised stability could lead to aggregation and immunogenicity, thereby jeopardizing the efficacy and safety of mAbs. We studied the transportation of vials as well as ready-to-administer infusion bags with blinatumomab, tocilizumab, and daratumumab. The methodology involved the measurement of both temperature and mechanical shock during drone transport. Moreover, the analytical techniques High Performance Size-Exclusion Chromatography (HP-SEC), Dynamic Light Scattering (DLS), Light Obscuration (LO), Micro-Flow Imaging (MFI), and Nanoparticle Tracking Analysis (NTA) were employed to comprehensively assess the presence of aggregates and particle formation. The key findings revealed no significant differences between car and drone transport, indicating that the stability of mAbs in both vials and infusion bags was adequately maintained during drone transport. This suggests that medical drones are a viable and reliable means for the inter-hospital transport of mAbs, paving the way for more efficient and predictable logistics in healthcare delivery.

LC-MS Characterization and Stability Assessment Elucidate Correlation Between Charge Variant Composition and Degradation of Monoclonal Antibody Therapeutics.

Aggregation stability of monoclonal antibody (mAb) therapeutics is influenced by many critical quality attributes (CQA) such as charge and hydrophobic variants in addition to environmental factors. In this study, correlation between charge heterogeneity and stability of mAbs for bevacizumab and trastuzumab has been investigated under a variety of stresses including thermal stress at 40°C, thermal stress at 55°C, shaking (mechanical), and low pH. Size- and charge-based heterogeneities were monitored using analytical size exclusion chromatography(SEC) and cation exchange chromatography(CEX), respectively, while dynamic light scattering was used to assess changes in hydrodynamic size. CEX analysis revealed an increase in cumulative acidic content for all variants of both mAbs post-stress treatment attributed to increased deamidation. Higher charge heterogeneity was observed in variants eluting close to the main peak than the ones eluting further away (25-fold and 42-fold increase in acidic content for main and B1 of bevacizumab and 19-fold for main of trastuzumab, respectively, under thermal stress; 50-fold increase in acidic for main and B1 of bevacizumab and 10% rise in basic content of main of trastuzumab under pH stress). Conversely, variants eluting far away from main exhibit greater aggregation as compared to close-eluting ones. Aggregation kinetics of variants followed different order for the different stresses for both mAbs (2nd order for thermal and pH stresses and 0th order for shaking stress). Half-life of terminal charge variants of both mAbs was 2- to 8-fold less than main indicating increased degradation propensity.