Low Molecular Weight Impurities Research Articles

Influence of Chemical Treatment on the Physical and Mechanical Properties of Bamboo Fibers as Potential Reinforcement for Polymer Composites

ABSTRACT The growing concern for environmentally friendly biomaterials has spurred interest in natural fibers as a viable alternative to synthetic fibers in various industrial applications. Chemical treatment of natural fibers is necessary in cases where it is desired to enhance their properties and make them more suitable for various industrial applications. These treatments can remove impurities, increase fiber roughness for better adhesion, and modify surface chemistry, ultimately improving characteristics like tensile strength, thermal stability, and compatibility with matrix materials in composites. In this study, chemical treatments, including Sodium Hydroxide, Ammonium Hydroxide, and Potassium Permanganate, were applied to the Bamboo fibers to modify the fiber structure. The tensile strength, morphological characteristics, and degree of crystallinity of the treated fibers were examined. Results revealed that 5% Sodium Hydroxide treatment for 2 hours significantly improved tensile strength and crystallinity compared to other treatments. Chemical composition analysis, XRD, and FTIR analyses confirmed the removal of impurities and amorphous substances. Density measurements indicated a reduction in volume due to the elimination of low molecular weight impurities, leading to increased fiber density. This research provides valuable insights for the development of sustainable biomaterials in the construction and automotive industries such as wall panels, dashboards and door panels.

Harnessing ceramic hydroxyapatite as an effective polishing strategy to remove product- and process-related impurities in bispecific antibody purification

Bispecific antibody (bsAb), a novel therapeutic modality, provides excellent treatment efficacy, yet poses numerous challenges to downstream process development, which are mainly due to the intricate diversity of bsAb structures and impurity profiles. Ceramic hydroxyapatite (CHT), a mixed-mode medium, allows proteins to interact with its calcium sites (C-sites) through metal affinity and/or its phosphate sites (P-sites) through cation exchange interactions. This dual-binding capability potentially offers unique bind and elute behaviours for different proteins of interest, resulting in optimal product purity when suitable elution conditions are employed. In this study, the effectiveness of CHT as a polishing step for bsAb purification was investigated across three model molecules and benchmarked against the traditional cation exchange chromatography (CEX). For both asymmetric and symmetric IgG-like bsAb post Protein A eluates, at least 97% product purity was achieved after CHT polishing. CHT delivered a superior aggregate clearance to CEX, resulting in low high molecular weight (HMW) impurities (0.5%) and low process-related impurities in the product pools. Moreover, CHT significantly mitigated "chromatography-induced aggregation" whereas eightfold more HMW was generated by CEX. This study illustrated the developability of CHT in effectively eliminating low molecular weight (LMW) impurities through post-load-wash (PLW) optimization, resulting in an additional reduction of up to 48% in LMW impurities. A mechanistic explanation regarding the performance of impurity removal and mitigation of the chromatography-induced aggregation by CHT was proposed, illustrating unique CHT capability is potentially driven by C-site cooperation, of which effectiveness could depend on the bsAb composition and size.Graphical abstract

Downstream process intensification for biotechnologically generated hyaluronic acid: Purification and characterization

Hyaluronic acid (HA), an anionic, non-sulfated glycosaminoglycan, has several clinical applications. This study examines several downstream methods for purifying HA with maximum recovery and purity. Following the fermentation of Streptococcus zooepidemicus MTCC 3523 to produce HA, the broth was thoroughly purified to separate cell debris and insoluble impurities using a filtration procedure and a variety of adsorbents for soluble impurities. Nucleic acids, proteins with high molecular weight, were successfully removed from the broth using activated carbons and XAD-7 resins. In contrast, insoluble and low molecular weight impurities were removed using diafiltration, with HA recovery of 79.16% and purity close to 90%. Different analytical and characterization procedures such as Fourier transform-infrared spectroscopy, X-ray diffraction, nuclear magnetic resonance, and scanning electron microscopy validated the presence, purity, and structure of HA. Microbial HA showed activity in tests for 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical-scavenging (4.87 ± 0.45kmolTE/g), total antioxidant capacity (13.32 ± 0.52%), hydroxyl radical-scavenging (32.03 ± 0.12%), and reducing power (24.85 ± 0.45%). The outcomes showed that the precipitation, adsorption, and diafiltration processes are suitable for extracting HA from a fermented broth under the chosen operating conditions. The HA produced was of pharmaceutical grade for non-injectable applications.

Modified method for obtaining phycocyanine concentrate of Arthrospira platensis biomass

The biomass of Arthrospira platensis cyanobacteria is a source of bioactive compounds such as chlorophylls, carotenoids, and, particularly, phycobiliproteins: C-phycocyanin and allophycocyanin. The wide range of biological activity shown by extracts with a high content of phycocyanins determines the prospects for their use as dietary supplements and ingredients of special foods. For food purposes, the degree of purity of phycocyanin concentrates, determined by the ratio of optical densities of their aqueous solutions at two wavelengths, namely D620/D280, must be greater than 0.7. Most methods for obtaining phycocyanin concentrates include laborious steps of fractional ammonium sulphate precipitation of protein from A. platensis biomass extracts followed by removal of salts solution. The use of membrane technology, specifically microfiltration, makes it possible to significantly intensify and simplify the process of obtaining phycocyanin concentrates. The aim of this research was to modify the method for obtaining a high-purity A. platensis phycocyanin concentrate by replacing the stage of ammonium sulfate precipitation of the protein by ultrafiltration of the extract followed by microfiltration. Material and methods. A sample of dry A. platensis biomass was used as a feedstock. Extraction of A. platensis biomass was carried out at a temperature of +40 °C for 3 h, the resulting suspension was centrifuged, and the supernatant was separated from the sediment. The obtained extract was subjected to ultrafiltration (membrane with a pore diameter of 30 kDa) followed by removal of the permeate containing low molecular weight impurities. The retentate was subjected to microfiltration (membrane with a pore size of 0.2 μm), concentrated by reverse osmosis and freeze-dried. Results. The content of C-phycocyanin and allophycocyanin in the dry concentrate was 42.0±1.3 and 7.0±0.3%, respectively, the degree of purity was 1.98. Conclusion. The scheme for obtaining A. platensis phycocyanin concentrate has been modified. A concentrate was obtained with a high degree of purity, allowing its use in food.

The Investigation the thermal characteristics of polyphenylene sulfides

The article presents the results of a study of the chemical structure, low molecular weight impurities and heat stability according to TGA/DTA of domestic polyphenylene sulfides (PPS) with different molecular weight and their imported analogues. It is shown that domestic samples are almost identical to imported analogues and can be used as a binder for highly filled reinforced polymer composite materials for structural purposes.

АНТИБАКТЕРИАЛЬНАЯ АКТИВНОСТЬ ПЧЕЛИНОГО МЁДА И ЕГО ПЕПТИДНЫХ ФРАКЦИЙ

Recently, the problem of bacteriological resistance has become more and more urgent. The antibiotics used in medical practice are constantly becoming obsolete due to the acquisition of resistance by pathogenic microorganisms, therefore there is an ever-increasing need to search for new antimicrobial drugs of various nature. A promising line of research in this area is the study of the antibacterial activity of protein molecules. Over the past two decades, a lot of data has accumulated showing the wide distribution of antimicrobial peptides among almost all living organisms. This article studied the antibacterial activity of peptide fractions weighing less than 5 kDa obtained from honey of two varieties — buckwheat and mixed origin, as well as the similar activity of various solutions of the same honey samples. The following methods were sequentially applied for isolation and purification of peptide fractions: ultrafiltration — to obtain a solution containing molecules with a mass of less than 5 kDa; ion exchange chromatography on DEAE-cellulose for purification of the peptide fraction from low molecular weight impurities (carbohydrates, pigments, phenols, etc.) and gel filtration on Sephadex G-25 to desalt the obtained peptide fraction from the buffer solution. Determination of antibacterial activity was carried out using the disk diffusion method with antibiotic-resistant strains of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella enteritis obtained from patients with various bacterial infections in chronic and acute forms. In the studied samples, the concentration of peptides, carbohydrates in ketosis and hydrogen peroxide was measured. The results obtained indicate that among the studied solutions of honey, buckwheat has the highest antibacterial activity, which is associated with an increased content of hydrogen peroxide. The study of the antagonistic activity of peptide fractions against test cultures showed that low molecular weight peptides obtained from the studied honey samples do not have an antibacterial effect against the used strains of microorganisms. Apparently, this is due to the absence of low molecular weight antimicrobial peptides in the obtained fractions. Probably, antimicrobial peptides weighing less than 5 kDa are completely absent in bee honey.

Composition of Low-Molecular Weight Impurities in Dimethylmethylphenylsiloxane Rubbers with Terminal Hydroxyl Groups

Composition of Low-Molecular Weight Impurities in Dimethylmethylphenylsiloxane Rubbers with Terminal Hydroxyl Groups

Study of Electrokinetic Properties of Magnetite – Silica Core – Shell Nanoparticles

In this work, the electrokinetic properties of Fe3O4 nanoparticles modified with various alkoxysilanes (tetraethoxysilane and 3-aminopropyltriethoxysilane) in various media were investigated. The determined values of the zeta potential of the Fe3O4/SiO2 samples indicate the complete coverage of nanoparticles with a tetraethoxysilane shell, as well as in the case of the Fe3O4/aminopropyltriethoxysilane. The data obtained on the zeta-potentials of modified nanoparticles with various ligands make it possible to predict the efficiency of subsequent functionalization by target molecules. A decisive role in the study of surface properties is played by cleaning from low molecular weight impurities that can screen the surface of nanoparticles or bind with an indifferent electrolyte. Thus, dispersion on a magnetic stirrer leads to an increase in the sorption capacity of the sample in comparison with ultrasonic dispersion, which causes irreversible destruction of the core-shell nanoparticle structure due to an increase in temperature and pressure in the cavities. This opens the prospective for practical application of modified nanoparticles for creation of tailored composite materials.

Simultaneous monitoring of multiple attributes of pyrrolobenzodiazepine antibody-drug conjugates by size exclusion chromatography – high resolution mass spectrometry

Antibody-drug conjugates (ADCs) are an emerging class of oncology treatments combining the unique specificity of monoclonal antibodies with the highly cytotoxic properties of small molecule compounds. Pyrrolobenzodiazepines (PBDs) are highly potent agents capable of inhibiting cellular DNA replication which leads to apoptosis. To ensure efficacy and patient safety upon administration of such toxic and heterogeneous molecules, their structure and quality attributes must be closely monitored. Size exclusion chromatography (SEC) is a powerful, fast and robust tool for the separation of compounds varying in molecular weight. When using volatile components in the chromatographic mobile phase, SEC has also been shown to be amenable for interfacing to mass spectrometry, providing potential for reliable identification of protein isoforms across the size variants present. Here, we present a SEC-MS method developed for the characterisation of PBD-based ADCs on the intact molecular level. We demonstrate that information on ADC monomers such as the glycoform distribution and the average drug-antibody ratio (DAR) can be obtained in 15 minutes of analysis time. Qualitative and quantitative information on low and high molecular weight impurities such as aggregates and fragments, fundamental for critical quality attribute analysis of biopharmaceuticals, can be generated simultaneously. SEC-MS enables the characterisation of multiple product quality attributes of complex biotherapeutics at the same time.

Development of a Technique for Obtaining Polysaccharides from Leaves of the Birch (Betula pendula Roth. and Betula pubescens Ehrh.)

The goal of the study was to develop a complex technique for the extraction and purification of polysaccharides from the leaves of birch (Betula pendula Roth., Betula pubescens Ehrh., family Betulaceae) (PSfB). The following characteristics have been used as criteria for estimating the effect of the parameters studied on the production of target compounds: the yield of PSfB (the gravimetric method), the protein content (spectrophotometry), the molecular weight distribution (high performance size-exclusion chromatography), and the degree of purification from low-molecular-weight impurities (LMWI). The following optimal parameters have been determined in experiments at different stages of PSfB production: the degree of grinding of a raw material 1.2–3 mm; the pH value of the extractant (purified water with the pH value of 6.5–7.0); the raw material : extractant ratio 1 : 20; the extraction temperature 50°C; the temperature and the degree of evaporation 50°C, four times; the concentrate : ethanol ratio 1 : 3; and the method of purification from LMWI by ultrafiltration. As a result of the work, an optimized method for obtaining PSfB has been developed, which affords a high yield without the loss of quality of the product (minimal admissible content of LMWI with the maximum content of high-molecular-weight fractions). The method is the basis for the development of laboratory regulations for obtaining an active pharmaceutical substance based on polysaccharides from birch leaves.

Composition of low molecular weight impurities in dimethylmethylpheylsiloxane rubbers with terminal hydroxyl groups

Synthesis of dimethylmethylphenylsiloxane rubbers with terminal hydroxyl groups by copolymerization of cyclosiloxanes can be accompanied by formation of low molecular weight compounds, impurities, which are difficultly removed (due to target product affinity) and can lead to rubber opalescence. Identification of the impurities (the content of each impurity ≥ 10 –4 wt.% in 1 μ l of the sample) can be carried out using a sensitive gas chromatography-mass spectrometric method. The method allows separation of the analyzed mixture into individual components on a capillary column. These components enter a mass analyzer and undergo electron ionization which leads to their dissociation into pairs «cation – neutral fragment». Only positively charged ions are observed in the mass spectra, which provides the possibility of predicting the structure of the analyzed siloxane impurities. Rearrangement of linear and cyclic molecules occurring upon mass spectrometric fragmentation result in their transformation into cyclic and bicyclic cations. The structure of the cations formed under ionization conditions depends on the number of silanol bonds and their location in the molecule. To prove the presence of compounds with hydroxyl groups in the composition of impurities and to demonstrate the effect of electron ionization on the fragmentation of compounds with silanol bonds, a sample of oligomethylsiloxane with terminal trimethylsiloxy groups was synthesized in a similar way using the copolymerization method. Compounds with silanol bonds present in the composition of the impurities appear as chromatographic asymmetric peaks with ascending and descending gently sloping zones. Those compounds are rearranged either when passing through a chromatographic column due to sorption-desorption and enter the detector in a modified form or drift through the column and undergo rearrangements already under conditions of electron ionization. The impurities contained linear and cyclic methylsiloxanes with silanol and phenyl bonds. The chromato-mass spectrometric method provide a reliable control of the composition and content of undesirable impurities which cause rubber opalescence.

A simple and novel sample preparation approach for effective characterization of antibody low molecular weight impurities by CE-SDS method

A simple and novel sample preparation approach for effective characterization of antibody low molecular weight impurities by CE-SDS method

Comparison of Strategies in Development and Manufacturing of Low Viscosity, Ultra-High Concentration Formulation for IgG1 Antibody

Monoclonal antibodies requiring higher doses for exerting therapeutic effect but having lower stability, are administered as dilute infusions, or as two (low concentration) injections both resulting in reduced patient compliance. Present research summarizes impact of manufacturing conditions on ultra-high concentration (≥150 mg/mL) IgG1 formulation, which can be administered as one subcutaneous injection. IgG1 was concentrated to ~200 mg/mL using tangential flow filtration (TFF). Alternatively, spray dried (SPD) and spray freeze dried (SFD) IgG1, was reconstituted in 30%v/v propylene glycol to form ultra-high concentration (~200 mg/mL) injectable formulation. Reconstituted, SPD and SFD IgG1 formulations, increased viscosity beyond an acceptable range for subcutaneous injections (<20 cP). Formulations developed by reconstitution of SPD IgG1, demonstrated increase in high and low molecular weight impurities, at accelerated and stressed conditions. Whereas, the stability data suggested reconstituted SFD IgG1 was comparable to control IgG1 formulation concentrated by TFF. Also, formulation of IgG1 diafiltered with proline using TFF, reduce viscosity from ~21.9 cP to ~11 cP at 25 °C and had better stability. Thus, conventional TFF technique stands to be one of the preferred methods for manufacturing of ultra-high concentration IgG1 formulations. Additionally, SFD could be an alternative method for long term storage of IgG1 in a dry powder state.

An alternative downstream process based on aqueous two-phase extraction for the purification of monoclonal antibodies

An alternative downstream process for the purification of a monoclonal antibody (mAb) based on aqueous two-phase extraction as clarification, capture and primary purification step was developed. For further purification unit operations which are commonly used for mAb platform processes were utilized. A diafiltration approach was used to combine virus inactivation and removal of phase forming components as well as low molecular weight impurities in one step followed by cation and anion exchange chromatography. Starting from cell containing cultivation broth, an overall mAb yield of 74 % was achieved within an application study. The process was optimized regarding mAb yield and the clearance of process related impurities like deoxyribonucleic acid as well as host cell proteins, which were removed to approximately 60 and 6000 ppm respectively. Critical product quality attributes, regarding glycosylation patterns, were also examined and remained unimpaired after the aqueous two-phase extraction. The alternative downstream process presented in this study offers great potential to improve mAb manufacturing.

Integrative strategy to determine residual proteins in cefaclor produced by immobilized penicillin G acylase

There is a growing trend in the pharmaceutical industry towards substituting conventional chemical synthesis routes of semi-synthetic β-lactam antibiotics (SSBAs) through environmentally sustainable enzymatic processes. These have advantages such as cost reduction in terms of solvent and waste treatment and time saving owing to fewer reaction steps. Penicillin G acylase (PGA) is an industrially important enzyme that is mainly used to catalyze the synthesis of SSBAs. In this study, we established an integrative strategy using three different analytical methods for determining the PGA-associated residual protein content, which is a critical quality issue in the end product. Cefaclor was taken as representative example of SSBAs. High-performance liquid chromatography coupled with fluorescence detection (HPLC-FD) allowed the routine analysis of PGA residual proteins and other low molecular weight (MW) impurities with high detection specificity and sensitivity, comparable to those of the Bradford assay and microfluidic protein chip electrophoresis. However, these latter two methods were superior for quantitative and qualitative analysis, respectively, and should be regarded as necessary adjuncts to the HPLC-FD method. By combining the three methods, trace levels of residual proteins were detected in four (out of 13) cefaclor bulk samples from two different manufacturers, with a major protein MW of ∼63 kDa. This suggests that the higher MW PGA subunit tends to persist in the end product. The integrative determination strategy described here can be used to evaluate SSBA bulk samples and monitor the process of SSBA manufacturing by enzymatic methods, especially in terms of inter-batch consistency and process stability.

DEVELOPMENT OF METHODS FOR OBTAINING POLYSACCHARIDES FROM BIRCH LEAVES (BETULA PENDU-LA ROTH., BETULA PUBESCENS EHRH.)

The development of a complex technique for the extraction and purification of polysaccharides from birch leaves (Betula pendula Roth.,Betula pubescens Ehrh.) (PSfB) was the purpose of this study. The following characteristics were used as criteria for evaluating the effect of the parameters studied on the production of target substances: yield of PSfB (gravimetric method), protein content (spectrophotometric method), molecular weight distribution (high performance exclusion chromatography) and degree of purification from low molecular weight impurities (LMWI) (IR) spectroscopy).During the experiment, the optimal parameters were determined at various stages of obtaining the PSfB: degree of raw materials grinding – 1.2–3 mm, pH of the extractant (purified water with pH=7), the ratio of raw materials : extractant (1 : 20), extraction temperature (50 °C), temperature and degree of evaporation (50 °C; 4 times), the ratio of concentrate : ethanol (1 : 3) and the method of purification from the LMWI (ultrafiltration).The result of this work is an optimized method for obtaining PSfB, allowing to achieve high yield without loss of product quality (minimum allowable content of low molecular weight impurities with the highest content of high molecular weight fractions).This technique is the basis for the development of laboratory regulations for obtaining an active pharmaceutical substance based on polysaccharides from birch leaves.

Soy and Rapeseed Protein Hydrolysis by the Enzyme Preparation Protosubtilin

A comparative study of soybean and rapeseed protein hydrolysis by protosubtilin, an original Russian enzyme preparation widely used in animal feed production, has been performed. SDS-PAG electrophoresis, HPLC, and mass spectrometry have been employed to analyze the obtained products. The soybean protein isolate used for hydrolysate production was obtained from a commercial supplier, and rapeseed proteins were prepared from the meal by alkali extraction. Low molecular weight impurities were removed by ultrafiltration. The degree of protein hydrolysis has been shown to depend on the substrate-to-enzyme preparation ratio, hydrolysis time, and protein concentration. Rapeseed protein hydrolysis by protosubtilin at an enzyme/protein ratio of 1: 20 and hydrolysis time of 20 h resulted in complete cleavage of the proteins present in the raw material and the accumulation of oligopeptides (molecular weight less than 14 kDa) and free amino acids, which accounted for 53 and 8% of the initial protein weight, respectively. In contrast to rapeseed proteins, soybean proteins showed considerable gelling at the initial stages of hydrolysis, and the formation of insoluble hydrolysis-resistant fragments was observed. The soluble part of the hydrolysate contained short oligopeptides and free amino acids, which accounted for 13% of the initial protein weight only.

Characterization of product-related low molecular weight impurities in therapeutic monoclonal antibodies using hydrophilic interaction chromatography coupled with mass spectrometry

Traditional SDS-PAGE method and its modern equivalent CE-SDS method are both widely applied to assess the purity of therapeutic monoclonal antibody (mAb) drug products. However, structural identification of low molecular weight (LMW) impurities using those methods has been challenging and largely based on empirical knowledges. In this paper, we present that hydrophilic interaction chromatography (HILIC) coupled with mass spectrometry analysis is a novel and orthogonal method to characterize such LMW impurities present within a purified mAb drug product sample. We show here that after removal of N-linked glycans, the HILIC method separates mAb-related LMW impurities with a size-based elution order. The subsequent mass measurement from a high-resolution accurate mass spectrometer provides direct and unambiguous identification of a variety of low-abundance LMW impurities within a single LC–MS analysis. Free light chain, half antibody, H2L species (antibody possessing a single light chain) and protein backbone-truncated species can all be confidently identified and elucidated in great detail, including the truncation sites and associated post-translational modifications. It is worth noting that this study provides the first example where the H2L species can be directly detected in a mAb drug product sample by intact mass analysis without prior enrichment.

Optimization-based framework for resin selection strategies in biopharmaceutical purification process development.

This work addresses rapid resin selection for integrated chromatographic separations when conducted as part of a high‐throughput screening exercise during the early stages of purification process development. An optimization‐based decision support framework is proposed to process the data generated from microscale experiments to identify the best resins to maximize key performance metrics for a biopharmaceutical manufacturing process, such as yield and purity. A multiobjective mixed integer nonlinear programming model is developed and solved using the ε‐constraint method. Dinkelbach's algorithm is used to solve the resulting mixed integer linear fractional programming model. The proposed framework is successfully applied to an industrial case study of a process to purify recombinant Fc Fusion protein from low molecular weight and high molecular weight product related impurities, involving two chromatographic steps with eight and three candidate resins for each step, respectively. The computational results show the advantage of the proposed framework in terms of computational efficiency and flexibility. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:1116–1126, 2017

Optimization of Fenton Oxidation Process for the Degradation of Color Precursors in Raw Sugar Beet Juice

The degradation of tyrosine and ferulic acid, via the Fenton process, was investigated in sucrose solution and raw sugar beet juice. Central composite design and response surface methodology were used to evaluate the relationships between degradation of the color precursors and the process parameters (i.e., initial concentration of the color precursors, solution pH, dosages of Fenton’s reagent, and reaction time). According to the statistical analysis, the optimized conditions for each variable in aqueous sucrose solutions including an initial concentration of the color precursors equal to 45.0 mg L−1 were as follows: reaction time: 30 min, pH 5.8, Fe2+ dosage: 1.34 mM, and H2O2 dosage: 53.6 mM. Under these conditions, 61.8 % of the color precursors and less than 2.5 % of sucrose were degraded after 30 min of reaction. The highest decolorization efficiency of the raw juice was obtained about 55 % with the addition of 1.34 mM Fe2+ and 53.6 mM H2O2 at an initial pH of 6.2. Using the same conditions, approximately 50 % of tyrosine was degraded in treated juice with Fenton’s reagent. Gel permeation chromatography analysis confirmed that the concentration of low molecular weight impurities in decolorized juice was considerably reduced. According to the results, Fenton oxidation process can be considered as a promising method for degradation of the color precursors and preparation of raw sugar beet juice with low color content.