Structure and physicochemical properties of rice starch modified with dodecenyl succinic anhydride and its use for microencapsulating Pediococcus acidilactici probiotic.

Polysaccharides are used as wall materials to extend the shelf life of lactic acid bacteria. Ice crystal formation during freezing leads to probiotic death. We prepared a series of dodecenyl succinic anhydride (DDSA)-modified rice starches with varying degrees of substitution and compared their functional properties. Fourier-transform infrared spectroscopy, X-ray diffraction analysis, and nuclear magnetic resonance results confirmed successful DDSA modification and the disruption of the long-range ordering of starch molecules. The structural changes modified the physicochemical properties of starch. For example, the apparent viscosity and viscoelastic characteristics of modified rice starch increased, and its freeze-thaw stability and emulsion capacity were remarkably improved after DDSA modification. Moreover, the modified starches exhibited promising performance for microencapsulating Pediococcus acidilactici. This study describes a rice starch derivative with excellent physicochemical properties that can be used to enhance the storage stability of bioactive probiotics.

Synthesis and characterization of modified chitosan as a promising material for enterosorption of heavy metal ions.

Currently, an important ecological problem is environmental pollution and its negative impact on living organisms, the consequences of which are deterioration in general health and the manifestation of various diseases, poisoning, endo- and exotoxicosis. Enterosorption method was proposed as a promising method for removing toxic substances from the living organisms using enterosorbents which can absorb various toxic substances of endogenous and exogenous nature in the lumen of the gastrointestinal tract. It has been proposed to use polymer-containing enterosorbents for eliminating of heavy metals from the organism. The purpose of this research was to synthesize a quaternized derivative of chitosan, specifically N-(2-hydroxybenzyl)-N-ethyl-N-methyl chitosan chloride (Q-CHS). The synthesis of Q-CHS involved the formation of a Schiff base, followed by the quaternization of the amino group of chitosan (CHS). The structures of both pure CHS and quaternized CHS were studied using various physico-chemical methods, including FTIR, NMR, XRD, SEM, DSC and TGA analyses in order to determine the structure and confirm the formation of the final product.

Improving Self-Healing Dental-Restorative Materials with Functionalized and Reinforced Microcapsules.

Dental resin composites are widely used in clinical settings but often face longevity issues due to the development and accumulation of microcracks, which eventually lead to larger cracks and restoration failure. The incorporation of microcapsules into these resins has been explored to introduce self-healing capability, potentially extending the lifespan of the restorations. This study aims to enhance the performance of self-healing dental resins by optimizing the microcapsules-resin matrix physicochemical interactions. Poly(urea-formaldehyde) (PUF) microcapsules were reinforced with melamine and subsequently subjected to surface functionalization with 3-aminopropyltriethoxysilane (APTES) and (3-mercaptopropyl)trimethoxysilane (MPTMS). Additionally, microcapsules were functionalized with a bilayer approach, incorporating tetraethyl orthosilicate (TEOS) with either APTES or MPTMS. X-ray photoelectron spectroscopy (XPS) and thermogravimetric analysis (TGA) confirmed an increased Si:C ratio from 0.006 to 0.165. The functionalization process did not adversely affect the structure of the microcapsules or their healing agent volume. Compared to PUF controls, the functionalized microcapsules demonstrated enhanced healing efficiency, with TEOS/MPTMS-functionalized microcapsules showing the highest performance, showing a toughness recovery of up to 35%. This work introduces a novel approach to functionalization of microcapsules by employing advanced silanizing agents such as APTES and MPTMS, and pioneering bilayer functionalization protocols through their combination with TEOS.

Phase transition and gel properties of chemically modified cassava starch in choline acetate and water mixtures.

This work studied the phase transition and gel properties of cassava starch in aqueous choline acetate ([Ch][OAc]) solution at different [Ch][OAc]:water weight ratios. The paste viscosity and gel strength followed a similar pattern to the starch phase transition temperature, increasing at a 2:3 [Ch][OAc]:water ratio and then decreasing at 3:2 and 4:1 ratios. However, the mobility of free water in the starch gel decreased as the [Ch][OAc]:water ratio increased. At the same [Ch][OAc]:water ratios, acetylated cassava starch (ACS) underwent phase transition more easily than native cassava starch (NCS), leading to greater granule destruction. Nevertheless, ACS gels displayed more viscous-dominated rheological behavior, lower paste viscosity, viscoelasticity, and weaker water-holding capacity (WHC) than NCS gels. In contrast, cross-linked cassava starch (CCS) gels had higher paste viscosity, gel viscoelasticity, and WHC. However, at a 4:1 [Ch][OAc]:water ratio, the viscoelasticity of CCS gel was lower than NCS gel, and the differences in WHC were minimal, likely due to the incomplete phase transition of especially CCS under this condition. Our findings show that starch chemical modification significantly affects phase transition behavior and gel properties in [Ch][OAc]:water mixtures, with outcomes influenced by the viscosity of the aqueous [Ch][OAc] solution and the interaction between [Ch][OAc] and water.

Modifying Naturally Occurring, Nonmammalian-Sourced Biopolymers for Biomedical Applications.

Natural biopolymers have a rich history, with many uses across the fields of healthcare and medicine, including formulations for wound dressings, surgical implants, tissue culture substrates, and drug delivery vehicles. Yet, synthetic-based materials have been more successful in translation due to precise control and regulation achievable during manufacturing. However, there is a renewed interest in natural biopolymers, which offer a diverse landscape of architecture, sustainable sourcing, functional groups, and properties that synthetic counterparts cannot fully replicate as processing and sourcing of these materials has improved. Proteins and polysaccharides derived from various sources (crustaceans, plants, insects, etc.) are highlighted in this review. We discuss the common types of polysaccharide and protein biopolymers used in healthcare and medicine, highlighting methods and strategies to alter structures and intra- and interchain interactions to engineer specific functions, products, or materials. We focus on biopolymers obtained from natural, nonmammalian sources, including silk fibroins, alginates, chitosans, chitins, mucins, keratins, and resilins, while discussing strategies to improve upon their innate properties and sourcing standardization to expand their clinical uses and relevance. Emphasis will be placed on methods that preserve the structural integrity and native biological functions of the biopolymers and their makers. We will conclude by discussing the untapped potential of new technologies to manipulate native biopolymers while controlling their secondary and tertiary structures, offering a perspective on advancing biopolymer utility in novel applications within biomedical engineering, advanced manufacturing, and tissue engineering.

The emulsifying capacity and stability of potato proteins and peptides: A comprehensive review.

The potato has recently attracted more attention as a promising protein source. Potato proteins are commonly extracted from potato fruit juice, a byproduct of starch production. Potato proteins are characterized by superior techno-functional properties, such as water solubility, gel-forming, emulsifying, and foaming properties. However, commercially isolated potato proteins are often denatured, leading to a loss of these functionalities. Extensive research has explored the influence of different conditions and techniques on the emulsifying capacity and stability of potato proteins. However, there has been no comprehensive review of this topic yet. This paper aims to provide an in-depth overview of current research progress on the emulsifying capacity and stability of potato proteins and peptides, discussing research challenges and future perspectives. This paper discusses genetic diversity in potato proteins and various methods for extracting proteins from potatoes, including thermal and acid precipitation, salt precipitation, organic solvent precipitation, carboxymethyl cellulose complexation, chromatography, and membrane technology. It also covers enzymatic hydrolysis for producing potato-derived peptides and methods for identifying potato protein-derived emulsifying peptides. Furthermore, it reviews the influence of factors, such as physicochemical properties, environmental conditions, and food-processing techniques on the emulsifying capacity and stability of potato proteins and their derived peptides. Finally, it highlights chemical modifications, such as acylation, succinylation, phosphorylation, and glycation to enhance emulsifying capacity and stability. This review provides insight into future research directions for utilizing potato proteins as sustainable protein sources and high-value food emulsifiers, thereby contributing to adding value to the potato processing industry.

Comparison of Transgenerational Neurotoxicity between Pristine and Amino-Modified Nanoplastics in C. elegans.

Increasing evidence has suggested that nanoplastic pollution has become a global concern. More importantly, transgenerational toxicity can be induced by nanoplastics at predicted environmentally relevant doses (ERDs). Considering that amino modification could increase nanoplastic toxicity, we compared transgenerational neurotoxicity between pristine polystyrene nanoparticle (PS-NP) and amino-modified PS-NP (NH2-PS-NP) in Caenorhabditis elegans. At 0.1-10 µg/L, NH2-PS-NP caused more severe transgenerational toxicity on locomotion and neuronal development. Accompanied with a difference in transgenerational neuronal damage, compared to PS-NP (10 µg/L), NH2-PS-NP (10 µg/L) induced more severe transgenerational activation of mec-4, crt-1, itr-1, and tra-3, which are required for the induction of neurodegeneration. Moreover, NH2-PS-NP (10 µg/L) caused more severe transgenerational inhibition in expressions of mpk-1, jnk-1, dbl-1, and daf-7 than PS-NP (10 µg/L), and RNA interference (RNAi) of these genes conferred susceptibility to the toxicity of PS-NP and NH2-PS-NP on locomotion and neuronal development. NH2-PS-NP (10 µg/L) further caused more severe transgenerational activation of germline ligand genes (ins-3, ins-39, daf-28, lin-44, egl-17, efn-3, and lag-2) than PS-NP (10 µg/L), and RNAi of these ligand genes caused resistance to the toxicity of PS-NP and NH2-PS-NP on locomotion and neuronal development. Our results highlighted more severe exposure risk of amino-modified nanoplastics at ERDs in causing transgenerational neurotoxicity in organisms.

A thiopyridine-bound mirror-image copper center in an artificial non-heme metalloenzyme.

Artificial metalloenzymes, in which a metal complex and protein matrix are combined, have been synthesized to catalyze stereoselective reactions using the chiral environment provided by the protein cavity. Artificial metalloenzymes can be engineered by the chemical modification and mutagenesis of the protein matrix. We developed artificial non-heme metalloenzymes using a cupin superfamily protein (TM1459) with a 4-His tetrad-metal-binding motif. The Cu-bound H52A/C106D mutant with 3-His triad showed a S-enantioselective Michael addition of nitromethane to α,ß-unsaturated ketone, 2-aza-chalcone 1. In this study, we demonstrated a chemical modification near the copper-binding site of this mutant to reverse its enantioselectivity. For chemical modification, the amino acid on the Si-face of the binding state of 1 to the copper center was replaced with Cys, followed by reaction with 4,4'-dithiopyridine (4-PDS) to form S-(pyridin-4-ylthio)cysteine (Cys-4py). Cu-bound I49C-4py/H52A/C106D showed reversal of the enantioselectivity from S-form to R-form (ee = 71%, (R)). The effect of steric hindrance of the amino acids at position 49 on enantioselectivity was investigated using I49X/H52A/C106D mutants (X = A, C, I, F, and W). Additionally, chemical modification with 2,2'-dithiopyridine (2-PDS) produced I49-2py/H52A/C106D, which showed lower R-enantioselectivity than I49-4py/H52A/C106D. Among the mutants, the 4py-modification on the Si-face was the most effective in reversing the enantioselectivity. By tuning the Re-face side, the H54A mutation introduced into the I49C-4py/H52A/C106D increased the R-enantioselectivity (ee = 88%, (R)). X-ray crystallography revealed a coordinated structure with ligation of thiopyridine in Cu-bound I49C-4py/H52A/H54A/C106D.

Flaxseed (Linum usitatissimum) mucilage: A versatile stimuli-responsive functional biomaterial for pharmaceuticals and healthcare.

The present review is novel as it discusses the main findings of researchers on the topic and their implications, as well as highlights the emerging research in this particular area and its future prospective. The seeds of Flax (Linum usitatissimum) extrude mucilage (FSM) that has a diverse and wide range of applications, especially in the food industry and as a pharmaceutical ingredient. FSM has been blended with several food and dairy products to improve gelling ability, optical properties, taste, and user compliance. The FSM is recognized as a foaming, encapsulating, emulsifying, suspending, film-forming, and gelling agent for several pharmaceutical preparations and healthcare materials. Owing to stimuli (pH) -responsive swelling-deswelling characteristics, high swelling indices at different physiological pHs of the human body, and biocompatibility, FSM is considered a smart material for intelligent, targeted, and controlled drug delivery applications through conventional and advanced drug delivery systems. FSM has been modified through carboxymethylation, acetylation, copolymerization, and electrostatic complexation to get the desired properties for pharma, food, and healthcare products. The present review is therefore devoted to the isolation techniques, structural characterization, highly valuable properties for food and pharmaceutical industries, preclinical and clinical trials, pharmacological aspects, biomedical attributes, and patents of FSM.

Biomedical potentials of alginate via physical, chemical, and biological modifications.

Alginate is a linear polysaccharide with a modifiable structure and abundant functional groups, offers immense potential for tailoring diverse alginate-based materials to meet the demands of biomedical applications. Given the advancements in modification techniques, it is significant to analyze and summarize the modification of alginate by physical, chemical and biological methods. These approaches provide plentiful information on the preparation, characterization and application of alginate-based materials. Physical modification generally involves blending and physical crosslinking, while chemical modification relies on chemical reactions, mainly including acylation, sulfation, phosphorylation, carbodiimide coupling, nucleophilic substitution, graft copolymerization, terminal modification, and degradation. Chemical modified alginate contains chemically crosslinked alginate, grafted alginate and oligo-alginate. Biological modification associated with various enzymes to realize the hydrolysis or grafting. These diverse modifications hold great promise in fully harnessing the potential of alginate for its burgeoning biomedical applications in the future. In summary, this review provides a comprehensive discussion and summary of different modification methods applied to improve the properties of alginate while expanding its biomedical potentials.

Advancements in Non-Addictive Analgesic Diterpenoid Alkaloid Lappaconitine: A Review.

The perennial herb Aconitum sinomontanum Nakai (Ranunculaceae) has been utilized as a traditional oriental medicine in China for numerous years. The principal pharmacological constituent of A. sinomontanum, lappaconitine (LA), exhibits analgesic, anti-inflammatory, anti-tumor, anti-arrhythmic, and anti-epileptic activities. Due to its potent efficacy and non-addictive nature, LA is widely utilized in the management of cancer pain and postoperative analgesia. This review encompasses the research advancements pertaining to LA including extraction methods, separation techniques, pharmacological properties, chemical modifications, and clinical applications. Additionally, it offers insights into the potential applications and current challenges associated with LA to facilitate future research endeavors.

Chemical modification of hyaluronan oligosaccharides differentially modulates hyaluronan-hyaladherin interactions.

The glycosaminoglycan hyaluronan (HA) is a ubiquitous, non-sulfated polysaccharide with diverse biological roles mediated through its interactions with HA-binding proteins (HABPs). Most HABPs belong to the Link module superfamily, including the major HA receptor, CD44, and secreted protein TSG-6, which catalyzes the covalent transfer of Heavy Chains (HC) from inter-α-inhibitor (IαI) onto HA. The structures of the HA-binding domains (HABD) of CD44 (HABD_CD44) and TSG-6 (Link_TSG6) have been determined and their interactions with HA extensively characterized. The mechanisms of binding are different, with Link_TSG6 interacting with HA primarily via ionic and CH-π interactions, whereas HABD_CD44 binds solely via hydrogen bonds and van der Waals forces. Here we exploit these differences to generate HA oligosaccharides, chemically modified at their reducing ends, that bind specifically and differentially to these target HABPs. Hexasaccharides (HA6AN) modified with 2- or 3-aminobenzoic acid or 2-amino-4-methoxybenzoic acid (HA6-2AA, HA6-3AA, HA6-2A4MBA, respectively) had increased affinities for Link_TSG6 compared to unmodified HA6AN. These modifications did not increase the affinity for CD44_HABD. A model of HA6-2AA (derived from the solution dynamic 3D structure of HA4-2AA) was docked into the Link_TSG6 structure, providing evidence that the 2AA-carboxyl forms a salt bridge with Arginine-81. These modeling results informed a 2nd series of chemical modifications for HA oligosaccharides, which again showed differential binding to the two proteins. Several modifications to HA4 and HA6 were found to convert the oligosaccharide into substrates for HC-transfer, whereas unmodified HA4 and HA6 are not. This study has generated valuable research tools to further understand HA biology.

Ultrasonic-assisted extraction, analysis and properties of purple mangosteen scarfskin polysaccharide and its acetylated derivative.

Purple mangosteen scarfskin polysaccharide has many important physiological functions, but its preparation method, structure, and function need further exploration. A polysaccharide was obtained from mangosteen scarfskin by ultrasonic-assisted extraction and purified. On this basis, its structure and physicochemical properties were investigated. The Congo red experiment was used to determine whether it has a triple helix conformation. The structure of purple mangosteen scarfskin polysaccharide was further analyzed by infrared spectroscopy and nuclear magnetic analysis. The antioxidant activities of the above three polysaccharides were studied by related experiments. It was found that the monosaccharide composition of purple mangosteen scarfskin polysaccharide mainly contained a large amount of arabinose, a small amount of rhamnoose and a very small amount of galacturonic acid, and its core main chain was composed of 1,4-α-arabinose. It did not have this spatial configuration. After the acetylation of purple mangosteen scarfskin polysaccharide, the acetylated derivative with a degree of substitution of 0.33 was obtained. It was found that they had certain scavenging and inhibiting effects on hydroxyl radicals and lipid peroxidation, and their activities were related to the concentration of polysaccharides. Meanwhile, the antioxidant activity of the polysaccharide was significantly enhanced after the modified treatment of acetylation, which indicated that chemical modification could effectively improve some activities of polysaccharide. The above studies provided some reference value for the further research and development of purple mangosteen scarfskin polysaccharide.

The selective chemical modification of the 6-amino group of adenosine of the premature termination codon induces readthrough to produce full-length peptide in the reconstituted E. Coli translation system.

Nonsense mutations in the coding region turn amino acid codons into termination codons, resulting in premature termination codons (PTCs). In the case of the in-frame PTC, if translation does not stop at the PTC but continues to the natural termination codon (NTC) with the insertion of an amino acid, known as readthrough, the full-length peptide is formed, albeit with a single amino acid mutation. We have previously developed the functionality-transfer oligonucleotide (FT-Probe), which forms a hybrid complex with RNA of a complementary sequence to transfer the functional group, resulting in modification of the 4-amino group of cytosine or the 6-amino group of adenine. In this study, the FT-Probe was used to chemically modify the adenosines of the PTC (UAA, UAG, and UGA) of mRNA, which were assayed for the readthrough in a reconstituted Escherichia coli translation system. The third adenosine-modified UAA produced three readthrough peptides incorporating tyrosine, glutamine and lysine at the UAA site. It should be noted that the additional modification with a cyclodextrin only induced glutamine incorporation. The adenosine modified UGA induced readthrough very efficiently with selective tryptophan incorporation. Readthrough of the modified UGA is caused by inhibition of the RF2 function. This study has demonstrated that the chemical modification of the adenosine 6-amino group of the PTC is a strategy for effective readthrough in a prokaryotic translation system.

Formulations based on pullulan and a derivative as coating material for the food sector.

Carboxymethylated derivatives of pullulan (PU) were synthesized and evaluated as coating for the postharvest preservation of blueberries. Carboxymethylpullulan was obtained by etherification reaction with the substitution degrees of 0.52, 0.34, and 0.26 for CMP1, CMP2, and CMP3 respectively. Infrared spectroscopy and nuclear magnetic resonance results showed characteristic signals of the carbonyl group belonging to the carboxymethyl group. Thermal analysis showed that CMP1, CMP2, and CMP3 derivatives presented thermal stability values of 209.91 C, 214.73 C, and 225.52 °C, respectively, and were lower with respect to PU with Td of 238.84 °C. Furthermore, an increase in the glass transition temperature due to carboxymethylation was determined. The chemical modification decreased the contact angle with respect to PU (71.34°) with values for CMP1, CMP2, and CMP3 of 39.89°, 53.72° and 60.61°, respectively. The carboxymethylation also increased the water vapor permeability and mechanical properties of the films. In addition, it was found that the CMP molecules affected the optical properties. The application of CMP-based coatings reduced the mass loss and ripening rate of blueberries compared to native pullulan, therefore, packaging from CMP molecules could be used as a coating capable of delaying ripening and extending the shelf life of fruits.

Antioxidant activity of polysaccharide from Garcinia mangostana rind and their derivatives.

BACKGROUND: Polysaccharide from Garcinia mangostana rind has many biological activities and deserves further research. METHODS: The antioxidant properties of UAEE-GMRP, UAEE-GMRP-1 A, CM-30, and Ac-30 were evaluated through two different antioxidant activity experimental systems. RESULTS: The four polysaccharides had a better scavenging effect on hydroxyl radicals, while their inhibitory effect on lipid peroxidation was relatively weak. However, overall, the four polysaccharides showed a certain degree of potential application in the two antioxidant experiments mentioned above, especially the chemically modified polysaccharides from Garcinia mangostana rind, which effectively improved their antioxidant activity. This also indicates that chemical modification is a better method to improve polysaccharide activity. In addition, in these two antioxidant exploration experiments, carboxymethylated polysaccharide showed stronger activity compared to the other three polysaccharides. CONCLUSION: The carboxymethylation modification may have great potential for application.

A Visual Compendium of Principal Modifications within the Nucleic Acid Sugar Phosphate Backbone.

Nucleic acid chemistry is a huge research area that has received new impetus due to the recent explosive success of oligonucleotide therapy. In order for an oligonucleotide to become clinically effective, its monomeric parts are subjected to modifications. Although a large number of redesigned natural nucleic acids have been proposed in recent years, the vast majority of them are combinations of simple modifications proposed over the past 50 years. This review is devoted to the main modifications of the sugar phosphate backbone of natural nucleic acids known to date. Here, we propose a systematization of existing knowledge about modifications of nucleic acid monomers and an acceptable classification from the point of view of chemical logic. The visual representation is intended to inspire researchers to create a new type of modification or an original combination of known modifications that will produce unique oligonucleotides with valuable characteristics.

Modified Halloysite as an Adsorbent for the Removal of Cu(II) Ions and Reactive Red 120 Dye from Aqueous Solutions.

The adsorption of copper ions and Reactive Red 120 azo dye (RR-120) as models of water pollutants on unmodified halloysite (H-NM), as well as halloysites modified with sulfuric acid (H-SA) and (3-aminopropyl)triethoxysilane (H-APTES), was investigated. The results showed that adsorption of both the adsorbates was pH-dependent and increased with the increase in halloysite dosage. The adsorption kinetics were evaluated and the results demonstrated that the adsorption followed the pseudo-second-order model. The adsorption isotherms of Cu(II) ions and RR-120 dye on the halloysites were described satisfactorily by the Langmuir model. The maximum adsorption capacities for the Cu(II) ions were 0.169, 0.236, and 0.507 mmol/g, respectively, for H-NM, H-SA, and H-APTES indicating that the NH2-functionalization rather than the surface area of the adsorbents was responsible for the enhanced adsorption. The adsorption capacities for RR-120 dye were found to be 9.64 µmol/g for H-NM, 75.76 µmol/g for H-SA, and 29.33 µmol/g for H-APTES. The results demonstrated that APTES-functionalization and sulfuric acid activation are promising modifications, and both modified halloysites have good application potential for heavy metals as well as for azo dye removal.

Oxidation-induced starch molecular degradation: A comprehensive kinetic investigation using NaClO/NaBr/TEMPO system.

Starch degradation often coincides with its chemical modification, and understanding how chemical modification influences starch degradation is vital for determining the properties of the resultant modified products. This work investigates the effect of oxidation on starch molecular degradation, examining factors such as oxidation degree, reaction kinetics, and degradation patterns during 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated starch oxidation under varying conditions, including reaction time, pH, temperature, and concentrations of NaBr, TEMPO, and NaClO. Results emphasize that extended reaction durations primarily lead to ß-elimination, causing α-1,4 linkage cleavages. pH 8.5 favored non-selective oxidation, while pH 11 enhanced ß-elimination, both slowing the reaction rate and severely damaging starch chains (Mw of 8.8 × 105 g/mol and 7.2 × 105 g/mol, respectively). Elevated temperature from 0 to 30 °C significantly expedited both selective and non-selective oxidation, dramatically reducing molecular mass to 8.1 × 105 g/mol. Increasing concentrations of NaBr and TEMPO boost the reaction rate with minimal impact on molecular mass. Meanwhile, increasing NaClO concentration from 0.2 to 2.2 mmol/g-starch not only affects the reaction rate but also reinforces ß-elimination, enhancing molecular degradation. This study is insightful for starch modification to achieve desired oxidation levels and chain lengths by controlling reaction conditions, offering potential advancements in oxidized starch-based materials like nano micelles.

Role of Charge Heterogeneity on Physical Stability of Monoclonal Antibody Biotherapeutic Products.

PURPOSE: Chemical modifications in monoclonal antibodies can change hydrophobicity, charge heterogeneity as well as conformation, which eventually can impact their physical stability. In this study, the effect of the individual charge variants on physical stability and aggregation propensity in two different buffer conditions used during downstream purification was investigated. METHODS: The charge variants were separated using semi-preparative cation exchange chromatography and buffer exchanged in the two buffers with pH 6.0 and 3.8. Subsequently each variant was analysed for size heterogeneity using size exclusion chromatography and dynamic light scattering, conformational stability, colloidal stability, and aggregation behaviour under accelerated stability conditions. RESULTS: Size variants in each charge variant were similar in both pH conditions when analyzed without extended storage. However, conformational stability was lower at pH 3.8 than pH 6.0. All charge variants showed similar apparent melting temperature at pH 6.0. In contrast, at pH 3.8 variants A3, A5, B2, B3 and B4 display lower Tm, suggesting reduced conformational stability. Further, A2, A3 and A5 exhibit reduced colloidal stability at pH 3.8. In general, acidic variants are more prone to aggregation than basic variants. CONCLUSION: Typical industry practice today is to examine in-process intermediate stability with acidic species and basic species taken as a single category each. We suggest that perhaps stability evaluation needs to be performed at specie level as different acidic or basic species have different stability and this knowledge can be used for clever designing of the downstream process to achieve a stable product.