Exclusion High Performance Liquid Chromatography Research Articles

High-Density Lipoprotein Function in Cardiovascular Disease and Diabetes Mellitus.

Cardiovascular disease (CVD) is the most common cause of death worldwide.1 Major CVD risk factors are hypertension, smoking, physical inactivity, abnormal glucose levels/diabetes mellitus, and dyslipidemia. Among these, dyslipidemia characterized by a low level of HDL-C (high-density lipoprotein cholesterol) is strongly and inversely correlated with CVD risk,2–4 and low HDL-C levels is part of the atherogenic dyslipidemia complex associated with diabetes mellitus.5 These observations triggered intense interest in increasing HDL-C levels for therapeutic intervention of CVD.6,7 However, several recent lines of evidence now suggest that the association between HDL-C levels and CVD status may be indirect or more complicated than previously recognized.7 Thus, human genetic studies demonstrate that genetically altered HDL-C levels do not necessarily translate to an altered risk of CVD.8–12 Phase III clinical trials of drugs that elevate HDL-C, such as niacin and CETP (cholesteryl ester transfer protein) inhibitors, also have largely failed to reduce CVD events in statin-treated subjects with established CVD.13–15 For several CETP inhibitors, improvement in CVD prevention was unsuccessful because of off-target effects (torcetrapib) or lack of efficacy (dalcetrapib and evacetrapib).14,16–18 The exception is the recent REVEAL trial (Randomized Evaluation of the Effects of Anacetrapib Through Lipid-Modification), which included more subjects and was performed for longer than previous CETP inhibitor trials. The REVEAL demonstrated that the CETP inhibitor anacetrapib exhibited beneficial effects on cardiovascular outcomes on top of those of statin therapy, although the risk reduction was moderate19,20 and might have been due, at least in part, to a reduction in non-HDL-C rather than elevated HDL-C.21 Considering these cumulative observations, it remains uncertain whether increased HDL-C directly impacts atherosclerosis and the …

Radiolabeling and Analysis of Labeled Proteins.

Protein-based preparations for biomedical research, and radiolabeled pharmaceuticals for nuclear medical diagnostics and therapy require highest chemical, radiochemical, and in particular protein purity. For detection of contaminations most commonly size exclusion high-performance liquid chromatography and polyacrylamide gel analysis in combination with silver staining is used. Here we describe a by far more sensitive radiolabeling method for the detection of traces of contaminating proteins.

Synthesis and Characterization of Fatty Acid Grafted Chitosan Polymer and Their Nanomicelles for Nonviral Gene Delivery Applications.

The aim of this study was to synthesize and characterize fatty acid-grafted-chitosan (fatty acid-g-CS) polymer and their nanomicelles for use as carriers for gene delivery. CS was hydrophobically modified using saturated fatty acids of increasing fatty acyl chain length. Carbodiimide along with N-hydroxysuccinimide was used for coupling carboxyl group of fatty acids with amine groups of CS. Proton nuclear magnetic resonance and Fourier transform infrared spectroscopy were used to quantify fatty acyl substitution onto CS backbone. The molecular weight distribution of the synthesized polymers was determined using size exclusion high performance liquid chromatography and was found to be in range of the parent CS polymer (∼50 kDa). The critical micelle concentration (cmc) of the polymers was determined using pyrene as a fluorescent probe. The cmc was found to decrease with an increase in fatty acyl chain length. The amphiphilic fatty acid-g-CS polymers self-assembled in an aqueous environment to form nanomicelles of ∼200 nm particle size and slightly positive net charge due to the cationic nature of free primary amino groups on CS molecule. These polymeric nanomicelles exhibited excellent hemo- and cytocompatibility, as evaluated by in vitro hemolysis and MTT cell viability assay, respectively, and showed superior transfection efficiency compared to unmodified chitosan and naked DNA. The surface of these nanomicelles can be further modified with ligands allowing for selective targeting, enhanced cell binding, and internalization. These nanomicelles can thus be exploited as potential nonviral gene delivery vectors for safe and efficient gene therapy.

Optimization of an anti-HER2 nanobody expression using the Taguchi method

ABSTRACTDespite being widely used in immunotherapy of cancer, whole antibodies are limited by several disadvantages. This has led to the advent of novel biomolecules such as nanobodies. Taguchi method is a statistical experimental design to study the effect of multiple variables in biological processes. In an effort to overexpress a recombinant anti-human epidermal growth factor receptor type 2 (HER2) nanobody, we performed a detailed study to find optimal condition of temperature, induction, culture media, vector, and host strain, using Taguchi methodology. A total of 16 various experiments were designed. Total protein of the formulated cultures were assessed by Bradford test and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by size exclusion high performance liquid chromatography to quantify the relative concentration of the nanobody in different expression settings. Western blotting was performed to confirm the expression of the anti-HER2 nanobody. When, individually, optimum parameters determined by Taguchi were applied, including SHuffle strain cultured in LB medium, induced with 0.4 mM isopropyl-β-D-thio-galactoside for 18 h at 24°C, production yield further increased by about 9% (25.4 mg/L), compared to the highest expression setting. Flow cytometry and enzyme-linked immunosorbent assay result indicated improved protein binding in optimized conditions. Overall, our findings provide a basis for further investigations on economical production of recombinant nanobodies to improve production yield and activity.

Correlation between Dynamic Light Scattering and Size Exclusion High Performance Liquid Chromatography for Monitoring the Effect of pH on Stability of Biopharmaceuticals.

Aggregate formation is a major problem affecting both safety and efficacy of biopharmaceuticals and is associated with protein immunogenicity. Size exclusion high performance liquid chromatography (SE-HPLC) has always been the gold standard technique for detection and determination of protein aggregates. However, large protein aggregates may be filtered off and build up on top of the column leading to deterioration in column performance. Moreover, low-affinity protein aggregates may dissociate during analysis and thus not detected. On the other hand, dynamic light scattering (DLS) is a simple and non-destructive technique that can detect high molecular weight physical and chemical aggregates in their native environment. Here, three model biopharmaceutical proteins of different physicochemical properties were selected; quadrivalent human papillomavirus virus like particles vaccine (HPV VLP, physically assembled subunit vaccine, 55kDa), pegylated Interferon (PegIFN, pegylated non-glycosylated protein, 31.3kDa) and Pegylated Erythropoietin (PegEPO, pegylated and glycosylated protein, 60kDa). Samples were subjected to forced degradation conditions previously shown to lead to aggregate formation (pH 4.0, 8.0 and 10.0, at 37°C for 24h) and samples were analyzed using DLS and SE-HPLC. Generally, good agreement between the results of DLS and SE-HPLC was noted, regardless of the differences in physicochemical properties of the studied biopharmaceuticals. Results showed that aggregate formation was not detected in some cases by SE-HPLC and the decrease in the concentration of the monomeric forms indicated that such aggregates might have been filtered off the column. Although no single techniques can reveal all aspects of protein stability, DLS can serve as a screening tool to detect aggregate formation and cross-validate SE-HPLC results during batch release testing. Owing to its simplicity and low-sample volume requirements, DLS can be used even by hospital pharmacists to confirm absence of protein aggregates immediately before drug administration.

Study of N-vinylpyrrolidone-N-vinylformamide copolymers labelled with indium-113m.

The procedure of the directed synthesis of N-vinylpyrrolidone-N-vinylformamide (VP-VFA) copolymers with grafted iminodiacetate (IDA) chelating units is presented. The methods for labelling resulting conjugates with indium-113m were developed. The metal-copolymer conjugates were characterized by different physicochemical methods, including IR and NMR, viscometry, light scattering, and exclusion high-performance liquid chromatography. Parameters of radiochemical synthesis of the conjugates labelled with indium-113m were optimized. It was shown that the VP-VFA-IDA copolymer firmly binds indium-113m both in the acid and alkaline solutions, with pH of the reaction mixture having almost no effect on the complexation. VP-VFA-IDA-In conjugates were found to be unstable in histidine challenge reaction.

High-throughput analysis of sub-visible mAb aggregate particles using automated fluorescence microscopy imaging.

Aggregation of therapeutic proteins is a major concern as aggregates lower the yield and can impact the efficacy of the drug as well as the patient's safety. It can occur in all production stages; thus, it is essential to perform a detailed analysis for protein aggregates. Several methods such as size exclusion high-performance liquid chromatography (SE-HPLC), light scattering, turbidity, light obscuration, and microscopy-based approaches are used to analyze aggregates. None of these methods allows determination of all types of higher molecular weight (HMW) species due to a limited size range. Furthermore, quantification and specification of different HMW species are often not possible. Moreover, automation is a perspective challenge coming up with automated robotic laboratory systems. Hence, there is a need for a fast, high-throughput-compatible method, which can detect a broad size range and enable quantification and classification. We describe a novel approach for the detection of aggregates in the size range 1 to 1000μm combining fluorescent dyes for protein aggregate labelling and automated fluorescence microscope imaging (aFMI). After appropriate selection of the dye and method optimization, our method enabled us to detect various types of HMW species of monoclonal antibodies (mAbs). Using 10μmolL-1 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate (Bis-ANS) in combination with aFMI allowed the analysis of mAb aggregates induced by different stresses occurring during downstream processing, storage, and administration. Validation of our results was performed by SE-HPLC, UV-Vis spectroscopy, and dynamic light scattering. With this new approach, we could not only reliably detect different HMW species but also quantify and classify them in an automated approach. Our method achieves high-throughput requirements and the selection of various fluorescent dyes enables a broad range of applications.

Protein interactions during flour mixing using wheat flour with altered starch

Wheat grain proteins responses to mixing and thermal treatment were investigated using Mixolab-dough analysis systems with flour from two cultivars, Ventura-26 (normal amylose content) and Ventura-19 (reduced amylose content). Size exclusion high performance liquid chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) analysis revealed that, stress associated and metabolic proteins largely interacted with dough matrix of Ventura-26 after 26min (56°C); gliadins, avenin-like b proteins, LMW-GSs, and partial globulins showed stronger interactions within the dough matrix of Ventura-26 at 32min/C3 (80°C), thereafter, however, stronger protein interactions were observed within the dough matrix of Ventura-19 at 38min/C4 (85°C) and 43min (80°C). Thirty-seven proteins associated with changes in dough matrix due to reduced amylose content were identified by mass spectrometry and mainly annotated to the chromosome group 1, 4, and 6. The findings provide new entry points for modifying final product attributes.

Simultaneous determination of nitrated and oligomerized proteins by size exclusion high-performance liquid chromatography coupled to photodiode array detection

Chemical modifications such as nitration and cross-linking may enhance the allergenic potential of proteins. The kinetics and mechanisms of the underlying chemical processes, however, are not yet well understood. Here, we present a size-exclusion chromatography/spectrophotometry method (SEC-HPLC-DAD) that allows a simultaneous detection of mono-, di-, tri-, and higher protein oligomers, as well as their individual nitration degrees (NDs). The ND results of proteins from this new method agree well with the results from an alternative well-established method, for the analysis of tetranitromethane (TNM)- and nitrogen dioxide and ozone (NO2/O3)-nitrated protein samples. Importantly, the NDs for individual oligomer fractions can be obtained from the new method, and also, we provide a proof of principle for the calculation of the concentrations for individual protein oligomer fractions by their determined NDs, which will facilitate the investigation of the kinetics and mechanism for protein tyrosine nitration and cross-linking.

An improved size exclusion-HPLC method for molecular size distribution analysis of immunoglobulin G using sodium perchlorate in the eluent

Size exclusion (SE) high performance liquid chromatography (HPLC) is widely used for the molecular size distribution (MSD) analyses of various therapeutic proteins. We report development and validation of a SE-HPLC method for MSD analyses of immunoglobulin G (IgG) in products using a TSKgel SuperSW3000 column and eluting it with 0.4M NaClO4, a chaotropic salt, in 40mM phosphate buffer, pH 6.8. The chromatograms show distinct peaks of aggregates, tetramer, and two dimers, as well as the monomer and fragment peaks. In addition, the method offers about half the run time (12min), better peak resolution, improved peak shape and more stable base-line compared to HPLC methods reported in the literature, including that in the European Pharmacopeia (EP). A comparison of MSD analysis results between our method and the EP method shows interactions between the protein and the stationary phase and partial adsorption of aggregates and tetramer on the stationary phase, when the latter method is used. Thus, the EP method shows lower percent of aggregates and tetramer than are actually present in the products. In view of the fact that aggregates have been attributed to playing a critical role in adverse reactions due to IgG products, our observation raises a major concern regarding the actual aggregate content in these products since the EP method is widely used for MSD analyses of IgG products. Our method eliminates (or substantially reduces) the interactions between the proteins and stationary phase as well as the adsorption of proteins onto the column. Our results also show that NaClO4 in the eluent is more effective in overcoming the protein/column interactions compared to Arg-HCl, another chaotropic salt. NaClO4 is shown not to affect the molecular size and relative distribution of different molecular forms of IgG. The method validated as per ICH Q2(R1) guideline using IgG products, shows good specificity, accuracy, precision and a linear concentration dependence of peak areas for different molecular forms. In summary, our method gives more reliable results than the SE-HPLC methods for MSD analyses of IgG reported in the literature, including the EP, particularly for aggregates and tetramer. The results are interpreted in terms of ionic (polar) and hydrophobic interactions between the stationary phase and the IgG protein.

In vitro evaluation of concentration, labeling effectiveness and stability for 131 I-labeled radioimmunoassay ligand using real-time detection technology.

Radioimmunoassay belongs to the analytical method enabling highly specific and sensitive quantification of molecules. The verification of the real-time radioimmunoassay technology usefulness for ligand-quality characteristics evaluation such as concentration, influence of radiolabeling on binding affinity and stability was estimated. The anti-epidermal growth factor receptor antibody 131 I-cetuximab was employed as the ligand antibody. The concentration of 131 I-cetuximab was derived from the shape of binding curves coming from the ligand-receptor interaction. The binding curves also allowed the estimation of 131 I-cetuximab binding affinity for different radiolabeling procedures (incubation times 1, 5, and 10minutes) in stability testing up to 96hours at 4°C. The stability testing also included comparative analysis by size exclusion high-performance liquid chromatography. The assessment of cetuximab concentrations using real-time method showed acceptable accordance between real and calculated values. The real-time method revealed that 1-minute radiolabeling proved to be the optimal incubation time for direct radioiodination of cetuximab. Stability testing showed the significant change in radioligand affinity by one order at the longest incubation times (72 and 96hours). Characterization of stability and binding behavior of radiolabeled monoclonal antibodies by the verified real-time method before use in other assays may be employed to eliminate variability and suboptimal antibody performance.

Cross-Contamination From Vial Caps for SEC Analysis Determined by MALDI-TOF Mass Spectrometry.

Cross-contamination from the polymeric material of the vial caps used in size exclusion chromatography (SEC) and high-performance liquid chromatographyautosamplers in the low molar mass (LMM) range (<2000 Da) of polymer samples was demonstrated with the help of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. It is shown that polyethylene (PE) caps, if exposed to either short or long periods intetrahydrofuran solvent, might extract oligomers that contaminate the LMM end of the SEC chromatograms. On the other hand, polytetrafluoroethylene (PTFE)/silicone caps have shown to be inert if used properly; namely, the PTFE part in contact with the sample solution. However, care should be taken if accidentally the silicon part is exposed to the solution because silicone oligomer peaks have been detected by MALDI-TOF MS analysis that might mislead the results.

Protein-transitions in and out of the dough matrix in wheat flour mixing

Sequential protein behavior in the wheat dough matrix under continuous mixing and heating treatment has been studied using Mixolab-dough samples from two Australian wheat cultivars, Westonia and Wyalkatchem. Size exclusion high performance liquid chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) analysis indicated that 32min (80°C) was a critical time point in forming large protein complexes and loosing extractability of several protein groups like y-type high molecular weight glutenin subunits (HMW-GSs), gamma-gliadins, beta-amylases, serpins, and metabolic proteins with higher mass. Up to 32min (80°C) Westonia showed higher protein extractability compared to Wyalkatchem although it was in the opposite direction thereafter. Twenty differentially expressed proteins could be assigned to chromosomes 1D, 3A, 4A, 4B, 4D, 6A, 6B, 7A and 7B. The results expanded the range of proteins associated with changes in the gluten-complex during processing and provided targets for selecting new genetic variants associated with altered quality attributes of the flour.

Improving the Digestibility of Lentil Flours and Protein Isolate and Characterization of Their Enzymatically Prepared Hydrolysates

Raw and cooked lentil flours and protein isolate were digested using one-step-multi-enzyme, two-step-sequential multi-enzyme or pre-hydrolyzed with one-step-single-enzyme systems. In vitro protein digestibility values ranged between 22.3 and 94.4%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed minimal loss of intact protein and more extensive digestion after actinidin and acid-protease pre-hydrolysis, respectively. Size exclusion high-performance liquid chromatography analysis confirmed greater loss of high molecular mass polypeptides after acid-protease pre-hydrolysis compared to the other enzymes. Additional digestion of pre-hydrolyzed samples using trypsin-α-chymotrypsin-peptidase resulted in further digestion and release of lower molecular mass peptides. The study demonstrates how different processing treatments and the use of enzymes (alone/in combination) influence lentil protein digestion.

Bio-assisted potentiometric multisensor system for purity evaluation of recombinant protein A

Recombinant proteins became essential components of drug manufacturing. Quality control of such proteins is routine task, which usually requires a lot of time, expensive reagents, specialized equipment and highly educated personnel. In this study we propose a new concept for protein purity evaluation that is based on application of bio-assisted potentiometric multisensor system. The model object for analysis was recombinant protein A from Staphylococcus aureus (SpA), which is commonly used for monoclonal antibody purification. SpA solutions with different amount of host cell related impurities (Escherichia coli, bacterial lysate) were analyzed. Two different bio-transducers were employed: proteinase K from Tritirachium album and baker's yeast Saccharomyces cerevisiae. It was shown that both bio-transducers are able to induce changes in pure and lysate-contaminated SpA samples. Different products of yeast digestion and proteolysis with proteinase of pure SpA and lysate were detected with size exclusion high-performance liquid chromatography (SE-HPLC). The induced changes of chemical composition are detectible with potentiometric multisensor system and can be related to SpA purity through projection on latent structures (PLS) regression technique. The proposed method allows for estimation of the impurity content with 12% accuracy using proteinase K and 16% accuracy using baker's yeast. The suggested approach could be useful for early contamination warning at initial protein purification steps. The analysis requires no expensive materials and equipment, no bio-material immobilization, and its duration time is comparable with other commonly used methods like chromatography or electrophoresis though the main part of this time is related to the sample preparation.

Abiotic stress induced changes in protein quality and quantity of two bread wheat cultivars

Protein quantity and quality of wheat flour have been found to be influenced by abiotic stress conditions. The aim of this study was to determine the effect of heat, drought and cold stress on protein quantity and quality using reverse phase and size exclusion high performance liquid chromatography in two bread wheat cultivars. Heat stress caused a significant increase in protein content of both cultivars, while drought stress caused a significant protein increase in Duzi. Sodium dodecyl sulphate (SDS) soluble large monomeric proteins were significantly increased by heat stress in both cultivars, and SDS insoluble large polymeric proteins were significantly increased by heat and drought stress in Kariega. SDS insoluble small polymeric proteins were significantly decreased by all three stress conditions in Duzi and heat stress in Kariega. Heat and drought stress had similar effects on protein, but cold stress caused different effects. GluD1 subunit 2 was least influenced by stress conditions, but GluD1 subunit 12 was significantly reduced by all stress conditions. GluA1 subunit 2* and GluB1 subunit 17 were significantly increased by heat and drought in Kariega. Heat stress had the largest effect on protein quality and quantity, and there was a large genotype effect.

Single-step purification of ovalbumin from egg white using aqueous biphasic systems

The ability of aqueous biphasic systems (ABS) composed of polyethylene glycols of different molecular weights (PEG 400, 600 and 1000) and buffered aqueous solutions of potassium citrate/citric acid (pH 5.0–8.0) to selectively extract ovalbumin from egg white was here investigated. Phase diagrams, tie-lines and tie-line lengths were determined at 25°C and the partitioning of ovalbumin in these systems was then evaluated. Aiming at optimizing the selective extraction of ovalbumin in the studied ABS, factors such as pH, PEG molecular weight and amount of the phase-forming components were initially investigated with pure commercial ovalbumin. In all ABS, it was observed a preferential partitioning of ovalbumin to the polymer-rich phase, with extraction efficiencies higher than 90%. The best ABS were then applied in the purification of ovalbumin from the real egg white matrix. In order to ascertain on the ovalbumin purity and yield, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion high performance liquid chromatography (SE-HPLC) analyses were conducted, confirming that the isolation/purification of ovalbumin from egg white was completely achieved in a single-step with a recovery yield of 65%. The results obtained show that polymer-salt-based ABS allow the selective extraction of ovalbumin from egg white with a simpler approach and better performance than previously reported. Finally, it is shown that ovalbumin can be completely recovered from the PEG-rich phase by an induced precipitation using an inexpensive and sustainable separation platform which can be easily applied on an industrial scale.

Electrochemical destruction of trans-cinnamic acid by advanced oxidation processes: kinetics, mineralization, and degradation route.

Acidic solutions of trans-cinnamic acid at pH3.0 have been comparatively treated by anodic oxidation with electrogenerated H2O2 (AO-H2O2), electro-Fenton (EF), and photoelectro-Fenton (PEF). The electrolytic experiments were carried out with a boron-doped diamond (BDD)/air-diffusion cell. The substrate was very slowly abated by AO-H2O2 because of its low reaction rate with oxidizing •OH produced from water discharge at the BDD anode. In contrast, its removal was very rapid and at similar rate by EF and PEF due to the additional oxidation by •OH in the bulk, formed from Fenton's reaction between cathodically generated H2O2 and added Fe2+. The AO-H2O2 treatment yielded the lowest mineralization. The EF process led to persistent final products like Fe(III) complexes, which were quickly photolyzed upon UVA irradiation in PEF to give an almost total mineralization with 98% total organic carbon removal. The effect of current density and substrate concentration on all the mineralization processes was examined. Gas chromatography-mass spectrometry (GC-MS) analysis of electrolyzed solutions allowed identifying five primary aromatics and one heteroaromatic molecule, whereas final carboxylic acids like fumaric, acetic, and oxalic were quantified by ion exclusion high-performance liquid chromatography (HPLC). From all the products detected, a degradation route for trans-cinnamic acid is proposed.

Improved Stability of a Model IgG3 by DoE-Based Evaluation of Buffer Formulations.

Formulating appropriate storage conditions for biopharmaceutical proteins is essential for ensuring their stability and thereby their purity, potency, and safety over their shelf-life. Using a model murine IgG3 produced in a bioreactor system, multiple formulation compositions were systematically explored in a DoE design to optimize the stability of a challenging antibody formulation worst case. The stability of the antibody in each buffer formulation was assessed by UV/VIS absorbance at 280 nm and 410 nm and size exclusion high performance liquid chromatography (SEC) to determine overall solubility, opalescence, and aggregate formation, respectively. Upon preliminary testing, acetate was eliminated as a potential storage buffer due to significant visible precipitate formation. An additional 24 full factorial DoE was performed that combined the stabilizing effect of arginine with the buffering capacity of histidine. From this final DoE, an optimized formulation of 200 mM arginine, 50 mM histidine, and 100 mM NaCl at a pH of 6.5 was identified to substantially improve stability under long-term storage conditions and after multiple freeze/thaw cycles. Thus, our data highlights the power of DoE based formulation screening approaches even for challenging monoclonal antibody molecules.

Polymeric Proteins Formation During Pasta-making with Einkorn (Triticum monococcum) and Semolina Mixtures and Effects on Cooking Behaviour and Acceptability

Pasta was produced in a pilot plant from semolina and semolina blended with increasing amounts of einkorn flour. According to size exclusion high-performance liquid chromatography (SE-HPLC), einkorn proteins interact with semolina proteins during pasta making, forming polymers of high molecular weight. Of these, the unextractable polymeric proteins (UPP) of pasta replaced with 50% einkorn were at significantly higher concentrations than in pasta made from semolina. The increase of S-S bonds and the decrease of –SH free groups in 50% einkorn pasta, with respect to that made of semolina, suggested that polymerization among the different class of proteins happen mainly through disulphide bonds. A decrease in stickiness and an increase in firmness in 50% einkorn pasta corresponded to the formation of large and insoluble protein aggregates.