Protein Purification Process Research Articles

Enhancing aggregate reduction using anion exchange hybrid filter in an immunocytokine diabody fusion protein purification process

Enhancing aggregate reduction using anion exchange hybrid filter in an immunocytokine diabody fusion protein purification process

Production of Tobacco Etch Virus Protease (TEV) Expressed in the Endotoxin-Free Bacillus subtilis and Its Application.

Tobacco Etch virus (TEV) protease is one of the most common tools for removing fusion tags, but no study has shown that TEV can be expressed at high levels in the GRAS host strain Bacillus subtilis and purified for further application. In this study, the fusion protein BsLysSN-TEV C/S-His-TEV consisting of a fusion tag, N-terminal domain of a lysyl-tRNA synthetase (BsLysSN) coded by B. subtilis lysS gene, placed at the N-terminus followed by an endoprotease TEV cleavage site and then the expression of this fusion protein in the cytoplasm of B. subtilis was investigated. The SDS-PAGE and Western-blot analysis demonstrated that His-TEV was overexpressed under the induction of IPTG. This result infers that His-TEV protease showed promising activity in the B. subtilis cytoplasm by the cleavage of the fusion protein. These cleavage products could be purified using the Ni-NTA column, which effectively cleaved the purified recombinant protein substrate, which can be applied in the protein purification process to remove the fusion tag. Significantly, since both His-TEV protease and the fusion recombinant protein substrate are expressed in the endotoxin-free host strain, the tag removal and purified product should be theoretically endotoxin-free, which could be a promising approach for producing therapeutic proteins and also for other relevant biomedical applications.

Application of flowsheet modeling for scheduling and debottlenecking analysis to support the development and scale-up of a plasma-derived therapeutic protein purification process

Plasma fractionation stands as a pivotal process for the production of therapeutic and diagnostic proteins, such as albumin and immunoglobulin G. Besides these two primary proteins in human plasma, numerous other proteins can be purified for therapeutic purposes. To support process development, a flowsheet modeling-based approach is utilized to improve production efficiency and productivity while minimizing the resource investments. The flowsheet model is first built to represent the baseline drug substance production process at pilot-scale, with operating parameters extrapolated from lab-scale experiments conducted at CSL Behring. To improve operational efficiency and save costs, throughput analysis is applied to enhance the batch throughput through new process design, scheduling, and bottleneck identification. Through implementing the strategies, the batch throughput could be increased by 47.2 % by introducing one additional operator and one buffer preparation tank into the process. Furthermore, after applying a new strategy involving multiple extractions of the initial material (paste), the batch throughput was doubled, with operating cost of goods reduced by 36.1 %. To assess the performance of the modified design and validate the model results, the pilot-scale experiments with two extractions were performed by CSL Behring and compared with model predictions, resulting in good agreement. This work demonstrates the potential of flowsheet modeling in facilitating process development from lab-scale to pilot-scale, fostering cost-effective and efficient production with limited resource investment.

Raman and NIR spectroscopy-based real-time monitoring of the membrane filtration process of a recombinant protein for the diagnosis of SARS-CoV-2

This research shows the detailed comparison of Raman and near-infrared (NIR) spectroscopy as Process Analytical Technology tools for the real-time monitoring of a protein purification process. A comprehensive investigation of the application and model development of Raman and NIR spectroscopy was carried out for the real-time monitoring of a process-related impurity, imidazole, during the tangential flow filtration of Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike protein. The fast development of Raman and NIR spectroscopy-based calibration models was achieved using offline calibration data, resulting in low calibration and cross-validation errors. Raman model had an RMSEC of 1.53 mM, and an RMSECV of 1.78 mM, and the NIR model had an RMSEC of 1.87 mM and an RMSECV of 2.97 mM. Furthermore, Raman models had good robustness when applied in an inline measurement system, but on the contrary NIR spectroscopy was sensitive to the changes in the measurement environment. By utilizing the developed models, inline Raman and NIR spectroscopy were successfully applied for the real-time monitoring of a process-related impurity during the membrane filtration of a recombinant protein. The results enhance the importance of implementing real-time monitoring approaches for the broader field of diagnostic and therapeutic protein purification and underscore its potential to revolutionize the rapid development of biological products.

Small ubiquitin-like modifier-tag and modified protein purification significantly increase the quality and quantity of recombinant African swine fever virus p30 protein.

African swine fever (ASF) is a highly virulent and contagious viral disease caused by the ASF virus (ASFV). It has a significant impact on swine production throughout the world, while existing vaccines and specific treatments remain ineffective. ASFV p30 is a potent antigenic protein that induces protective antibodies immediately after infection; however, most recombinant p30 is insoluble. This study aimed to improve the solubility, yield, and purity of recombinant p30 by tagging it with a small ubiquitin-like modifier (SUMO) and modifying the protein purification process. SUMO fused with ASFV p30 (SUMO-p30) and p30 alone were cloned and expressed in Escherichia coli. SUMO-p30 and p30 solubility and expression levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein purification was modified by combining ammonium sulfate precipitation method with affinity chromatography. In addition, large-scale production of all versions of p30 were compared using SDS-PAGE and western blotting, and the purified p30 was used to develop the indirect enzyme-linked immunosorbent assay (ELISA). The solubility and expression levels of SUMO-p30 were dramatically enhanced compared with that of p30. Modification of the purification process significantly increased purified and soluble SUMO-p30 and p30 yields by 6.59 and 1.02 μg/mL, respectively. Large-scale production confirmed that this procedure increased the quantity of recombinant p30 while maintaining protein purity and immunogenicity. The p30-based indirect ELISA was able to discriminate between positive and negative serum samples with statistically significant differences in mean optical density 450 values (p < 0.001). This study demonstrates the enhancement of solubility, purity, and yield of ASFV p30 expressed in E.coli by SUMO fusion tagging and combining ammonium sulfate precipitation with affinity chromatography for protein purification. These positive effects were sustained in large-scale production. Cleavage and removal of hexahistidine-SUMO tag from the fusion protein by protease may not be suitable when handling a large amount of the protein. However, the SUMO-fused p30 retained strong immunoreactivity to convalescent swine serum, indicating its application in immunization and diagnostic purposes. The expression and purification procedures in this study could be applied to increase solubility, quality, and quantity of other recombinant proteins as well.

Novel method for production and purification of untagged pneumococcal surface protein A from clade 1

Streptococcus pneumoniae can cause diseases with high mortality and morbidity. The licensed vaccines are based on capsular polysaccharides and induce antibodies with low cross reactivity, leading to restricted coverage of serotypes. For surpassing this limitation, new pneumococcal vaccines are needed for induction of broader protection. One important candidate is the pneumococcal surface protein A (PspA), which can be classified in 6 clades and 3 families. We have reported an efficient process for production and purification of untagged recombinant PspA from clade 4 (PspA4Pro). We now aim to obtain a highly pure recombinant PspA from clade 1 (PspA1) to be included, together with PspA4Pro, in a vaccine formulation to broaden response against pneumococci. The vector pET28a-pspA1 was constructed and used to transform Escherichia coli BL21(DE3) strain. One clone with high production of PspA1 was selected and adapted to high-density fermentation (HDF) medium. After biomass production in 6 L HDF using a bioreactor, the purification was defined after testing 3 protocols. During the batch bioreactor cultivation, plasmid stability remained above 90% and acetate formation was not detected. The final protein purification process included treatment with a cationic detergent after lysis, anion exchange chromatography, cryoprecipitation, cation exchange chromatography, and multimodal chromatography. The final purification process showed PspA1 purity of 93% with low endotoxin content and an overall recovery above 20%. The novel established process can be easily scaled-up and proved to be efficient to obtain a highly pure untagged PspA1 for inclusion in vaccine formulations.Key points• Purification strategy for recombinant PspA1 from Streptococcus pneumoniae• Downstream processing for untagged protein antigens, the case of PspA1• Purification strategy for PspA variants relies on buried amino acids in their sequences

Wheat flour-derived amyloid fibrils for efficient removal of organic dyes from contaminated water

Amyloid fibrils derived from different proteins have been proved as a promising material for adsorption of various pollutants from wastewater, which showed advantages of low cost and eco-friendliness. However, most of the amyloid fibrils derived from animal-based proteins with high environmental footprint, while more sustainable amyloid fibrils derived from plant materials are desirable. In this study, a plant-derived amyloid fibril was extracted from the commonly used wheat flour with a simple and scalable protein purification and fibrillization process. Interestingly, the amyloid fibrils showed good adsorption capacity towards typical organic dyes (Eosin Y (EY) and Congo red (CR)) from contaminated water. Adsorption kinetic analysis indicated the adsorption process to EY or CR by wheat flour amyloid well fitted with a pseudo-second-order model. The adsorption also followed a Langmuir isothermal model with adsorption capacities of 333 mg/g and 138 mg/g towards CR and EY, respectively. This work demonstrated the feasibility to utilize the plant-based amyloid fibril for organic dyes removal from contaminated water, which provided an affordable, sustainable and scalable tool for organic dyes removal from wastewater.Graphical

Removal of plant endogenous proteins from tobacco leaf extract by freeze–thaw treatment for purification of recombinant proteins

Plants are useful as a low-cost source for producing biopharmaceutical proteins. A significant hurdle in the production of recombinant proteins in plants, however, is the complicated process of removing plant-derived components. Removing endogenous plant proteins, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), a major photosynthetic plant enzyme that catalyzes photosynthesis through carboxylation and oxygenation, is important for the purification of recombinant plant proteins. In particular, RuBisCO accounts for 50% of the soluble leaf protein; thus, the removal of RuBisCO is critical for the purification of recombinant proteins from plant materials. An effective conventional method, known as freeze–thaw treatment, was developed for the removal of RuBisCO from Nicotiana benthamiana, which expresses recombinant green fluorescent protein (GFP). Crude extracts or supernatants were frozen at − 30 °C. Upon thawing, most of the RuBisCO was precipitated by centrifugation without significant inactivation and/or yield reduction of GFP. Based on the proteomics analysis, using this method, RuBisCO large and small subunits were reduced to approximately 10% and 20% of those of the unfrozen supernatant solutions, respectively, without the need for specific reagents or equipment. The proteomic analysis also revealed that many ribosomal proteins were removed from the extracts. This method improves the purification process of recombinant proteins from plant materials. Prolonged freezing damaged recombinant β-glucuronidase (GUS), suggesting that the applicability of this treatment should be carefully considered for each recombinant protein.

Breakthrough curve modeling for lysozyme by ion-exchange nanofiber membrane: Linear and nonlinear analysis

BackgroundNanofibers prepared by electrospinning technology are widely used as an adsorber in protein purification processes. However, the evaluation of dynamic adsorption performance has not been reported yet. MethodsLysozyme was subjected to assess dynamic adsorption of weak ion exchange nanofiber membranes by applying a continuous flow system. Different operating conditions were selected to study the lysozyme adsorption breakthrough curve, which included four factors: pH, feedstock lysozyme concentration, feed flow rate, and stacked membrane layers. The breakthrough curve was applied to analyze the performance for dynamic binding with lysozyme as a model protein. In addition, the adsorption models (Bohart-Adams, Thomas, Yoon-Nelson, and BDST) were fitted to the breakthrough curves using linear and nonlinear regression analysis. Significant findingsIt was found that the four operating parameters had considerable influences on the dynamic adsorption performance. Among these models, the breakthrough curve was well-fitted by using linear regression analysis of the Yoon-Nelson model. The breakthrough curve characteristics could be used to describe the dynamic binding performance of interested membranes to lysozyme molecules.

Extract2Chip-Bypassing Protein Purification in Drug Discovery Using Surface Plasmon Resonance.

Modern drug discovery relies on combinatorial screening campaigns to find drug molecules targeting specific disease-associated proteins. The success of such campaigns often relies on functional and structural information of the selected therapeutic target, only achievable once its purification is mastered. With the aim of bypassing the protein purification process to gain insights on the druggability, ligand binding, and/or characterization of protein-protein interactions, herein, we describe the Extract2Chip method. This approach builds on the immobilization of site-specific biotinylated proteins of interest, directly from cellular extracts, on avidin-coated sensor chips to allow for the characterization of molecular interactions via surface plasmon resonance (SPR). The developed method was initially validated using Cyclophilin D (CypD) and subsequently applied to other drug discovery projects in which the targets of interest were difficult to express, purify, and crystallize. Extract2Chip was successfully applied to the characterization of Yes-associated protein (YAP): Transcriptional enhancer factor TEF (TEAD1) protein-protein interaction inhibitors, in the validation of a ternary complex assembly composed of Dyskerin pseudouridine synthase 1 (DKC1) and RuvBL1/RuvBL2, and in the establishment of a fast-screening platform to select the most suitable NUAK family SNF1-like kinase 2 (NUAK2) surrogate for binding and structural studies. The described method paves the way for a potential revival of the many drug discovery campaigns that have failed to deliver due to the lack of suitable and sufficient protein supply.

Improvement of Selectivity and Antifouling Properties of Chitosan-modified Polyvinylidene Fluoride (PVDF) Membrane for Protein Filtration

&lt;p&gt;Protein with high purity is currently a valuable commercial commodity, prized for its high value and application in specialized functions. PVDF-based membrane technology has found widespread use in protein purification processes. However, the issue of fouling on the membrane surface frequently hampers the performance of PVDF membranes. The present study aims to enhance PVDF membranes' selectivity and antifouling properties through chitosan modification, thereby optimizing protein filtration. PVDF and PVDF/Chitosan membranes were fabricated using the phase inversion technique, followed by a comprehensive characterization of their surface properties, thermal attributes, and performance in bovine serum albumin (BSA) protein filtration. The collected data revealed notable increases in membrane hydrophilicity, porosity, and pure water flux due to the incorporation of chitosan. The membrane protein rejection capabilities were significantly enhanced to levels exceeding 90% with the introduction of 0.5% chitosan. Moreover, as the chitosan concentration increased, the membrane exhibited superior antifouling characteristics, with the 0.5% chitosan concentration yielding the highest Flux Recovery Ratio (FRR) value of 85.35%. Considering the significant improvement selectivity and antifouling properties in the combined system, the chitosan-modified PVDF membrane holds substantial promise in protein purification applications.&lt;/p&gt;

Improvement of Selectivity and Antifouling Properties of Chitosan-modified Polyvinylidene Fluoride (PVDF) Membrane for Protein Filtration

&lt;p&gt;Protein with high purity is currently a valuable commercial commodity, prized for its high value and application in specialized functions. PVDF-based membrane technology has found widespread use in protein purification processes. However, the issue of fouling on the membrane surface frequently hampers the performance of PVDF membranes. The present study aims to enhance PVDF membranes' selectivity and antifouling properties through chitosan modification, thereby optimizing protein filtration. PVDF and PVDF/Chitosan membranes were fabricated using the phase inversion technique, followed by a comprehensive characterization of their surface properties, thermal attributes, and performance in bovine serum albumin (BSA) protein filtration. The collected data revealed notable increases in membrane hydrophilicity, porosity, and pure water flux due to the incorporation of chitosan. The membrane protein rejection capabilities were significantly enhanced to levels exceeding 90% with the introduction of 0.5% chitosan. Moreover, as the chitosan concentration increased, the membrane exhibited superior antifouling characteristics, with the 0.5% chitosan concentration yielding the highest Flux Recovery Ratio (FRR) value of 85.35%. Considering the significant improvement selectivity and antifouling properties in the combined system, the chitosan-modified PVDF membrane holds substantial promise in protein purification applications.&lt;/p&gt;&lt;p&gt; &lt;/p&gt;&lt;p&gt;&lt;img src="/public/site/images/fitria/rsz_72110.png" alt="" /&gt;&lt;/p&gt;

Characterization of ionic strength for X-MuLV inactivation by low pH treatment for monoclonal antibody purification.

In the production of monoclonal antibodies (mAbs) intended for use in humans, it is a global regulatory requirement that the manufacturing process includes unit operations that are proven to inactivate or remove adventitious agents to ensure viral safety. Viral inactivation by low pH hold (LPH) is typically used to ensure this viral safety in the purification process of mAbs and other biotherapeutics derived from mammalian cell lines. To ascertain the effectiveness of the LPH step, viral clearance studies have evaluated LPH under worst-case conditions of pH above the manufacturing set point and hold duration at or below the manufacturing minimum. Highly acidic conditions (i.e., pH < 3.60) provide robust and effective enveloped virus inactivation but may lead to reduced product quality of the therapeutic protein. However, when viral inactivation is operated above pH 3.60 to ensure product stability, effective (>4 log10 reduction factor) viral inactivation may not be observed under these worst-case pH conditions in viral clearance studies. A multivariate design of experiments was conducted to further characterize the operating space for low pH viral inactivation of a model retrovirus, xenotropic murine leukemia virus (X-MuLV). The statistically designed experiment evaluated the effect of mAb isotype, pH, temperature, acid titrant, sodium chloride (NaCl) concentration, virus spike timing, and post-spike filtration on X-MuLV inactivation. Data from the characterization study were used to generate predictive models to identify conditions that reliably achieve effective viral inactivation at pH ≥ 3.60. Results of the study demonstrated that NaCl concentration has the greatest effect on virus inactivation in the range studied, and pH has a large effect when the load material has no additional NaCl. Overall, robust and effective inactivation of X-MuLV at pH 3.65-3.80 can be achieved by manipulating either the pH or the NaCl concentration of the load material. This study contributes to the understanding of ionic strength as an influential parameter in low pH viral inactivation studies.

Creation of a versatile automated two-step purification system with increased throughput capacity for preclinical mAb material generation

The ever-increasing speed of biotherapeutic drug discovery has driven the development of automated and high throughput purification capabilities. Typically, purification systems require complex flow paths or third-party components that are not found on a standard fast protein liquid chromatography instrument (FPLC) (e.g., Cytiva's ÄKTA) to enable higher throughput. In early mAb discovery there is often a trade-off between throughput and scale where a high-throughput process requires miniaturized workflows necessitating a sacrifice in the amount of material generated. At the interface of discovery and development, flexible automated systems are required that can perform purifications in a high-throughput manner, while also generating sufficient quantities of preclinical material for biophysical, developability, and preclinical animal studies. In this study we highlight the engineering efforts to generate a highly versatile purification system capable of balancing the purification requirements between throughput, chromatographic versatility, and overall product yields. We incorporated a 150 mL Superloop into an ÄKTA FPLC system to expand our existing purification capabilities. This allowed us to perform a range of automated two-step tandem purifications including primary affinity captures (protein A (ProA)/immobilized metal affinity chromatography (IMAC)/antibody fragment (Fab)) followed by secondary polishing with either size exclusion (SEC) or cation exchange (CEX) chromatography. We also integrated a 96 deep-well plate fraction collector into the ÄKTA FPLC system with purified protein fractions being analyzed by a plate based high performance liquid chromatography instrument (HPLC). This streamlined automated purification workflow allowed us to process up to 14 samples within 24 h, enabling purification of ∼1100 proteins, monoclonal antibodies (mAbs), and mAb related protein scaffolds during a 12-month period. We purified a broad range of cell culture supernatant volumes, between 0.1 and 2 L, with final purification yields up to 2 g. The implementation of this new automated, streamlined protein purification process greatly expanded our sample throughput and purification versatility while also enabling the accelerated production of greater quantities of biotherapeutic candidates for preclinical in vivo animal studies and developability assessment.

Study on the secondary structure and hydration effect of human serum albumin under acidic pH and ethanol perturbation with IR/NIR spectroscopy

Human serum albumin (HSA) is the most abundant protein in plasma and plays an essential physiological role in the human body. Ethanol precipitation is the most widely used way to obtain HSA, and pH and ethanol are crucial factors affecting the process. In this study, infrared (IR) spectroscopy and near-infrared (NIR) spectroscopy in combination with chemometrics were used to investigate the changes in the secondary structure and hydration of HSA at acidic pH (5.6–3.2) and isoelectric pH when ethanol concentration was varied from 0% to 40% as a perturbation. IR spectroscopy combined with the two-dimensional correlation spectroscopy (2DCOS) analysis for acid pH system proved that the secondary structure of HSA changed significantly when pH was around 4.5. What’s more, the IR spectroscopy and 2DCOS analysis showed different secondary structure forms under different ethanol concentrations at the isoelectric pH. For the hydration effect analysis, NIR spectroscopy combined with the McCabe–Fisher method and aquaphotomics showed that the free hydrogen-bonded water fluctuates dynamically, with ethanol at 0–20% enhancing the hydrogen-bonded water clusters, while weak hydrogen-bonded water clusters were formed when the ethanol concentration increased continuously from 20% to 30%. These measurements provide new insights into the structural changes and changes in the hydration behavior of HSA, revealing the dynamic process of protein purification, and providing a theoretical basis for the selection of HSA alcoholic precipitation process parameters, as well as for further studies of complex biological systems.

Amberlite XAD-4 is a convenient tool for removing Triton X-100 and Sarkosyl from protein solutions.

Amberlite has been shown to be an appropriate material for the adsorption of organic contaminants from aqueous solutions. In addition, Amberlite XAD-2 has been successfully used, as an alternative to Bio-Beads, to remove Triton X-100 from protein solutions, such as from samples of solubilized membrane proteins. However, Amberlite has not been tested as an adsorbent when a mixture of detergents is necessary to solubilize and refold a target protein. Here the authors show that Amberlite XAD-4 can be appropriately used to aid the purification process of proteins solubilized from inclusion bodies with the ternary detergent system consisting of Sarkosyl, Triton X-100 and CHAPS.

1Progress, applications, challenges and prospects of protein purification technology.

Protein is one of the most important biological macromolecules in life, which plays a vital role in cell growth, development, movement, heredity, reproduction and other life activities. High quality isolation and purification is an essential step in the study of the structure and function of target proteins. Therefore, the development of protein purification technologies has great theoretical and practical significance in exploring the laws of life activities and guiding production practice. Up to now, there is no forthcoming method to extract any proteins from a complex system, and the field of protein purification still faces significant opportunities and challenges. Conventional protein purification generally includes three steps: pretreatment, rough fractionation, and fine fractionation. Each of the steps will significantly affect the purity, yield and the activity of target proteins. The present review focuses on the principle and process of protein purification, recent advances, and the applications of these technologies in the life and health industry as well as their far-reaching impact, so as to promote the research of protein structure and function, drug development and precision medicine, and bring new insights to researchers in related fields.

Production and Evaluation of Chicken Egg Yolk Immunoglobulin (IgY) against Human and Simian Rotaviruses.

Producing specific antibodies in chickens is an attractive approach for diagnosis or therapeutic applications. Besides the high immunoglobulin Y (IgY) yield transferred to the egg yolk and its suitability for large-scale production, such an approach is more bioethical for animal maintenance. The IgY technology offers new possibilities for application in human and veterinary diagnostics and therapeutics, including strategies for treating severe intestinal diseases in children, particularly in emerging countries. Herein, we describe the production and purification of polyclonal antibodies against rotavirus group A (RVA) in immunised hens aiming at its application in prophylaxis and treatment of rotavirus-induced diarrhoea. For this purpose, we inoculated Rhodia laying chickens (Gallus gallus domesticus) with two or three doses of RVA combined with adjuvants or only adjuvants (control group). As the egg-laying period began, the yolk protein purification processes yielded a high concentration of specific IgY, the highest titre resulting from the group of hens that received three doses of the immunogen. The purified IgY blocked the functional activity of RVA in MA-104 cells, thus confirming the neutralisation ability. Therefore, anti-RVA IgY could be a promising candidate for pre- and post-exposure prevention or treatment of rotavirus-induced diarrhoea.

REVOLVER: A low-cost automated protein purifier based on parallel preparative gravity column workflows

Protein purification is a ubiquitous procedure in biochemistry and the life sciences, and represents a key step in the protein production pipeline. The need for scalable and parallel protein purification systems is driven by the demands for increasing the throughput of recombinant protein characterization. Therefore, automating the process to simultaneously handle multiple samples with minimal human intervention is highly desirable, yet there are only a handful of such systems that have been developed, all of which are closed source and expensive. To address this challenge, we present REVOLVER, a 3D-printed programmable protein purification system based on gravity-column workflows and controlled by Arduino boards that can be built for under $130 USD. REVOLVER takes a cell lysate sample and completes a full protein purification process with almost no human intervention and yields results indistinguishable from those obtained by an experienced biochemist when purifying a real-world protein sample. We further present and describe MULTI-VOLVER, a scalable version of the REVOLVER that allows for parallel purification of up to six samples and can be built for under $250 USD. Both systems can help accelerate protein purification and ultimately link them to bio-foundries for protein characterization and engineering.

Investigation of protective effect of ethanol on the natural structure of protein with infrared spectroscopy

The stability of biological drugs with protein as an active substance depends heavily on the retention of natural protein structure during freeze-drying. Stabilizers have become important substances in the process of protein freeze-drying. In order to further understand the mechanism of the interaction between protein and stabilizers, human serum albumin (HSA) and simple hydroxyl compound ethanol were used as models. Infrared (IR) spectroscopy combined with chemometrics was implemented to investigate the changes of secondary structure and hydration of HSA when different concentrations of ethanol were considered as interference. Through the analysis of the protein secondary structure and hydrated layer, we found that the addition of ethanol-d6 increased the α-helix of HSA and reduced the disordered structure. The hydrogen bond structure around HSA was enhanced and intermolecular aggregation was reduced through the action of the water molecules. The hypothesis was verified by circular dichroism (CD) and transmission electron microscopy (TEM) observation by adding different concentrations of ethanol-d6. It was found that a small amount of ethanol could protect the native conformation of HSA. In conclusion, this study revealed the mechanism of ethanol as a protein protector, provided a new idea for protein purification process and a theoretical basis for biomolecular interaction.