Purification Process for a Secreted Protein Produced in Insect Cells.

The Baculovirus Expression Vector System (BEVS) is used with cultured insect cells to produce a wide variety of heterologous proteins, which can be secreted into the culture medium during the transient infection process (Smith et al. Mol Cell Biol 12:2156-2165, 1983). When the infection process is complete, centrifugation is often used to separate the desired protein from the spent insect cells. The desired product in the harvested supernatant is contaminated with baculovirus, amino acids, lipids, detergents, oils, lysed cells from the infection process, genomic DNA from the insect cells, and proteases due to the lytic nature of the baculovirus infection process and many other contaminants (Ikonomou et al. Appl Microbiol Biotechnol 62:1-20, 2003). All these contaminants that are present in the centrifuged supernatant with the desired secreted protein make the initial chromatographic capture step critical for effective purification of the desired protein. A purification scheme will be outlined for a slightly acidic secreted protein using cation exchange chromatography (Lundanes et al. Chromatography: basic principles, sample preparations and related methods, 1st edn. Wiley, 2013).

One-Step Purification Strategy for Cowpea Chlorotic Mottle Virus-Like Particles Produced by the IC-BEVS.

Virus-like particles (VLP) of the cowpea chlorotic mottle virus (CCMV), a plant virus, have been shown to be safe and noncytotoxic vehicles for delivering various cargos, including nucleic acids and peptides, and as scaffolds for presenting epitopes. Thus, CCMV-VLP have acquired increasing attention to be used in fields such as gene therapy, drug delivery, and vaccine development. Regardless of their production method, most reports purify CCMV-VLP through a series of ultracentrifugation steps using sucrose density gradient ultracentrifugation, which is a complex and time-consuming process. Here, the use of anion exchange chromatography is described as a one-step protocol for purification of CCMV-VLP produced by the insect cell-baculovirus expression vector system (IC-BEVS).

Anion Exchange Chromatography-Mass Spectrometry to Characterize Proteoforms of Alpha-1-Acid Glycoprotein during and after Pregnancy.

Alpha-1-acid glycoprotein (AGP) is a heterogeneous glycoprotein fulfilling key roles in many biological processes, including transport of drugs and hormones and modulation of inflammatory and immune responses. The glycoform profile of AGP is known to change depending on (patho)physiological states such as inflammatory diseases or pregnancy. Besides complexity originating from five N-glycosylation sites, the heterogeneity of the AGP further expands to genetic variants. To allow in-depth characterization of this intriguing protein, we developed a method using anion exchange chromatography (AEX) coupled to mass spectrometry (MS) revealing the presence of over 400 proteoforms differing in their glycosylation or genetic variants. More precisely, we could determine that AGP mainly consists of highly sialylated higher antennary structures with on average 16 sialic acids and 0 or 1 fucose per protein. Interestingly, a slightly higher level of fucosylation was observed for AGP1 variants compared to that of AGP2. Proteoform assignment was supported by integrating data from complementary MS-based approaches, including AEX-MS of an exoglycosidase-treated sample and glycopeptide analysis after tryptic digestion. The developed analytical method was applied to characterize AGP from plasma of women during and after pregnancy, revealing differences in glycosylation profiles, specifically in the number of antennae, HexHexNAc units, and sialic acids.

Integrated Platform for Large-Scale Quantitative Profiling of Phosphotyrosine Signaling Complexes Based on Cofractionation/Mass Spectrometry and Complex-Centric Algorithm.

The scarcity and dynamic nature of phosphotyrosine (pTyr)-modified proteins pose a challenge for researching protein complexes with pTyr modification, which are assembled through multiple protein-protein interactions. We developed an integrated complex-centric platform for large-scale quantitative profiling of pTyr signaling complexes based on cofractionation/mass spectrometry (CoFrac-MS) and a complex-centric algorithm. We initially constructed a trifunctional probe based on pTyr superbinder (SH2-S) for specifically binding and isolation of intact pTyr protein complexes. Then, the CoFrac-MS strategy was employed for the identification of pTyr protein complexes by integrating ion exchange chromatography in conjunction with data independent acquisition mass spectrometry. Furthermore, we developed a novel complex-centric algorithm for quantifying protein complexes based on the protein complex elution curve. Utilizing this algorithm, we effectively quantified 216 putative protein complexes. We further screened 21 regulated pTyr protein complexes related to the epidermal growth factor signal. Our study engenders a comprehensive framework for the intricate examination of pTyr protein complexes and presents, for the foremost occasion, a quantitative landscape delineating the composition of pTyr protein complexes in HeLa cells.

Online Mixed-Bed Ion Exchange Chromatography for Native Top-Down Proteomics of Complex Mixtures.

Native top-down mass spectrometry (nTDMS) allows characterization of protein structure and noncovalent interactions with simultaneous sequence mapping and proteoform characterization. The majority of nTDMS studies utilize purified recombinant proteins, with significant challenges hindering application to endogenous systems. To perform native top-down proteomics (nTDP), where endogenous proteins from complex biological systems are analyzed by nTDMS, it is essential to separate proteins under nondenaturing conditions. However, it remains difficult to achieve high resolution with MS-compatible online chromatography while preserving protein tertiary structure and noncovalent interactions. Herein, we report the use of online mixed-bed ion exchange chromatography (IEC) to enable separation of endogenous proteins from complex mixtures under nondenaturing conditions, preserving noncovalent interactions for nTDP analysis. We have successfully detected large proteins (>146 kDa) and identified endogenous metal-binding and oligomeric protein complexes in human heart tissue lysate. The use of a mixed-bed stationary phase allowed retention and elution of proteins over a wide range of isoelectric points without altering the sample or mobile phase pH. Overall, our method provides a simple online IEC-MS platform that can effectively separate proteins from complex mixtures under nondenaturing conditions and preserve higher-order structure for nTDP applications.

[Recent progress of chromatographic techniques for antibody purification].

Antibody drugs are becoming increasingly popular in disease diagnosis, targeted therapy, and immunoprevention owing to their characteristics of high targeting ability, strong specificity, low toxicity, and mild side effects. The demand for antibody drugs is steadily increasing, and their production scale is expanding. Upstream cell culture technology has been greatly improved by the high-capacity production of monoclonal antibodies. However, the downstream purification of antibodies presents a bottleneck in the production process. Moreover, the purification cost of antibodies is extremely high, accounting for approximately 50%-80% of the total cost of antibody production. Chromatographic technology, given its selectivity and high separation efficiency, is the main method for antibody purification. This process usually involves three stages: antibody capture, intermediate purification, and polishing. Different chromatographic techniques, such as affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, and temperature-responsive chromatography, are used in each stage. Affinity chromatography, mainly protein A affinity chromatography, is applied for the selective capture and purification of antibodies from raw biofluids or harvested cell culture supernatants. Other chromatographic techniques, such as ion-exchange chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography, are used for intermediate purification and antibody polishing. Affinity biomimetic chromatography and hydrophobic charge-induction chromatography can produce antibodies with purities comparable with those obtained through protein A chromatography, by employing artificial chemical/short peptide ligands with good selectivity, high stability, and low cost. Temperature-responsive chromatography is a promising technique for the separation and purification of antibodies. In this technique, antibody capture and elution is controlled by simply adjusting the column temperature, which greatly eliminates the risk of antibody aggregation and inactivation under acidic elution conditions. The combination of different chromatographic methods to improve separation selectivity and achieve effective elution under mild conditions is another useful strategy to enhance the yield and quality of antibodies. This review provides an overview of recent advances in the field of antibody purification using chromatography and discusses future developments in this technology.

Optimization of High-Activity earthworm fibrinolytic enzyme extraction methods and protein component analysis.

OBJECTIVE: Optimize the extraction process of earthworm fibrinolytic enzyme. METHODS: Chinese common earthworms underwent a series of purification processes, including grinding, salting out, hydrophobic medium chromatography, ammonium sulfate precipitation, and ion exchange chromatography, to obtain purified earthworm fibrinolytic enzyme. RESULTS: Utilizing Pheretima aspergillum as the starting material, we discovered that the specific activity of lumbrokinase extracted via ammonium sulfate precipitation was 58 U/mg, noticeably surpassing that achieved through heat precipitation and ethanol precipitation methods. After undergoing two rounds of chromatographic separations employing hydrophobic affinity chromatography and anion exchange chromatography, the specific activity of the lumbrokinase protein soared to 9267 U/mg, significantly exceeding the 3,178 U/mg specific activity attained through industrial extraction methods. DISCUSSION: The development of a novel crude extraction method for lumbrokinase protein can significantly boost its activity and purity. The discovery of a high-efficiency purification method and the identification of protein components within highly active lumbrokinase pave the way for further investigations into these proteins.

Separation of full, empty, and partial adeno-associated virus capsids via anion-exchange chromatography with continuous recycling and accumulation.

The field of recombinant adeno-associated virus (rAAV) gene therapy has attracted increasing attention over decades. Within the ongoing challenges of rAAV manufacturing, the co-production of impurities, such as empty and partial capsids containing no or truncated transgenes, poses a significant challenge. Due to their potential impact on drug efficacy and clinical safety, it is imperative to conduct comprehensive monitoring and characterization of these impurities prior to the release of the final gene therapy product. Nevertheless, existing analytical techniques encounter notable limitations, encompassing low throughput, long turnaround times, high sample consumption, and/or complicated data analysis. Chromatography-based analytical methods are recognized for their current Good Manufacturing Practice (cGMP) alignment, high repeatability, reproducibility, low limit of detection, and rapid turnaround times. Despite these advantages, current anion exchange high pressure liquid chromatography (AEX-HPLC) methods struggle with baseline separation of partial capsids from full and empty capsids, resulting in inaccurate full-to-empty capsid ratio, as partial capsids are obscured within peaks corresponding to empty and full capsids. In this study, we present a unique analytical AEX method designed to characterize not only empty and full capsids but also partial capsids. This method utilizes continuous N-Rich chromatography with recycling between two identical AEX columns for the accumulation and isolation of partial capsids. The development process is comprehensively discussed, covering the preparation of reference materials representing full (rAAV-LacZ), partial (rAAV-GFP), and empty (rAAV-empty) capsids, N-rich method development, fraction analysis, determination of fluorescence response factors between capsid variants, and validation through comparison with other comparative techniques.

Production and Purification of Adeno-Associated Viral Vectors (AAVs) Using Orbitally Shaken HEK293 Cells.

Here, we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium using orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI)-mediated transfection of a three-plasmid system and is specified for production in milliliter-to-liter scales. After PEI and plasmid DNA (pDNA) complex formation, the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 7-day batch process, cell cultures are further processed using a set of methods for cell lysis and vector recovery. Methods for the purification of viral particles are described, including immunoaffinity and anion-exchange chromatography, ultrafiltration, as well as digital PCR to quantify the concentration of vector particles.

Purification and Quality Control of Recombinant Proteins Expressed in Mammalian Cells: A Practical Review.

In the recent years, there has been a rapid development of new technologies and strategies when it comes to protein purification and quality control (QC), but the basic technologies for these processes go back a long way, with many improvements over the past few decades. The purpose of this chapter is to review these approaches, as well as some other topics such as the advantages and disadvantages of various purification methods for intracellular or extracellular proteins, the most effective and widely used genetically engineered affinity tags, solubility-enhancing tags, and specific proteases for removal of nontarget sequences. Affinity chromatography (AC), like Protein A or G resins for the recovery of antibodies or Fc fusion proteins or immobilized metals for the recovery of histidine-tagged proteins, will be discussed along with other conventional chromatography techniques: ion exchange (IEC), hydrophobic exchange (HEC), mixed mode (MMC), size exclusion (SEC), and ultrafiltration (UF) systems. How to select and combine these different technologies for the purification of any given protein and the minimal criteria for QC characterization of the purity, homogeneity, identity, and integrity of the final product will be presented.

Synthesis of weak cation exchange/C18 bifunctional magnetic polymers for pretreatment and determination of glufosinate and its two metabolites in plasma samples.

This study focuses on the purification and detection of glufosinate (GLUF) and its metabolites N-acetyl GLUF and MPP in plasma samples. A Dikma Polyamino HILIC column was used for the effective retention and separation of GLUF and its metabolites, and the innovative addition of a low concentration of ammonium fluoride solution to the mobile phase effectively improved the detection sensitivity of the target analytes. Monodisperse core-shell weak cation exchange (WCX)/C18 bifunctional magnetic polymer composites (Fe3O4@WCX/C18) were prepared in a controllable manner, and their morphology and composition were fully characterized. The Fe3O4@WCX/C18 microspheres were used as a magnetic solid-phase extraction (MSPE) adsorbent for the sample purification and detection of GLUF and its metabolites in plasma samples combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The purification conditions of Fe3O4@WCX/C18 microspheres for GLUF and its metabolites in spiked plasma samples were optimized to achieve the best MSPE efficiency. The purification mechanisms of the target analytes in plasma samples include electrostatic attraction and hydrophobic interactions. Furthermore, the effect of the molar ratio of the two functional monomers 4-VBA and 1-octadecene in the adsorbent was optimized and it shows that the bifunctional components WCX/C18 have a synergistic effect on the determination of GLUF and its metabolites in plasma samples. In addition, the present study compared the purification performance of the Fe3O4@WCX/C18 microsphere-based MSPE method with that of the commercial Oasis WCX SPE method, and the results showed that the Fe3O4@WCX/C18 microsphere-based MSPE method established in this work had a stronger ability to remove matrix interferences. Under optimal purification conditions, the recoveries of GLUF and its metabolites in plasma were 87.6-111 % with relative standard deviations (RSDs) ranging from 0.2 % to 4.8 %. The limits of detection (LODs, S/N≥3) and limits of quantification (LOQs, S/N≥10) were 0.10-0.18 µg/L and 0.30-0.54 µg/L, respectively. The MSPE-LC-MS/MS method developed in this study is fast, simple, accurate and sensitive and can be used to confirm GLUF intoxication based not only on the detection of the GLUF prototype but also on the detection of its two metabolites.

Titer and charge-based heterogeneity multiattribute monitoring of mAbs in cell culture harvest using 2D ProA CEX MS.

Robust monitoring of heterogeneity in biopharmaceutical development is crucial for producing safe and efficacious biotherapeutic products. Multiattribute monitoring (MAM) has emerged as an efficient tool for monitoring of mAb heterogeneities like deamidation, sialylation, glycosylation, and oxidation. Conventional biopharma analysis during mAb development relies on use of one-dimensional methods for monitoring titer and charge-based heterogeneity using non-volatile solvents without direct coupling with mass spectrometry (MS). This approach requires analysis of mAb harvest by ProA for titer estimation followed by separate cation exchange chromatography (CEX) analysis of the purified sample for estimating charge-based heterogeneity. This can take up to 60-90 min due to the required fraction collection and buffer exchange steps. In this work, a native two-dimensional liquid chromatography (2DLC) mass spectrometry method has been developed with Protein A chromatography in the first dimension for titer estimation and cation exchange chromatography (CEX) in the second dimension for charge variant analysis. The method uses volatile salts for both dimensions and enables easy coupling to MS. The proposed 2DLC method exhibits a charge variant profile that is similar to that observed via the traditional methods and takes only 15 min for mass identification of each variant. A total of six charge variants were separated by the CEX analysis after titer estimation, including linearity assessment from 5 µg to 160 µg of injected mAb sample. The proposed method successfully estimated charge variants for the mAb innovator and 4 of its biosimilars, showcasing its applicability for biosimilarity exercises. Hence, the 2D ProA CEX MS method allows direct titer and charge variant estimation of mAbs in a single workflow.

A method for the quantitative analysis of polar anionic pesticides in milk/infant formula, cereals and fruit and vegetables using ion chromatography coupled to tandem mass spectrometry.

Polar pesticides such as anionic or ionisable compounds have always provided a challenge for analytical chemists. Methods of analysis have been developed using a range of techniques including normal phase chromatography, ion-pairing, derivatisation and HILIC or multi-mode chromatography. These work well with some of these compounds but, except for HILIC, all of them have their limitations and none of them cover the range required by legislation. Some of these compounds, glyphosate, chlorate and phosphonic acid, are found regularly in a range of food matrices, and therefore reliable methods of analysis are essential. This study describes an ion chromatography method with tandem mass spectrometry detection which not only covers the full range of compounds required by legislation but also can be expanded to include other anionic or ionisable pesticides and metabolites. These include glyphosate and its metabolites, glufosinate and its metabolites, ethephon and its metabolites as well as fosetyl aluminium, chlorate and perchlorate. The method is fully validated according to the performance criteria from the SANTE guidelines for the analysis of pesticides in food and feed over a wide range of matrices, including milk, infant formula, cereals and fruits and vegetables. Over 300 food samples have analysed as part of our routine monitoring program.

Multifunctional-separation-mode ion chromatography method for determining major metabolites during multiple parallel fermentation of rice wine.

Facile and effective analysis methods are desirable for elucidating the behaviours of metabolites during fermentation reactions. Herein, a multifunctional-separation-mode ion chromatography (MFS-IC) method was developed for the simultaneous monitoring of major metabolites during multiple parallel fermentation, including those related to central carbon metabolism (saccharification, glycolysis, alcoholic fermentation, and the tricarboxylic acid (TCA) cycle). The use of two types of sulfo-modified size-exclusion columns and phthalic acid as the eluent allowed the separation of oligosaccharides (disaccharides, trisaccharides, and tetrasaccharides), glucose, pyruvate, and major organic acids during the TCA cycle (cis-aconitate, citrate, iso-citrate, malate, fumarate, and succinate but not α-ketoglutarate) from other non-target analytes. The MFS-IC method was successfully applied to monitoring the major metabolites in the rice wine brewing process. This approach can contribute to an improved understanding of metabolite behaviour during fermentation without requiring the use of expensive advanced instrumentation methods such as liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry.

Characterization of Complex Proteoform Mixtures by Online Nanoflow Ion-Exchange Chromatography-Native Mass Spectrometry.

The characterization of proteins and complexes in biological systems is essential to establish their critical properties and to understand their unique functions in a plethora of bioprocesses. However, it is highly difficult to analyze low levels of intact proteins in their native states (especially those exceeding 30 kDa) with liquid chromatography (LC)-mass spectrometry (MS). Herein, we describe for the first time the use of nanoflow ion-exchange chromatography directly coupled with native MS to resolve mixtures of intact proteins. Reference proteins and protein complexes with molecular weights between 10 and 150 kDa and a model cell lysate were separated using a salt-mediated pH gradient method with volatile additives. The method allowed for low detection limits (0.22 pmol of monoclonal antibodies), while proteins presented nondenatured MS (low number of charges and limited charge state distributions), and the oligomeric state of the complexes analyzed was mostly kept. Excellent chromatographic separations including the resolution of different proteoforms of large proteins (>140 kDa) and a peak capacity of 82 in a 30 min gradient were obtained. The proposed setup and workflows show great potential for analyzing diverse proteoforms in native top-down proteomics, opening unprecedented opportunities for clinical studies and other sample-limited applications.

Impact of drug-linker on method selection for analytical characterization and purification of antibody-drug conjugates.

In addition to traditional characterisation methods of hydrophobic interaction (HIC) and reverse phase (RP) chromatography, an anion exchange chromatography (AIEX) was developed to analyse and purify antibody drug conjugates (ADCs). Since different drug antibody ratio (DAR) species may impact biological activity, therapeutic index, PK parameters or even potential immunogenicity, homogenous ADC DAR demands have been significantly increasing. To accelerate linker designs, drug screening and ADC DAR purification for in vitro and in vivo studies, we built the analytical toolbox including HIC, RP, AIEX, icIEF, SEC, and MS for downstream ADC DAR purification using HIC and AIEX. The established analytical methods can quickly assess the quality of ADC DAR profiles and provide important information to select the proper ADC DAR purification method. Since drug-linker structures can significantly affect ADC physicochemical properties, and highly impact on selections of analytical methods, we applied both HIC and AIEX characterisation and purification platforms to achieve ADC DAR homogenous. Our experiments also implied that unlike HIC, AIEX could be used to separate DAR4 positional isomers.

Strong cation-exchange combined with mass spectrometry reveals the glycoform heterogeneity of sialylated glycoproteins.

Sialylation is an important modification of proteins, related to protein life and bioactivity. However, the evaluation of sialylation is only based on the average molecular composition by peptide mapping and glycan profiling because sialylated proteins are usually too heterogeneous to obtain good quality mass spectra by conventional intact mass analysis methods. In this study, a simple strong cation exchange-mass spectroscopy (SCX-MS) method was developed for intact mass analysis of sialylated glycoproteins. The developed SCX-MS method provided good separation for sialylated glycoproteins and had an inherent characteristic of native MS. Thus, the intact mass analysis of highly heterogeneous glycoprotein, which cannot be obtained by reversed-phase liquid chromatography (RPLC)-MS and size exclusion chromatography (SEC)-MS methods, can be well analyzed using the current SCX-MS method. First, the method was developed and optimized using the etanercept monomer. Conditions including MS parameters, flow rate, and gradient were investigated. Then, the developed method was used to analyze a new recombinant vaccine, protein 1. Similar to the etanercept monomer, the intact molecular information of protein 1, which cannot be obtained by RPLC-MS and SEC-MS, can be achieved using SCX-MS. Combined with information obtained on peptide mapping and glycan profiles obtained by LC-MS, the new vaccine was well characterized. Finally, the SCX-MS method was used to quickly evaluate the batch-to-batch reproducibility of protein 1. It was much faster than peptide mapping and glycan profiling methods and can provide information complementary to these strategies. It should be useful for many applications where speed and comprehensive characterization are required, such as recombinant sialylated vaccines and fusion proteins.

Polymeric monolithic columns based on natural wood for rapid purification of targeted protein.

With multiscale hierarchical structure, wood is suitable for a range of high-value applications, especially as a chromatographic matrix. Here, we have aimed to provide a weak anion-exchange polymeric monolithic column based on natural wood with high permeability and stability for effectively separating the targeted protein. The wood-polymeric monolithic column was synthesized by in situ polymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in wood, and coupled with diethylaminoethyl hydrochloride. The wood-polymeric monolithic column can be integrated with fast-protein liquid chromatography for large-scale protein purification. According to the results, the wood-polymeric monolithic column showed high hydrophilicity, permeability and stability. Separation experiments verified that the wood-polymeric monolithic column could purify the targeted protein (spike protein of SARS-COV-2 and ovalbumin) from the mixed proteins by ion exchange, and the static adsorption capacity was 33.04 mg mL-1 and the dynamic adsorption capacity was 24.51 mg mL-1. In addition, the wood-polymerized monolithic column had good stability, and a negligible decrease in the dynamic adsorption capacity after 20 cycles. This wood-polymerized monolithic column can provide a novel, efficient, and green matrix for monolithic chromatographic columns.

Preparation of carboxyl-functionalized silica core-shell microspheres and their applications in weak cation exchange chromatography, heavy metal removal, and lysozyme enrichment.

Chromatography is a technique of separation based on adsorption and/or interaction of target molecules with stationary phases. Herein, we report the design and fabrication of BTDA@SiO2 core-shell microspheres as a new class of stationary phase and demonstrate its impressive performance for chromatographic separations. The silica microspheres of BTDA@SiO2 were synthesized by in situ method with 1,3,5-benzenetricarboxaldehyde and 3,5-diaminobenzoic to separate peptides and proteins on high-performance liquid chromatography. The BTDA@SiO2 core-shell structure has a high specific surface area and retention factor of 4.27 and 8.31 for anionic and cationic peptides, respectively. The separation factor and resolution were high as well. A typical chromatogram illustrated nearly baseline resolution of the two peptides in less than 3 min. The BTDA@SiO2 was also highly stable in the pH range of 1 to 14. Furthermore, the prepared BTDA@SiO2 core-shell material not only be used for chromatographic separation but also as heavy metal removal from water. Using a BTDA@SiO2, we also achieved a lysozyme enrichment with a maximum saturated adsorption capacity reaching 714 mg/g. In summary, BTDA@SiO2 has great application prospects and significance in separation and purification systems.

Purified Pyropia yezoensis Pigment Extract-Based Tandem Dye Synthesis.

Red phycoerythrin (R-PE) is a highly valuable protein found in an edible seaweed, Pyropia yezoensis. It is used extensively in biotechnological applications due to its strong fluorescence and stability in diverse environments. However, the current methods for extracting and purifying R-PE are costly and unsustainable. The aim of the present study was to enhance the financial viability of the process by improving the extraction and purification of R-PE from dried P. yezoensis and to further enhance R-PE value by incorporating it into a tandem dye for molecular biology applications. A combination of ultrafiltration, ion exchange chromatography, and gel filtration yielded concentrated (1 mg·mL-1) R-PE at 99% purity. Using purified PE and Cyanine5 (Cy5), an organic tandem dye, phycoerythrin-Cy5 (PE-Cy5), was subsequently established. In comparison to a commercially available tandem dye, PE-Cy5 exhibited 202.3% stronger fluorescence, rendering it suitable for imaging and analyzes that require high sensitivity, enhanced signal-to-noise ratio, broad dynamic range, or shorter exposure times to minimize potential damage to samples. The techno-economic analysis confirmed the financial feasibility of the innovative technique for the extraction and purification of R-PE and PE-Cy5 production.