Design of Gd3+-immobilized two-dimensional magnetic magadiite nanosheets for highly selective enrichment of phosphopeptides.

Exfoliated magadiite nanosheets embedded with Fe3O4 were constructed. Advantage was taken of the strong coordination between the silanol groups in magadiite nanosheets and the Gd3+ ion to prepare the final adsorbent, Gd3+-immobilized magnetic magadiite nanosheets. The adsorbent with two-dimensional (2D) morphology offered high surface area and abundant Gd3+ contents for phosphopeptides enrichment, on which Fe3O4 with positive electricity incorporated the magnetic properties. Combining with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF-MS), the method showed low detection limit (0.05 fmol). The feasibility of using the 2D nanocomposite for phosphopeptides enrichment was demonstrated using mixtures of ß-casein and bovine serum albumin (1:5000). The standard deviation of captured phosphopeptides in three repeated experiments were in the range 0.15-0.42 (< 0.5% RSD). Further evaluation revealed that the nanocomposite was capable of enriching phosphopeptides from non-fat milk, human saliva, and serum. A novel Gd3+-immobilized two-dimensional magnetic magadiite nanosheets-based enrichment platform was designed. The developed material was employed as the adsorbent for the selective enrichment of phosphopeptides by coupling with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The material was successfully applied to enrich phosphopetides from standard peptide mixtures, nonfat milk, human saliva, and serum.

Raman spectroscopy as a process analytical technology to investigate biopharmaceutical freeze concentration processes.

Freezing processes are a well-established unit operation in the biopharmaceutical industry to increase the shelf-life of protein-based drugs. While freezing reduces degradation reaction rates, it may also exert stresses such as freeze concentration. Macroscopic freeze concentration in large-scale freezing processes has been described thoroughly by examination of frozen bulk material, but the transient process leading to such freeze concentration profiles has not been monitored yet for biopharmaceutical solutions. In this study, Raman spectroscopy as a process analytical technology is demonstrated for model formulations containing monoclonal antibodies (mAbs) or bovine serum albumin (BSA) in varying concentrations of sucrose and buffer salts. Therefore, a Raman probe was immersed into a bulk volume at different heights, monitoring the freeze concentration in the liquid phase during the freezing processes. Partial least square regression models were used to quantitatively discriminate between the protein and excipients simultaneously. The freeze concentration profiles were dependend on freezing temperature and formulation with freeze concentrations up to 2.4-fold. Convection currents at the bottom of the freezing container were observed with a maximum height of 1 mm. Furthermore, freeze concentration was correlated with the sucrose concentration in a formulation. Analysis of the freeze concentration slope indicated diffusion from the bottom to the top of the container. In summary, Raman spectroscopy is a valuable tool for process validation of freeze concentration simulations and to overcome scale-dependent challenges.

Cobalt-Iron Double Ion-Bovine Serum Albumin Chelation-Assisted Thermo-Sensitive Surface-Imprinted Nanocage with High Specificity.

To develop multifunctional protein imprinted materials, a cobalt-iron double ion-BSA directional chelation-assisted thermo-sensitive surface-imprinted hollow nanocage (Co-Fe@CBMA-MIPs) with excellent specificity is developed on the surface of ZIF-67@Co-Fe in this study by synergizing the advantages of surface imprinting, metal ion chelation, anti-protein adsorption segments, and thermo-sensitive components. Beyond previous research, well-designed multifunctional protein-imprinted materials possess high binding capacity, fast adsorption kinetics, and outstanding selectivity. When the adsorption is carried out at 32 °C, the adsorption capacity of Co-Fe@CBMA-MIPs for BSA reaches 520.35 mg/g within 50 min. The imprinting factor is 8.55. The selectivity factors of Co-Fe@CBMA-MIPs for HSA, Bhb, OVA, and Lyz are 3.72, 6.09, 4.10, and 8.41, respectively. More significantly, Co-Fe@CBMA-MIPs could specifically recognize BSA from mixed proteins and actual samples and exhibit excellent repeated use stability. Based on the above advantages, the development of this research provides an effective means to improve the recognition specificity of molecularly imprinted polymers.

Cationic Covalent Organic Framework as an Ion Exchange Material for Efficient Adsorptive Separation of Biomolecules.

Although covalent organic frameworks (COFs) have earned significant interest in separation applications, the use of COFs in biomolecule separation remains unexplored. We examined the ionic COF Py-BPy2+-COF as an ion exchange material for biomolecule separation. After characterizing the properties of the synthesized COF with a variety of techniques, binding experiments with both large and small biomolecules were performed. High adsorption capacities of amino acids with different hydrophobicity and charge, as well as proteins of different isoelectric points and molecular weights, were determined in batch equilibrium experiments. Desorption experiments with mixtures of model proteins demonstrated an ability to successfully separate one protein from another with the selectivity hypothesized to be a combination of the isoelectric point, hydrophobicity, and ability to penetrate the crystalline material. Overall, the results demonstrated that Py-BPy2+-COF can be exploited as a robust crystalline anion exchange biomolecule separation material.

Facile synthesis of bifunctional polymer monolithic column for tunable and specific capture of glycoproteins and phosphoproteins.

Efficiently tunable capture of the glycosylated/phosphorylated proteins is critical to meet the need of in-depth glycoproteome and phosphoproteome studies. Reported here is a new bifunctional polymer monolithic column by introducing benzeneboronic acid and phosphonic acid onto monolithic column (denoted as poly (EDMA-co-VPBA-co-VPA) monolith) for tunable and specific enrichment of glycoproteins and phosphoproteins via switching different mobile phases. Based on boronate affinity and immobilized metal affinity, the as-prepared poly (EDMA-co-VPBA-co-VPA) monolith exhibited superior performance in selective separation of small molecules and biomacromolecules containing cis-diol/phosphate groups or not. And the frontal chromatography analysis showed that the binding capacity of the poly (EDMA-co-VPBA-co-VPA) monolith towards horseradish peroxidase (HRP, glycoprotein) or ß-casein (phosphoprotein) is four-fold higher than that of bovine serum albumin (BSA, non-glycosylated/phosphorylated protein). Furthermore, combined with mass spectrometry identification, the successful application in specific enrichment of glycopeptides/phosphopeptides from tryptic digests of HRP/ß-casein and direct capture of low abundant endogenous phosphopeptides from human serum proved great practicability in complex samples. This study provides a novel insight for fabricating the monolithic columns with multifunctionalization to facilitate further post-translational modification (PTM)-proteomics development.

Protein Adsorption on Core-shell Particles: Comparison of Capto™ Core 400 and 700 Resins.

Structural and functional characteristics of the two core-shell resins Capto™ Core 400 and 700, which are useful for the flow-through purification of bioparticles such as viruses, viral vectors, and vaccines, are compared using bovine serum albumin (BSA) and thyroglobulin (Tg) as models for small and large protein contaminants. Both resins are agarose-based and contain an adsorbing core surrounded by an inert shell. Although shell thicknesses are comparable (3.6 and 4.2 µm for Capto Core 400 and 700, respectively), the two resins differ substantially in pore size (pore radii of 19 and 50 nm, respectively). Because of the smaller pores and higher surface area, the BSA binding capacity of Capto Core 400 is approximately double that of Capto Core 700. However, for the much larger Tg, the attainable capacity is substantially larger for Capto Core 700. Mass transfer in both resins is affected by diffusional resistances through the shell and within the adsorbing core. For BSA, core and shell effective pore diffusivities are about 0.25 × 10-7 and 0.6 × 10-7 cm2/s, respectively, for Capto Core 400, and about 1.6 × 10-7 and 2.6 × 10-7 cm2/s, respectively, for Capto Core 700. These values decrease dramatically for Tg to 0.022 × 10-7 and 0.088 × 10-7 cm2/s and to 0.13 × 10-7 and 0.59 × 10-7 cm2/s for Capto Core 400 and 700, respectively. Adsorbed Tg further hinders diffusion of BSA in both resins. Column measurements show that, despite the higher static capacity of Capto Core 400 for BSA, the dynamic binding capacity is greater for Capto Core 700 as a result of its faster kinetics. However, some of this advantage is lost if the feed is a mixture of BSA and Tg since, in this case, Tg binding leads to greater diffusional hindrance for BSA.

A Simple Method for In-Depth Proteome Analysis of Mammalian Cell Culture Conditioned Media Containing Fetal Bovine Serum.

A conditioned medium of a cell culture is widely used for various biological applications and frequently analyzed to characterize the functional proteins responsible for observed biological functions. However, a large number of abundant proteins in fetal bovine serum (FBS), usually included in the conditioned medium of a mammalian cell culture medium, hampers in-depth proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For a deep proteomic analysis of a conditioned medium by LC-MS/MS, we developed a simple albumin depletion approach coupled with data-independent acquisition (DIA)-mode LC-MS/MS for the conditioned medium of mammalian cells in this study. The results showed that this approach enabled the detection of more than 3700 cell-derived proteins in the cell culture supernatant containing FBS. We further demonstrated the potency of this approach by analyzing proteins in the conditioned media of HeLa cells with and without tumor necrosis factor (TNF) stimulation: >40 differentially accumulated proteins, including four cytokines, upon TNF stimulation were identified in the culture media, which were hardly detected by conventional proteome approaches in the literature.

Preparation of Fe3O4@PMAA@Ni Microspheres towards the Efficient and Selective Enrichment of Histidine-Rich Proteins.

Magnetic material is considered to as a major concern material for the enrichment of histidine-rich proteins (His-proteins) via metal-ion affinity. In this work, magnetic polymer microspheres with core-shell structure (Fe3O4@PMAA@Ni) were successfully prepared via reflux-precipitation polymerization followed by in situ reduction and growth of Ni2+. The obtained Ni nanofoams with flower-like structure and uniform pore size (3.34 nm) provided numerous binding sites for His-proteins. The adsorption performance of Fe3O4@PMAA@Ni microspheres for His-proteins was estimated via selectively separating bovine hemoglobin (BHb) and bovine serum albumin (BSA) from a matrix composed of BHb, BSA, and lysozyme (LYZ). The results indicated that Fe3O4@PMAA@Ni microspheres could efficiently and selectively separate His-proteins from the matrix, with a maximum adsorption capacity of ∼2660 mg/g for BHb. Moreover, Fe3O4@PMAA@Ni microspheres exhibited good stability and recyclability for BHb separation over seven cycles. Therefore, this work reported a novel and facile strategy to prepare core-shell Fe3O4@PMAA@Ni microspheres, which was promising for practical applications of His-protein separation and purification in proteomics.

Feasibility of Human Platelet Lysate as an Alternative to Foetal Bovine Serum for In Vitro Expansion of Chondrocytes.

Osteoarthritis (OA) is a degenerative joint disease that affects a lot of people worldwide. Current treatment for OA mainly focuses on halting or slowing down the disease progress and to improve the patient's quality of life and functionality. Autologous chondrocyte implantation (ACI) is a new treatment modality with the potential to promote regeneration of worn cartilage. Traditionally, foetal bovine serum (FBS) is used to expand the chondrocytes. However, the use of FBS is not ideal for the expansion of cells mean for clinical applications as it possesses the risk of animal pathogen transmission and animal protein transfer to host. Human platelet lysate (HPL) appears to be a suitable alternative to FBS as it is rich in biological factors that enhance cell proliferation. Thus far, HPL has been found to be superior in promoting chondrocyte proliferation compared to FBS. However, both HPL and FBS cannot prevent chondrocyte dedifferentiation. Discrepant results have been reported for the maintenance of chondrocyte redifferentiation potential by HPL. These differences are likely due to the diversity in the HPL preparation methods. In the future, more studies on HPL need to be performed to develop a standardized technique which is capable of producing HPL that can maintain the chondrocyte redifferentiation potential reproducibly. This review discusses the in vitro expansion of chondrocytes with FBS and HPL, focusing on its capability to promote the proliferation and maintain the chondrogenic characteristics of chondrocytes.

Platelet-Rich and Platelet-Poor Plasma Might Play Supportive Roles in Cancer Cell Culture: A Replacement for Fetal Bovine Serum?

BACKGROUND: Platelet-Rich (PRP) and Platelet-Poor plasma (PPP) are widely used in research and clinical platforms mainly due to their capacities to enhance cell growth. Although the short half-life (5 days) and the high price of platelet products pose challenges regarding their usage, they maintain the growth regulatory functions for weeks. Thus, we aimed to assess the supplementary values of these products in human CCRF- CEM cancer cells. Mechanistically, we also checked if the PRP/PPP treatment enhances YKL-40 expression as a known protein regulating cell growth. METHODS: The PRP/PPP was prepared from healthy donors using manual stepwise centrifugation and phase separation. The viability of the cells treated with gradient PRP/PPP concentrations (2, 5, 10, and 15%) was measured by the MTT assay. The YKL-40 mRNA and protein levels were assessed using qRT-PCR and western blotting. The data were compared to FBS-treated cells. RESULTS: Our findings revealed that the cells treated by PRP/PPP not only were morphologically comparable to those treated by FBS but also showed greater viability at the concentrations of 10 and 15%. Moreover, it was shown that PRP/PPP induce cell culture support, at least in part, via inducing YKL-40 expression at both mRNA and protein levels in a time- and dose-dependent manner. CONCLUSION: Collectively, by showing cell culture support comparable to FBS, the PRP/PPP might be used as good candidates to supplement the cancer cell culture and overcome concerns regarding the use of FBS as a non-human source in human cancer research.

A whole area scanning-enabled direct-counting strategy for studying blocking efficiency in mitigating protein-solid surface binding.

The study of protein-solid surface binding as well as blocking efficiency of blocking agents plays an important role in the development of high-performance immunoassays. Although conventional colorimetric based assays are widely employed to monitor protein non-specific binding on the surface of microplate wells and evaluate the performance of blocking agents, there is still a great need to develop new methods to achieve the same goal from a new perspective. In this study, an innovative whole area scanning (WAS)-enabled direct-counting strategy was developed and validated through studying the blocking efficiency of different blocking agents on the non-specific binding of streptavidin-alkaline phosphatase conjugate (Strep-ALP, a model protein) to the surface of 96-well microplates. After non-specific binding of Strep-ALP in wells with or without blocking agents' treatment and loading of ELF™ 97 phosphate (ELFP), ALP in Strep-ALP conjugates converts ELFP to water-insoluble ELF™ 97 alcohol (ELFA), which precipitates locally, self-assembles into large needle structures, and glows green fluorescence upon excitation. After quenching the reaction, WAS of the whole wells allows us to directly count the number of individual fluorescent precipitates, which can be used to calculate and compare the blocking efficiency of three commonly used blocking agents (BSA, casein, and dry milk) based on mitigating the non-specific binding of Strep-ALP. WAS-enabled counting of individual needle-type precipitates opens a new avenue to investigate protein-solid surface binding as well as the efficiency of blocking agents with high sensitivity.

Effect of pore structure on protein adsorption mechanism on ion exchange media: A preliminary study using low field nuclear magnetic resonance.

The adsorption process of bovine serum albumin (BSA), ovalbumin (OVA) and human immunoglobulin G (IgG) on agarose ion-exchange media Q Sepharose FF and two dextran-grafted agarose media including Q Sepharose XL and Capto Q were studied using low field nuclear magnetic resonance (NMR). The T2 relaxation time was found directly proportional to the pore size and diminished after protein adsorbed, therefore, a theoretical model describing the relationship between protein binding amount and T2 relaxation signals was established. The model parameters, a, which reflects the contact area between the adsorbed protein and media surface, and the δ, which defined as the ratio of the protein volume to the pore volume after adsorption, were found to describe the pore occupation states of proteins in media with different pore structures very well. For small proteins, such as BSA and OVA, monolayer adsorption occurred on Q Sepharose FF, which has no dextran chains. Therefore, the adsorbed protein only occupied 49.05% of the pore volume for BSA and 25.51% for OVA, and contact area of each protein on the media were also low, suggesting mostly monolayer adsorption occurred. In the contrast, their adsorption to Q Sepharose XL and Capto Q with dextran chains tended to form multilayer adsorption, thus higher contact area was obtained and the pore volumes were almost 100% occupied. For large protein, such as IgG, the adsorption to all these three media was similar and about 30% of the pore volume were occupied, probably due to the similar restriction for IgG to entering the media pore. Results of this study will help to elucidate the relationship between protein adsorption and pore size variation, which present the significance of low field NMR in understanding protein adsorption mechanism.

Comparison of separation modes for microchip electrophoresis of proteins.

Separation of a set of model proteins was tested on a microchip electrophoresis analytical platform capable of sample injection by two different electrokinetic mechanisms. A range of separation modes-microchip capillary zone electrophoresis, microchip micellar electrokinetic chromatography, and nanoparticle-based sieving-was tested on glass and polydimethylsiloxane/glass microchips and with silica-nanoparticle colloidal arrays. The model proteins calmodulin (18 kiloDalton), bovine serum albumin (66 kDa), and concanavalin (106 kDa) were labeled with Alexa Fluor 647 for laser-induced fluorescence detection. The best separation and resolution were obtained in a silica-nanoparticle colloidal array chip.

Controlled radical polymerization of hydrophilic and zwitterionic brush-like polymers from silk fibroin surfaces.

Bombyx mori silk fibroin is a fibrous protein whose tunable properties and biocompatibility have resulted in its utility in a wide-variety of applications, including as drug delivery vehicles, wound dressings, and tissue engineering scaffolds. Control of protein and cell attachment is vital to the performance of biomaterials, but silk fibroin is mostly hydrophobic and interacts nonspecifically with cells and proteins. Silk functionalised with hydrophilic polymers reduces attachment, but the low number of reactive sites makes achieving a uniform conjugation a persistent challenge. This work presents a new approach to grow brush-like polymers from the surface of degradable silk films, where the films were enriched with hydroxyl groups, functionalised with an initiator, and finally reacted with acrylate monomers using atom transfer radical polymerisation. Two different routes to hydroxyl enrichment were investigated, one involving reaction with ethylene oxide (EO) and the other using a two-step photo-catalysed oxidation reaction. Both routes increased surface hydrophilicity, and hydrophilic monomers containing either uncharged (poly(ethylene glycol), PEG) pendant groups or zwitterionic pendant groups were polymerised from the surfaces. The initial processing of the films to induce beta sheet structures was found to impact the success of the polymerizations. Compared to the EO modified or unmodified silk surfaces, the oxidation reaction resulted in more polymer conjugation and the surfaces appear more uniform. Mesenchymal stem cell and protein attachment were the lowest on polymers grown from oxidised surfaces. PEG-containing brush-like polymers displayed lower protein attachment than surfaces conjugated with PEG using a previously reported "grafting to" method, but polymers containing zwitterionic side chains displayed both the lowest contact angles and the lowest cell and protein attachment. This finding may arise from the interactions of the zwitterionic pendant groups through their permanent dipoles and is an important finding because PEG is susceptible to oxidative damage that can reduce efficacy over time. These modified silk materials with lower cell and protein attachments are envisioned to find utility when enhanced diffusion around surfaces is required, such as in drug delivery implants.

Distribution of Protein Precipitation Capacity within Variable Proanthocyanidin Fingerprints.

Proanthocyanidins (PAs) are highly bioactive plant specialized metabolites. One of their most characteristic features is their ability to precipitate proteins. In this study, eleven plant species were used to study the structure-activity patterns between PAs and their protein precipitation capacity (PPC) with bovine serum albumin. To obtain a comprehensive selection of PAs with highly variable procyanidin to prodelphinidin ratios and mean degree of polymerizations, nearly 350 subfractions were produced from the eleven plant species by semi-preparative liquid chromatography. Their PA composition was defined by tandem mass spectrometry and high-resolution mass spectrometry, and their PPC was measured with a turbidimetry-based well-plate reader assay. The distribution of the PPC within plant species varied significantly. The mean degree of polymerization of the PAs had a strong correlation with the PPC (r = 0.79). The other structural features were significant from the PPC point of view as well, but they contributed to the PPC in different ways in different plant species. Retention time, prodelphinidin proportion, and mean degree of polymerization explained 64% of the measured variance of the PPC.

A comparison of data quality using quartz vs. sapphire cell windows in analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) cells use either quartz or sapphire windows as end caps for the cell housing. Current generation sapphire windows are not recommended for absorbance data collection below 235 nm, because the window material shows a precipitous drop in transmittance at low wavelengths due to impurities in the sapphire. Quartz windows can be used below 235 nm as they do not exhibit adverse transmittance at low wavelengths. In this study, we demonstrate the optical properties of new generation sapphire windows and compare them to those of quartz windows across a wide range of wavelengths and present the results of sedimentation velocity experiments on BSA using both types of windows using data collected at both the 280 nm absorbance maxima as well as the 230-240 nm (closer to the peptide bond maximum). Our results show that the quartz and new generation sapphire windows deliver identical results in absorbance mode. We also demonstrate that quartz windows suffer significant mechanical deformation while spinning at very high speeds, while sapphire windows do not. This renders Rayleigh interference mode data collected at high speeds using quartz windows much noisier than with sapphire windows-which we have quantified by measuring how the signal to noise ratio of Fourier transformed Rayleigh interference scans degrades at high speed. Thus, we conclude that new-generation sapphire windows can be used for all AUC experiments through almost the entire mid UV range-obviating the need for quartz windows, unless wavelengths below 220 nm must be accessed.

Gold nanoparticle-glutathione-functionalized porous graphene oxide-based hydrophilic beads for the selective enrichment of N-linked glycopeptides.

A three-dimensional structured porous graphene oxide-polyethylenimine bead (pGP) is synthesized for immobilizing gold nanoparticles and modifying glutathione molecules (denoted as pGP/AuG). The pGP/AuG has open pore structure, honeycomb-like channels, and excellent hydrophilicity. By taking advantages of the porous structure, abundant binding sites, and multivalent interactions between glycopeptides and both glutathione molecules and free amino groups, the pGP/AuG is adopted to the selective enrichment of N-linked glycopeptides with low limit of detection (2 fmol), high enrichment selectivity (1:500), binding capacity (333.3 mg/g), recovery yield (91.3 ± 2.1%), and repeatability (< 6.0% RSD) using matrix-assisted laser desorption/ionization time of flight mass spectrometry detection method. Furthermore, the practical applicability of pGP/AuG is evaluated, in which 209 N-glycosylated peptides corresponding to 128 N-glycosylated proteins are identified from 1 µL human serum in three independent analysis procedures, suggesting the great potential for application in glycoproteome fields.Graphical abstract Schematic presentation of preparation for porous graphene oxide-based hydrophilic beads (pGP/AuG) with honeycomb-like microstructure. The pGP/AuG was successfully used for enriching and identifying glycopeptides from actual biological sample.

Polyoxometalate-functionalized macroporous microspheres for selective separation/enrichment of glycoproteins.

Glycoproteins always participate in various biological processes. Selective separation and enrichment of glycoproteins are of great significance for the research of pathogenesis. Herein, macroporous polymer microspheres were fabricated, and further functionalized by polyoxometalate. Thus, a simple, efficient and highly selective approach was constructed for glycoprotein enrichment from a complex matrix. The as-prepared material shows promise as a potential adsorbent in bio-separation and downstream clinical applications.

Using modern approaches to sedimentation velocity to detect conformational changes in proteins.

It has been known for decades that proteins undergo conformational changes in response to binding ligands. Such changes are usually accompanied by a loss of entropy by the protein, and thus conformational changes are integral to the thermodynamics of ligand association. Methods to detect these alterations are numerous; here, we focus on the sedimentation velocity (SV) mode of AUC, which has several advantages, including ease of use and rigorous data-selection criteria. In SV, it is assumed that conformational changes manifest primarily as differences in the sedimentation coefficient (the s-value). Two methods of determining s-value differences were assessed. The first method used the widely adopted c(s) distribution to gather statistics on the s-value differences to determine whether the observed changes were reliable. In the second method, a decades-old technique called "difference SV" was revived and updated to address its viability in this era of modern instrumentation. Both methods worked well to determine the extent of conformational changes to three model systems. Both simulations and experiments were used to explore the strengths and limitations of the methods. Finally, software incorporating these methodologies was produced.

Bovine serum albumin and myoglobin separation by size exclusion SMB.

The separation of the proteins Bovine Serum Albumin (BSA) and Myoglobin (Mb) was achieved by Size-Exclusion Simulated Moving Bed (SE-SMB) and performed experimentally in the FlexSMB® unit, an SMB unit designed and built in the Laboratory of Separation and Reaction Engineering. Before accomplishing the separation experiments in the mentioned unit, separation regions were computed by simulation based on a phenomenological mathematical model to determine appropriate operating conditions. The developed model was validated in advance, against fixed-bed dynamic adsorption experimental results, for pure component and binary mixtures. Then the SMB experiments were carried out, and purities of the Mb on the extract and BSA on the raffinate streams were 98% and 96%, respectively. The achieved recoveries were 80% of Mb on the extract and 94% of BSA on the raffinate. Lastly, productivities of 6.4 gprotein⋅lads-1⋅day-1 for the extract and 28.8 gprotein⋅lads-1⋅day-1 for the raffinate were obtained.