Decisional tool development and application for techno-economic analysis of fungal laccase production.

Textile and medical effluents causing bioaccumulation and biomagnification have been successfully biodegraded by fungal laccases. Here, a decision-making tool was developed and applied to evaluate 45 different laccase production strategies which determined the best potential source from a techno-economical perspective. Laccase production cost was calculated with a fixed output of 109 enzymatic units per batch (USD$per109U) and a sensitivity analysis was performed. Results indicate that optimization of enzymatic kinetics for each organism is essential to avoid exceeding the fermentation time point at which production titer reaches its peak and, therefore, higher production costs. Overall, the most cost-effective laccase-producing strategy was obtained when using Pseudolagarobasidium acaciicola with base production cost of USD $42.46 per 109 U. This works serves as platform for decision-making to find the optimal laccase production strategy based on techno-economic parameters.

Strategies for surface coatings of implantable cardiac medical devices.

Cardiac medical devices (CMDs) are required when the patient's cardiac capacity or activity is compromised. To guarantee its correct functionality, the building materials in the development of CMDs must focus on several fundamental properties such as strength, stiffness, rigidity, corrosion resistance, etc. The challenge is more significant because CMDs are generally built with at least one metallic and one polymeric part. However, not only the properties of the materials need to be taken into consideration. The biocompatibility of the materials represents one of the major causes of the success of CMDs in the short and long term. Otherwise, the material will lead to several problems of hemocompatibility (e.g., protein adsorption, platelet aggregation, thrombus formation, bacterial infection, and finally, the rejection of the CMDs). To enhance the hemocompatibility of selected materials, surface modification represents a suitable solution. The surface modification involves the attachment of chemical compounds or bioactive compounds to the surface of the material. These coatings interact with the blood and avoid hemocompatibility and infection issues. This work reviews two main topics: 1) the materials employed in developing CMDs and their key characteristics, and 2) the surface modifications reported in the literature, clinical trials, and those that have reached the market. With the aim of providing to the research community, considerations regarding the choice of materials for CMDs, together with the advantages and disadvantages of the surface modifications and the limitations of the studies performed.

Laccase-luminol chemiluminescence system: an investigation of substrate inhibition.

Chemiluminescence (CL) reactions are widely used for the detection and quantification of many types of analytes. Laccase has previously been proposed in CL reactions; however, its light emission behaviour has not been characterized. This study was conducted to characterize the laccase-luminol system, determine its kinetic parameters, and analyze the effects of protein and OH- concentration on the CL signal. Laccase from Coriolopsis gallica was combined with different concentrations of luminol (125 nM to 4 mM), and the enzyme kinetics were evaluated using diverse kinetic models. The laccase-luminol system was able to produce CL without an intermediate molecule, but it exhibited substrate-inhibition behaviour. A two-site random model was used and suggested that when the first luminol molecule was bound to the active site, laccase affinity for the second luminol molecule was increased. This inhibition effect could be avoided using a low luminol concentration. At 5 µM luminol concentration, 1 mg/ml (0.13 U) laccase is needed to achieve nearly 90% of the maximum CL signal, suggesting that the available luminol could not bind to all active sites. Furthermore, the concentration of NaOH negatively affected the CL signal. The laccase-luminol system represents an alternative to existing CL systems, with potential uses in molecular detection and quantification.

Phytochemical Profile and Antioxidant and Antiproliferative Activity of Sedum dendroideum on Pterygium Fibroblasts.

BACKGROUND: Sedum dendroideum has antioxidant effects that are beneficial for different diseases. We aimed to analyze the antiproliferative activity of S. dendroideum in human pterygium fibroblasts (HPFs). METHODS: HPFs were treated for 24 h with 0-1000 µg/mL of S. dendroideum lyophilized to analyze its effect on cell viability using the CellTiter assay. RNA from HPF treated with 250 µg/mL of S. dendroideum lyophilized was isolated, and the expression of VEGF and CTGF genes was evaluated by qPCR. A dermal fibroblast cell line (HDFa) was used as a healthy control. The total phenolic content, antioxidant activity, and chemical profile of S. dendroideum lyophilized were determined. RESULTS: Viability of HPF decreased after 24 h treatment of S. dendroideum in a dose-dependent manner. The expression of VEGF and CTGF significantly decreased (P < 0.01) in HPF treated with 250 µg/mL of S. dendroideum when compared with untreated HPF. The total phenolic concentration in the S. dendroideum lyophilized was 33.67 mg gallic acid equivalents (GAE)/g. Antioxidant activity was 384.49 mM Trolox equivalents/mL. The main phenolic compounds identified by HPLC analysis were the kaempferol-3-O-glycoside, kaempferol-3-O-rhamnoside, kaempferol-3-O-neohesperidoside-7-O-α-rhamnopyranoside, and kaempferol-3-O-glycoside-7-O-rhamnoside. CONCLUSIONS: S. dendroideum decreases the proliferation of HPF and the expression of VEGF and CTGF. The phenolic compound concentration, antioxidant activity, and phytochemical profile may play a role in these effects.

Purification of Modified Therapeutic Proteins Available on the Market: An Analysis of Chromatography-Based Strategies.

Proteins, which have inherent biorecognition properties, have long been used as therapeutic agents for the treatment of a wide variety of clinical indications. Protein modification through covalent attachment to different moieties improves the therapeutic's pharmacokinetic properties, affinity, stability, confers protection against proteolytic degradation, and increases circulation half-life. Nowadays, several modified therapeutic proteins, including PEGylated, Fc-fused, lipidated, albumin-fused, and glycosylated proteins have obtained regulatory approval for commercialization. During its manufacturing, the purification steps of the therapeutic agent are decisive to ensure the quality, effectiveness, potency, and safety of the final product. Due to the robustness, selectivity, and high resolution of chromatographic methods, these are recognized as the gold standard in the downstream processing of therapeutic proteins. Moreover, depending on the modification strategy, the protein will suffer different physicochemical changes, which must be considered to define a purification approach. This review aims to deeply analyze the purification methods employed for modified therapeutic proteins that are currently available on the market, to understand why the selected strategies were successful. Emphasis is placed on chromatographic methods since they govern the purification processes within the pharmaceutical industry. Furthermore, to discuss how the modification type strongly influences the purification strategy, the purification processes of three different modified versions of coagulation factor IX are contrasted.

Comparative Economic Analysis Between Endogenous and Recombinant Production of Hyaluronic Acid.

Hyaluronic acid (HA) is a biopolymer with a wide range of applications, mainly in the cosmetic and pharmaceutical sectors. Typical industrial-scale production utilizes organisms that generate HA during their developmental cycle, such as Streptococcus equi sub. zooepidemicus. However, a significant disadvantage of using Streptococcus equi sub. zooepidemicus is that it is a zoonotic pathogen, which use at industrial scale can create several risks. This creates opportunities for heterologous, or recombinant, production of HA. At an industrial scale, the recovery and purification of HA follow a series of precipitation and filtration steps. Current recombinant approaches are developing promising alternatives, although their industrial implementation has yet to be adequately assessed. The present study aims to create a theoretical framework to forecast the advantages and disadvantages of endogenous and recombinant strains in production with the same downstream strategy. The analyses included a selection of the best cost-related recombinant and endogenous production strategies, followed by a sensitivity analysis of different production variables in order to identify the three most critical parameters. Then, all variables were analyzed by varying them simultaneously and employing multiple linear regression. Results indicate that, regardless of HA source, production titer, recovery yield and bioreactor scale are the parameters that affect production costs the most. Current results indicate that recombinant production needs to improve current titer at least 2-fold in order to compete with costs of endogenous production. This study serves as a platform to inform decision-making for future developments and improvements in the recombinant production of HA.

Validation of aqueous two-phase extraction method.

Nowadays, consumer interest in food with natural ingredients has increased. This need has led to the research of new sources and green extraction methods. Betalains are compounds responsible for giving color to cacti fruits. The aim is to obtain low-sugar betacyanins extracts from jiotilla Escontria chiotilla using aqueous two-phase systems (ATPS) to color food with the extract. The effect of principal parameters of ATPS (Ethyl alcohol- KH2PO4/K2HPO4) as tie-line length (TL;40,50 and 70), phase volume ratios (Vr; 1 and 3) on the partitioning of betacyanins, betaxanthins, total sugars, reducing sugars, and antioxidant activity in the extract was evaluated. The yields were determined from the top and bottom phases of the aforementioned parameters. Multivariate analysis of variance (MANOVA, α = 0.05) showed that TLL and Vr were statistically significant (P < 0.05). The lowest bottom sugar yield (25.78 ± 3.14%) corresponds to TLL = 40, Vr = 3. Under these conditions, the corresponding value for betacyanins yield is 62.98±4.52%. For the first time, the ATPS was used to extract betacyanins from cactus fruit.•Escontria chiotilla, as a biological source, contained a high percent of betalains•Aqueous two-phase systems (ATPS) was statistically optimized•The developed method enriches the valorization of environmentally related plants waste materials.

Primary recovery of hyaluronic acid produced in Streptococcus equi subsp. zooepidemicus using PEG-citrate aqueous two-phase systems.

Given its biocompatibility, rheological, and physiological properties, hyaluronic acid (HA) has become a biomaterial of increasing interest with multiple applications in medicine and cosmetics. In recent decades, microbial fermentations have become an important source for the industrial production of HA. However, due to its final applications, microbial HA must undergo critical and long purification processes to ensure clinical and cosmetic grade purity. Aqueous two-phase systems (ATPS) have proven to be an efficient technique for the primary recovery of high-value biomolecules. Nevertheless, their implementation in HA downstream processing has been practically unexplored. In this work, polyethylene glycol (PEG)-citrate ATPS were used for the first time for the primary recovery of HA produced with an engineered strain of Streptococcus equi subsp. zooepidemicus. The effects of PEG molecular weight (MW), tie-line length (TLL), volume ratio (VR), and sample load on HA recovery and purity were studied with a clarified fermentation broth as feed material. HA was recovered in the salt-rich bottom phase, and its recovery increased when a PEG MW of 8000 g mol-1 was used. Lower VR values (0.38) favoured HA recovery, whereas purity was enhanced by a high VR (3.50). Meanwhile, sample load had a negative impact on both recovery and purity. The ATPS with the best performance was PEG 8000 g mol-1, TLL 43% (w/w), and VR 3.50, showing 79.4% HA recovery and 74.5% purity. This study demonstrated for the first time the potential of PEG-citrate ATPS as an effective primary recovery strategy for the downstream process of microbial HA.

Development of aqueous two-phase systems-based approaches for the selective recovery of metalloproteases and phospholipases A2 toxins from Crotalus molossus nigrescens venom.

Snake venoms are rich sources of proteins with potential biotechnological and pharmaceutical applications. Among them, metalloproteases (MPs) and phospholipases A2 (PLA2) are the most abundant. Their isolation involves a multistep chromatographic approach, which has proven to be effective, however implies high operating costs and long processing times. In this study, a cost-effective and simple method based on aqueous two-phase systems (ATPS) was developed to recover MPs and PLA2 from Crotalus molossus nigrescens venom. A system with PEG 400 g mol-1, volume ratio (VR) 1, tie line length (TLL) 25% w/w and pH 7 showed the best performance for PLA2 recovery. In systems with PEG 400 g mol-1, VR 1, TLL 15% w/w, pH 7 and 1 and 3% w/w of NaCl, selective recovery of MP subtype P-III was achieved; whereas, in a system with PEG 400 g mol-1, VR 1, TLL 25% w/w and pH 8.5, MP subtypes P-I and P-III were recovered. Due to their low costs, ethanol-salt systems were also evaluated, however, failed to differentially partition PLA2 and MPs. The use of ATPS could contribute to the simplification and cost reduction of protein isolation processes from snake venoms and other toxin fluids, as well as potentially aid their biochemical, proteomic and biological analyses.

Corrigendum: Immobilization of Growth Factors for Cell Therapy Manufacturing.

[This corrects the article DOI: 10.3389/fbioe.2020.00620.].

Immobilization of Growth Factors for Cell Therapy Manufacturing.

Cell therapy products exhibit great therapeutic potential but come with a deterring price tag partly caused by their costly manufacturing processes. The development of strategies that lead to cost-effective cell production is key to expand the reach of cell therapies. Growth factors are critical culture media components required for the maintenance and differentiation of cells in culture and are widely employed in cell therapy manufacturing. However, they are expensive, and their common use in soluble form is often associated with decreased stability and bioactivity. Immobilization has emerged as a possible strategy to optimize growth factor use in cell culture. To date, several immobilization techniques have been reported for attaching growth factors onto a variety of biomaterials, but these have been focused on tissue engineering. This review briefly summarizes the current landscape of cell therapy manufacturing, before describing the types of chemistry that can be used to immobilize growth factors for cell culture. Emphasis is placed to identify strategies that could reduce growth factor usage and enhance bioactivity. Finally, we describe a case study for stem cell factor.

Structure and functional properties of Capto™ Core 700 core-shell particles.

Capto™ Core 700 is a core-shell chromatographic support with an adsorbing core contained within an inert shell layer designed to purify larger biomolecules and bioparticles in a flow-through mode. The present study aims to characterize the structure and functional properties of this resin using bovine serum albumin (BSA, Mr~65 kDa) and thyroglobulin (Tg, Mr~660 kDa) as model impurity proteins. The functionalized adsorbing core and the inert shell have the same fibrous structure typical of agarose-based beads. The resin average bead size is 90.7 µm with a range of 50-130 µm, the shell thickness is 4.18 µm with a range of 3-6 µm and a standard deviation of 0.55 µm, and the pore radius, obtained by inverse size exclusion chromatography, is 50.4 ± 1.3 nm. Both proteins present highly favorable binding isotherms with maximum binding capacities of 55 and 105 mg/mL of total bead volume for BSA and Tg, respectively. The addition of 500 mM NaCl reduces the binding capacity by less than 50%, showing the ability of the resin to operate at high salt conditions. For both proteins, the effective pore diffusivity in the core is smaller than in the shell due to additional hindrance by bound protein in the core area. Effective pore diffusivities values in the core are 1.6 × 10-7 and 0.16 × 10-7 cm2/s for BSA and Tg, respectively. The DBC10% at 2 min residence time are 24 and 2 mg/mL for BSA and Tg, respectively. This study provides qualitative and quantitative information about Capto™ Core 700 resin. This information could be used to predict and optimize the purification of large biomolecules and bioparticle in route to the establishment of more effective downstream processes.

Laccases in Food Industry: Bioprocessing, Potential Industrial and Biotechnological Applications.

Laccase is a multi-copper oxidase that catalyzes the oxidation of one electron of a wide range of phenolic compounds. The enzyme is considered eco-friendly because it requires molecular oxygen as co-substrate for the catalysis and it yields water as the sole by-product. Laccase is commonly produced by fungi but also by some bacteria, insects and plants. Due it is capable of using a wide variety of phenolic and non-phenolic substrates, laccase has potential applications in the food, pharmaceutical and environmental industries; in addition, it has been used since many years in the bleaching of paper pulp. Fungal laccases are mainly extracellular enzyme that can be recovered from the residual compost of industrial production of edible mushrooms as Agaricus bisporus and Pleurotus ostreatus. It has also been isolated from microorganisms present in wastewater. The great potential of laccase lies in its ability to oxidize lignin, one component of lignocellulosic materials, this feature can be widely exploited on the pretreatment for agro-food wastes valorization. Laccase is one of the enzymes that fits very well in the circular economy concept, this concept has more benefits over linear economy; based on "reduce-reuse-recycle" theory. Currently, biorefinery processes are booming due to the need to generate clean biofuels that do not come from oil. In that sense, laccase is capable of degrading lignocellulosic materials that serve as raw material in these processes, so the enzyme's potential is evident. This review will critically describe the production sources of laccase as by-product from food industry, bioprocessing of food industry by-products using laccase, and its application in food industry.

Aqueous Two-Phase Systems at Large Scale: Challenges and Opportunities.

Aqueous two-phase systems (ATPS) have proved to be an efficient and integrative operation to enhance recovery of industrially relevant bioproducts. After ATPS discovery, a variety of works have been published regarding their scaling from 10 to 1000 L. Although ATPS have achieved high recovery and purity yields, there is still a gap between their bench-scale use and potential industrial applications. In this context, this review paper critically analyzes ATPS scale-up strategies to enhance the potential industrial adoption. In particular, large-scale operation considerations, different phase separation procedures, the available optimization techniques (univariate, response surface methodology, and genetic algorithms) to maximize recovery and purity and economic modeling to predict large-scale costs, are discussed. ATPS intensification to increase the amount of sample to process at each system, developing recycling strategies and creating highly efficient predictive models, are still areas of great significance that can be further exploited with the use of high-throughput techniques. Moreover, the development of novel ATPS can maximize their specificity increasing the possibilities for the future industry adoption of ATPS. This review work attempts to present the areas of opportunity to increase ATPS attractiveness at industrial levels.

Refolding of laccase from Trametes versicolor using aqueous two phase systems: Effect of different additives.

Protein refolding is a strategy used to obtain active forms of proteins from inclusion bodies. On its part, laccase is an enzyme with potential for different biotechnological applications but there are few reports regarding its refolding which in many cases is considered inefficient due to the poor obtained refolding yields. Aqueous Two-Phase Systems (ATPS) have been used for the refolding of proteins getting acceptable recovery percentages since PEG presents capacity to avoid protein aggregation. In this work, 48 PEG-phosphate ATPS were analyzed to study the impact of different parameters (i.e. tie line length (TLL), volume ratio (VR) and PEG molecular weight) upon the recovery and refolding of laccase. Additionally, since laccase is a metalloprotein, the use of additives (individually and in mixture) was studied with the aim of favoring refolding. Results showed that laccase presents a high affinity for the PEG-rich phase obtaining recovery values of up to 90%. Such affinity increases with increasing TLL and decreases when PEG molecular weight and VR increase. In denatured state, this PEG-rich phase affinity decreases drastically. However, the use of additives such as l-cysteine, glutathione oxidized, cysteamine and Cu+2 was critical in improving refolding yield values up to 100%. The best conditions for the refolding of laccase were obtained using the PEG 400gmol-1, TLL 45% w/w, VR 3 ATPS and a mixture of 2.5mM cysteamine with 1mM Cu+2. To our knowledge, this is the first time that the use of additives and the behavior of the mixture of such additives to enhance refolding performance in ATPS is reported.

PEGylated protein separation using different hydrophobic interaction supports: Conventional and monolithic supports.

Protein hydrophobicity can be modified after a PEGylation process. However, hydrophobic interaction chromatography (HIC) has been used to separate PEGylation reaction products less frequently than other techniques. In this context, chromatographic monoliths represent a good alternative to continue exploring the separation of PEGylated proteins with HIC. In this work, the separation of PEGylated proteins using C4 A monolith as well as Toyopearl Butyl 650C and Butyl Sepharose was analyzed. Three proteins were used as models: RNase A, ß-lactoglobulin, and lysozyme. All proteins were PEGylated in the N-terminal amino groups with 20 kDa methoxy poly(ethylene glycol) propionaldehyde. The concentration of ammonium sulfate (1 M) used was the same for all stationary phases. The results obtained demonstrated that the C4 A monolith could better resolve all protein PEGylation reaction mixtures, since the peaks of mono- and di-PEGylated proteins can be clearly distinguished in the chromatographic profiles. On the contrary, while using Butyl Sepharose media only the PEGylation reaction mixtures of RNase A could be partially separated at 35 and 45 CVs. PEGylated proteins of ß-lactoglobulin and lysozyme could not be resolved when Toyopearl Butyl 650C and Butyl Sepharose were used. It is then clear that monoliths are an excellent choice to explore the purification process of PEGylated proteins exploiting the advantages of HIC. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:702-707, 2016.

Intensified fractionation of brewery yeast waste for the recovery of invertase using aqueous two-phase systems.

The potential recovery of high-value products from brewery yeast waste confers value to this industrial residue. Aqueous two-phase systems (ATPS) have demonstrated to be an attractive alternative for the primary recovery of biological products and are therefore suitable for the recovery of invertase from this residue. Sixteen different polyethylene glycol (PEG)-potassium phosphate ATPS were tested to evaluate the effects of PEG molecular weight (MW) and tie-line length (TLL) upon the partition behavior of invertase. Concentrations of crude extract from brewery yeast waste were then varied in the systems that presented the best behaviors to intensify the potential recovery of the enzyme. Results show that the use of a PEG MW 400 g mol-1 system with a TLL of 45.0% (w/w) resulted in an invertase bottom phase recovery with a purification factor of 29.5 and a recovery yield of up to 66.2% after scaling the system to a total weight of 15.0 g. This represents 15.1 mg of invertase per mL of processed bottom phase. With these results, a single-stage ATPS process for the recovery of invertase is proposed.

Aldehyde PEGylation of laccase from Trametes versicolor in route to increase its stability: effect on enzymatic activity.

Laccase is a multicopper oxidase that catalyzes the oxidation of phenolic compounds. Laccase can be used in bioremediation, beverage (wine, fruit juice, and beer) processing, ascorbic acid determination, sugar beet pectin gelation baking, and as a biosensor. Recently, the antiproliferative activity of laccase toward tumor cells has been reported. Because of the potential applications of this enzyme, the efforts for enhancing and stabilizing its activity have increased. Thus, the PEGylation of laccase can be an alternative. PEGylation is the covalent attachment of one or more molecules of methoxy poly(ethylene glycol) (mPEG) to a protein. Normally, during the PEGylation reaction, the activity is reduced but the stability increases; thus, it is important to minimize the loss of activity. In this work, the effects of molar ratio (1:4, 1:8, and 1:12), concentration of laccase (6 and 12 mg/ml), reaction time (4 and 17 h), molecular weight, and type of mPEG (20, 30, 40 kDa and 40 kDa-branched) were analyzed. The activity was measured using three substrates: ABTS, 2,6-dimethoxyphenol, and syringaldazine. The best conditions for laccase PEGylation were 12 mg/ml of laccase, molar ratio 1:4, and 4 h reaction time. Under these conditions, the enzyme was able to maintain nearly 100% of its enzymatic activity with ABTS. The PEGylation of laccase has not been extensively explored, so it is important to analyze the effects of this bioconjugation in route to produce a robust modified enzyme.

Separation of PEGylated variants of ribonuclease A and apo-α-lactalbumin via reversed phase chromatography.

The covalent attachment of polyethylene glycol (PEG) molecules to pharmaceutical proteins, "PEGylation", often results in a population of conjugate species that includes differing numbers and locations of attached PEG chains. As some portion of this population may be biologically inactive, a challenging separation problem arises. An interesting alternative to the size-based resolution of these conjugates involves the use of reversed phase chromatography (RPC), treating the PEG moieties as hydrophobic purification tags. The use of RPC raises concerns about protein denaturation in the mobile and on the stationary phase. Here, the potential dual role of conjugated PEG chains as both group-specific separation tags and as steric or structural stabilizers in RPC was explored. In this work, RPC with C18-based media was used to resolve PEGylation number variants of ribonuclease A (RNase A) and apo-α-lactalbumin (apo-αLac) in a neutral pH mobile phase. While the attachment of 20kDa PEG molecules did not modify the structures of RNase A and apo-αLac, as confirmed by structural analysis using circular dichroism, exposure to the mobile phase modifier, acetonitrile, and to the C18 media during separation resulted in perturbations to both the secondary and tertiary structures of all species studied. RNase A experienced small perturbations that were mediated to some extent by PEGylation; these results were consistent with activity assays which showed that PEGylated RNase A species retained native-like activity after RPC separation. Apo-αLac, a more hydrophobic and less stable protein than RNase A, experienced extensive structural perturbations regardless of PEGylation state. The temperature of the mobile phase was found to strongly influence chromatographic separation of PEG-conjugates with conjugate species becoming more strongly retained with increasing temperature. This work shows that it is feasible to employ RPC with neutral pH mobile phases to resolve PEG conjugate number heterogeneity.

Hydrophobic interaction chromatography for purification of monoPEGylated RNase A.

The chromatographic methods used for the purification of PEGylated proteins are mainly Size Exclusion (SEC) and Ion Exchange Chromatography (IEX). Although the PEGylation affects the protein hydrophobicity, Hydrophobic Interaction Chromatography (HIC) has not been extensively applied for the separation of these proteins. Purification of monoPEGylated Ribonuclease A (RNase A) using HIC is studied in this work. The products of the PEGylation reaction of RNase A with 20 kDa methoxy-poly(ethylene glycol) were separated using three resins with different degrees of hydrophobicity: Butyl, Octyl and Phenyl sepharose. The effects of resin type, concentration and salt type (ammonium sulphate or sodium chloride), and gradient length on the separation performance were evaluated. Yield and purity were calculated using the plate model. Under all conditions assayed the native protein was completely separated from PEGylated species. The best conditions for the purification of monoPEGylated RNase A were: Butyl sepharose, 1 M ammonium sulphate and 35 column volumes (CVs); this resulted in a yield as high as 85% with a purity of 97%. The purity of monoPEGylated RNase A is comparable to that obtained when the separation is performed using SEC, but the yield increases from 65% with SEC to ~85% with HIC. This process represents a viable alternative for the separation of PEGylated proteins.