Chromatography affinity resin with photosynthetically-sourced protein A ligand.

Green, photosynthesizing plants can be proficiently used as cost-effective, single-use, fully biodegradable bioreactors for environmentally-friendly production of a variety of valuable recombinant proteins. Being near-infinitely scalable and most energy-efficient in generating biomass, plants represent profoundly valid alternatives to conventionally used stationary fermenters. To validate this, we produced a plastome-engineered tobacco bioreactor line expressing a recombinant variant of the protein A from Staphylococcus aureus, an affinity ligand widely useful in antibody purification processes, reaching accumulation levels up to ~ 250 mg per 1 kg of fresh leaf biomass. Chromatography resin manufactured from photosynthetically-sourced recombinant protein A ligand conjugated to agarose beads demonstrated the innate pH-driven ability to bind and elute IgG-type antibodies and allowed one-step efficient purification of functional monoclonal antibodies from the supernatants of the producing hybridomas. The results of this study emphasize the versatility of plant-based recombinant protein production and illustrate its vast potential in reducing the cost of diverse biotechnological applications, particularly the downstream processing and purification of monoclonal antibodies.

Proceedings of the 2023 Viral Clearance Symposium, Session 2: Viral Clearance Strategy and Case Studies.

This session deals with the rational design of viral clearance studies for biopharmaceuticals including recombinant proteins such as monoclonal antibodies and, as new in scope of the symposium, also viral clearance for adeno-associated viral (AAV) vectors. For recombinant proteins, large datasets were accumulated over the last decades and are intended to be used for accelerated product process development and streamlining of viral clearance studies. How to utilize prior knowledge in viral clearance validation and how it can be used in a risk assessment tool to decide whether additional virus clearance studies are necessary during product development is being addressed by three of the presentations of this session. This also includes an a priori intended design and generation of validation data for a new kind of detergent such as CG-110, to build up a platform dataset to be used as prior knowledge in future marketing application. Another presentation investigates the virus removal mechanism of a newly developed hydrophobic interaction chromatography (HIC) resin and demonstrates for highly hydrophobic antibodies appropriate reduction for a retrovirus and impurities in a defined process range in contrast to the moderate to poor virus reduction of recent HIC resins. The last two presentations deal with virus clearance approaches for AAV, which will become mandatory with approval of the ICH Q5A revision. Appropriate virus removal and virus inactivation procedures can be implemented into the manufacturing processes of AAV vectors including viral filtration, viral inactivation (e.g., heat inactivation), affinity chromatography, and anion-exchange chromatography with which it seems possible to achieve a good clearance for helper and also adventitious viruses. The heat treatment step can be even a robust step for adenovirus helper inactivation for AAV products when product characteristics and process conditions are understood.

Cu(II)-Loaded Polydopamine-Coated Urchin-like Titanate Microspheres as a High-Performance IMAC Adsorbent for Hemoglobin Separation.

Immobilized metal ion affinity chromatography (IMAC) adsorbents generally have excellent affinity for histidine-rich proteins. However, the leaching of metal ions from the adsorbent usually affects its adsorption performance, which greatly affects the reusable performance of the adsorbent, resulting in many limitations in practical applications. Herein, a novel IMAC adsorbent, i.e., Cu(II)-loaded polydopamine-coated urchin-like titanate microspheres (Cu-PDA-UTMS), was prepared via metal coordination to make Cu ions uniformly decorate polydopamine-coated titanate microspheres. The as-synthesized microspheres exhibit an urchin-like structure, providing more binding sites for hemoglobin. Cu-PDA-UTMS exhibit favorable selectivity for hemoglobin adsorption and have a desirable adsorption capacity towards hemoglobin up to 2704.6 mg g-1. Using 0.1% CTAB as eluent, the adsorbed hemoglobin was easily eluted with a recovery rate of 86.8%. In addition, Cu-PDA-UTMS shows good reusability up to six cycles. In the end, the adsorption properties by Cu-PDA-UTMS towards hemoglobin from human blood samples were analyzed by SDS-PAGE. The results showed that Cu-PDA-UTMS are a high-performance IMAC adsorbent for hemoglobin separation, which provides a new method for the effective separation and purification of hemoglobin from complex biological samples.

Affinity chromatography for virus-like particle manufacturing: Challenges, solutions, and perspectives.

The increasing medical application of virus-like particles (VLPs), notably vaccines and viral vectors, has increased the demand for commercial VLP production. However, VLP manufacturing has not yet reached the efficiency level achieved for recombinant protein therapeutics, especially in downstream processing. This review provides a comprehensive analysis of the challenges associated with affinity chromatography for VLP purification with respect to the diversity and complexity of VLPs and the associated upstream and downstream processes. The use of engineered affinity ligands and matrices for affinity chromatography is first discussed. Although several representative affinity ligands are currently available for VLP purification, most of them have difficulty in balancing ligand universality, ligand selectivity and mild operation conditions. Then, phage display technology and computer-assisted design are discussed as efficient methods for the rapid discovery of high-affinity peptide ligands. Finally, the VLP purification by affinity chromatography is analyzed. The process is significantly influenced by virus size and variation, ligand type and chromatographic mode. To address the updated regulatory requirements and epidemic outbreaks, technical innovations in affinity chromatography and process intensification and standardization in VLP purification should be promoted to achieve rapid process development and highly efficient VLP manufacturing, and emphasis is given to the discovery of universal ligands, applications of gigaporous matrices and platform technology. It is expected that the information in this review can provide a better understanding of the affinity chromatography methods available for VLP purification and offer useful guidance for the development of affinity chromatography for VLP manufacturing in the decades to come.

Quantum dot fluorescent microsphere-based immunochromatographic strip for detecting PRRSV antibodies.

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). Current vaccine prevention and treatment approaches for PRRS are not adequate, and commercial vaccines do not provide sufficient cross-immune protection. Therefore, establishing a precise, sensitive, simple, and rapid serological diagnostic approach for detecting PRRSV antibodies is crucial. The present study used quantum dot fluorescent microspheres (QDFM) as tracers, covalently linked to the PRRSV N protein, to develop an immunochromatography strip (ICS) for detecting PRRSV antibodies. Monoclonal antibodies against PRRSV nucleocapsid (N) and membrane (M) proteins were both coated on nitrocellulose membranes as control (C) and test (T) lines, respectively. QDFM ICS identified PRRSV antibodies under 10 min with high sensitivity and specificity. The specificity assay revealed no cross-reactivity with the other tested viruses. The sensitivity assay revealed that the minimum detection limit was 1.2 ng/mL when the maximum dilution was 1:2,048, comparable to the sensitivity of enzyme-linked immunosorbent assay (ELISA) kits. Moreover, compared to PRRSV ELISA antibody detection kits, the sensitivity, specificity, and accuracy of QDFM ICS after analyzing 189 clinical samples were 96.7%, 97.9%, and 97.4%, respectively. Notably, the test strips can be stored for up to 6 months at 4 °C and up to 4 months at room temperature (18-25 °C). In conclusion, QDFM ICS offers the advantages of rapid detection time, high specificity and sensitivity, and affordability, indicating its potential for on-site PRRS screening. KEY POINTS: • QDFM ICS is a novel method for on-site and in-lab detection of PRRSV antibodies • Its sensitivity, specificity, and accuracy are on par with commercial ELISA kits • QDFM ICS rapidly identifies PRRSV, aiding the swine industry address the evolving virus.

Insights into nucleoside hydrolase from Leishmania donovani inhibition: A new bioaffinity chromatography-based screening assay and docking studies.

Leishmaniasis, a group of neglected infectious diseases, encompasses a serious health concern, particularly with visceral leishmaniasis exhibiting potentially fatal outcomes. Nucleoside hydrolase (NH) has a fundamental role in the purine salvage pathway, crucial for Leishmania donovani survival, and presents a promising target for developing new drugs for visceral leishmaniasis treatment. In this study, LdNH was immobilized into fused silica capillaries, resulting in immobilized enzyme reactors (IMERs). The LdNH-IMER activity was monitored on-flow in a multidimensional liquid chromatography system, with the IMER in the first dimension. A C18 analytical column in the second dimension furnished the rapid separation of the substrate (inosine) and product (hypoxanthine), enabling direct enzyme activity monitoring through product quantification. LdNH-IMER exhibited high stability and was characterized by determining the Michaelis-Menten constant. A known inhibitor (1-(ß-d-Ribofuranosyl)-4-quinolone derivative) was used as a model to validate the established method in inhibitor recognition. Screening of three additional derivatives of 1-(ß-d-Ribofuranosyl)-4-quinolone led to the discovery of novel inhibitors, with compound 2a exhibiting superior inhibitory activity (Ki = 23.37 ± 3.64 µmol/L) compared to the employed model inhibitor. Docking and Molecular Dynamics studies provided crucial insights into inhibitor interactions at the enzyme active site, offering valuable information for developing new LdNH inhibitors. Therefore, this study presents a novel screening assay and contributes to the development of potent LdNH inhibitors.

Generation of affinity maps for thiazolidinediones with human serum albumin using affinity microcolumns. I. Studies of effects by glycation on multisite drug binding.

Human serum albumin (HSA) is known to undergo modifications by glucose during diabetes. This process produces glycated HSA that can have altered binding to some drugs. In this study, high-performance affinity microcolumns and competition studies were used to see how glycation affects the binding by two thiazolidinedione-class drugs (i.e., pioglitazone and rosiglitazone) at specific regions of HSA. These regions included Sudlow sites I and II, the tamoxifen and digitoxin sites, and a drug-binding site located in subdomain IB. At Sudlow site II, the association equilibrium constants (or binding constants) for pioglitazone and rosiglitazone with normal HSA were 1.7 × 105 M-1 and 2.0 × 105 M-1 at pH 7.4 and 37 °C, with values that changed by up to 5.7-fold for glycated HSA. Sudlow site I of normal HSA had binding constants for pioglitazone and rosiglitazone of 3.4 × 105 M-1 and 4.6 × 105 M-1, with these values changing by up to 1.5-fold for glycated HSA. Rosiglitazone was found to also bind a second region that had a positive allosteric effect on Sudlow site I for all the tested preparations of HSA (binding affinity, 1.1-3.2 × 105 M-1; coupling constant for Sudlow site I, 1.20-1.34). Both drugs had a strong positive allosteric effect on the tamoxifen site of HSA (coupling constants, 13.7-19.9 for pioglitazone and 3.7-11.5 for rosiglitazone). Rosiglitazone also had weak interactions at a site in subdomain IB, with a binding constant of 1.4 × 103 M-1 for normal HSA and a value that was altered by up to 6.8-fold with glycated HSA. Neither of the tested drugs had any significant binding at the digitoxin site. The results were used to produce affinity maps that described binding by these thiazolidinediones with HSA and the effects of glycation on these interactions during diabetes.

A review on magnetic beads-based SELEX technologies: Applications from small to large target molecules.

This review summarizes the stepwise strategy and key points for magnetic beads (MBs)-based aptamer selection which is suitable for isolating aptamers against small and large molecules via systematic evolution of ligands by exponential enrichment (SELEX). Particularities, if any, are discussed according to the target size. Examples targeting small molecules (<1000 Da) such as xenobiotics, toxins, pesticides, herbicides, illegal additives, hormones, and large targets such as proteins (biomarkers, pathogens) are discussed and presented in tabular formats. Of special interest are the latest advances in more efficient alternatives, which are based on novel instrumentation, materials or microelectronics, such as fluorescence MBs-SELEX or microfluidic chip system-assisted MBs-SELEX. Limitations and perspectives of MBs-SELEX are also reviewed. Taken together, this review aims to provide practical insights into MBs-SELEX technologies and their ability to screen multiple potential aptamers against targets from small to large molecules.

DIP-MS: ultra-deep interaction proteomics for the deconvolution of protein complexes.

Most proteins are organized in macromolecular assemblies, which represent key functional units regulating and catalyzing most cellular processes. Affinity purification of the protein of interest combined with liquid chromatography coupled to tandem mass spectrometry (AP-MS) represents the method of choice to identify interacting proteins. The composition of complex isoforms concurrently present in the AP sample can, however, not be resolved from a single AP-MS experiment but requires computational inference from multiple time- and resource-intensive reciprocal AP-MS experiments. Here we introduce deep interactome profiling by mass spectrometry (DIP-MS), which combines AP with blue-native-PAGE separation, data-independent acquisition with mass spectrometry and deep-learning-based signal processing to resolve complex isoforms sharing the same bait protein in a single experiment. We applied DIP-MS to probe the organization of the human prefoldin family of complexes, resolving distinct prefoldin holo- and subcomplex variants, complex-complex interactions and complex isoforms with new subunits that were experimentally validated. Our results demonstrate that DIP-MS can reveal proteome modularity at unprecedented depth and resolution.

Polylevodopa nanoplatform for lateral flow immunochromatography assay of SARS-CoV-2 and influenza A virus.

At the end of 2019, an unprecedented outbreak of novel coronavirus pneumonia ravaged the global landscape, inflicting profound harm upon society. Following numerous cycles of transmission, we find ourselves in an epoch where the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coexists alongside influenza viruses (Flu A). Swift and accurate diagnosis of SARS-CoV-2 and Flu A is imperative to stem the spread of these maladies and administer appropriate treatment. Presently, colloidal gold-based lateral flow immunoassays (Au-LFIAs) constructed through electrostatic adsorption are beset by challenges such as diminished sensitivity and feeble binding stability. In this context, we propose the adoption of black polylevodopa nanoparticles (PLDA NPs) featuring abundant carboxyl groups as labeling nanomaterials in LFIA to bolster the stability and sensitivity of SARS-CoV-2 antigens and influenza A virus identifications. The engineered PLDA-LFIAs exhibit the capacity to detect SARS-CoV-2 and Flu A within 30 min, boasting a detection threshold of 5 pg/ml for the SARS-CoV-2 antigen and 0.1 ng/ml for the Flu A H1N1 antigen, thereby underscoring their heightened sensitivity relative to Au-LFIAs. These PLDA-LFIAs hold promise for the early detection of SARS-CoV-2 and Flu A, underscoring the potential of PLDA NPs as a discerning labeling probe to heighten the sensitivity of LFIA across diverse applications.

A rapid one-step affinity purification of C-phycocyanin from Spirulina platensis.

The high-purity phycocyanin has a high commercial value. Most current purification methods of C-phycocyanin involve multiple steps, which are complicated and time-consuming. To solve the problem, this research was studied, and an efficient affinity chromatography purification for C-phycocyanin from Spirulina platensis was developed. Through molecular docking simulation, virtual screening of ligands was performed, and ursolic acid was identified as the specific affinity ligand, which coupled to Affi-Gel 102 gel via 1-ethyl (3-dimethylaminopropyl)-3-carbodiimide, hydrochloride as coupling agent. With this customized and synthesized resin, a high-efficiency one-step purification procedure for C-phycocyanin was developed and optimized, the purity was determined to be 4.53, and the yield was 69 %. This one-step purification protocol provides a new approach for purifying other phycobilin proteins.

Establishment and application of a screening method for α-glucosidase inhibitors based on dual sensing and affinity chromatography.

α-Glucosidase plays a direct role in the metabolic pathways of starch and glycogen, any dysfunction in its activity could result in metabolic disease. Concurrently, this enzyme serves as a target for diverse drugs and inhibitors, contributing to the regulation of glucose metabolism in the human body. Here, an integrated analytical method was established to screen inhibitors of α-glucosidase. This step-by-step screening model was accomplished through the biosensing and affinity chromatography techniques. The newly proposed sensing program had a good linear relationship within the enzyme activity range of 0.25 U mL-1 to 1.25 U mL-1, which can quickly identify active ingredients in complex samples. Then the potential active ingredients can be captured, separated, and identified by an affinity chromatography model. The combination of the two parts was achieved by an immobilized enzyme technology and a microdevice for reaction, and the combination not only ensured efficiency and accuracy for inhibitor screening but also eliminated the occurrence of false positive results in the past. The emodin, with a notable inhibitory effect on α-glucosidase, was successfully screened from five traditional Chinese medicines using this method. The molecular docking results also demonstrated that emodin was well embedded into the active pocket of α-glucosidase. In summary, the strategy provided an efficient method for developing new enzyme inhibitors from natural products.

Neuropeptidomics of Genetically Defined Cell Types in Mouse Brain.

Peptidomic techniques are powerful tools to identify peptides in a biological sample. In the case of brain, which contains a complex mixture of cell types, standard peptidomics procedures reveal the major peptides in a dissected brain region. It is difficult to obtain information on peptides within a specific cell type using standard approaches, unless that cell type can be isolated. This protocol describes a targeted peptidomic approach that uses affinity chromatography to purify peptides that are substrates of carboxypeptidase E (CPE), an enzyme present in the secretory pathway of neuroendocrine cells. Many CPE products function as neuropeptides and/or peptide hormones, and therefore represent an important subset of the peptidome. Because CPE removes C-terminal Lys and Arg residues from peptide processing intermediates, organisms lacking CPE show a large decrease in the levels of the mature forms of most neuropeptides and peptide hormones, and a very large increase in the levels of the processing intermediates that contain C-terminal Lys and/or Arg (i.e., the CPE substrates). These CPE substrates can be purified on an anhydrotrypsin-agarose affinity resin, which specifically binds peptides with C-terminal basic residues. When this method is used with mice lacking CPE activity in genetically defined cell types, it allows the detection of peptides specifically produced in that cell type.

Rapid and sensitive quantitation of amitraz in orange, tomato, and eggplant samples using immunochromatographic assay.

Amitraz (AMT) is a broad-spectrum formamidine insecticide and acaricide. In this study, we produced an anti-AMT monoclonal antibody (mAb) with high performance. The half-maximal inhibitory concentration of the anti-AMT mAb was 4.418 ng/mL, the cross reactivity with other insecticides was negligible, and an affinity constant was 2.06 × 109 mmol/L. Additionally, we developed an immunochromatographic assay for the rapid detection of AMT residues in oranges, tomatoes, and eggplants. The cut-off values were 2000 µg/kg in oranges and tomato samples and 1000 µg/kg in eggplant samples and the calculated limits of detection were 14.521 µg/kg, 6.281 µg/kg, and 3.518 µg/kg in oranges, tomatoes, and eggplants, respectively, meeting the detection requirements for AMT in fruits and vegetables. The recovery rates ranged between 95.8 % and 105.2 %, consistent with the recovery rates obtained via LC-MS/MS. Our developed immunochromatographic assay can effectively, accurately, and rapidly determine AMT residues in oranges, tomatoes, and eggplants.

One-Step Affinity Purification of Leucine-Rich α2-Glycoproteins from Snake Sera and Characterization of Their Phospholipase A2-Inhibitory Activities as ß-Type Phospholipase A2 Inhibitors.

Snakes contain three types of phospholipase A2 (PLA2)-inhibitory proteins in their blood, PLIα, ß, and γ, which protect them from their own venom, PLA2. PLIß is the snake ortholog of leucine-rich α2 glycoprotein (LRG). Since autologous cytochrome c (Cyt c) serves as an endogenous ligand for LRG, in this study, we purified snake LRGs from various snake serum samples using Cyt c affinity chromatography. All purified snake LRGs were found to be dimers linked by disulfide bonds. Laticauda semifasciata and Naja kaouthia LRGs showed no inhibitory activity against L. semifasciata PLA2 and weak inhibitory activity against Gloydius brevicauda basic PLA2. Elaphe climacophora PLIß had weaker inhibitory activity against G. brevicauda basic PLA2 than G. brevicauda and Elaphe quadrivirgata PLIs, which are abundant in blood and known to neutralize G. brevicauda basic PLA2. Protobothrops flavoviridis LRG showed no inhibitory activity against basic venom PLA2, PL-X, or G. brevicauda basic PLA2. Binding analysis of P. flavoviridis LRG using surface plasmon resonance showed very strong binding to snake Cyt c, followed by that to horse Cyt c, weak binding to yeast Cyt c, and no binding to P. flavoviridis PL-X or BPI/II. We also deduced the amino acid sequences of L. semifasciata and P. flavoviridis LRG by means of cDNA sequencing and compared them with those of other known sequences of PLIs and LRGs. This study concluded that snake LRG can potentially inhibit basic PLA2, but, whether it actually functions as a PLA2-inhibitory protein, PLIß, depends on the snake.

Functional Green-Emitting Mn2+-doped Zinc Germanate Persistent Luminescent Nanoparticles for Dual-Mode Immunochromatographic Detection.

Immunochromatography is a commonly used immediate detection technique, using signal labels to generate detection signals for rapid medical diagnosis. However, its detection sensitivity is affected by background fluorescence caused by the excitation light source. We have developed an immunochromatographic test strip using Zn2GeO4:Mn2+ (ZGM) persistent luminescent nanoparticles (PLNPs) for immediate fluorescence detection and highly sensitive persistent luminescence (PersL) detection without background fluorescence interference. ZGM emits a strong green light when exposed to ultraviolet (UV) excitation, and its green PersL can persist for over 30 min after the excitation light is turned off. We modified the surface of ZGM with heparin-binding protein (HBP) antibodies to create immunochromatographic test strips for the detection of HBP as the target analyte. Under UV excitation, the chromatography test paper can be visually observed at concentrations as low as 25 ng/mL. After the excitation light source is switched off, PersL can achieve a detection limit of 4.7 ng/mL without background interference. This dual-mode immunochromatographic detection, based on ZGM, shows great potential for in vitro diagnostic applications.

Quantitative immunochromatographic assay for rapid and cost-effective on-site detection of benzo[a]pyrene in oilfield chemicals.

Contamination of oilfield chemicals (OFCs) by benzo[a]pyrene (B[a]P) is increasingly becoming a severe environmental security issue. There is an urgent need to develop a rapid and accurate method for B[a]P detection in OFCs. In this study, B[a]P hapten was designed using computer aided molecular design. A high-affinity, specific, and matrix-insensitive monoclonal antibody (mAb) with IC50 values of 6.77 ng/mL was obtained. Based on this mAb, we developed a rapid gold nanoparticle-based immunochromatographic strip assay (GICA) with double T-line mode for on-site detection of B[a]P in OFCs samples. The GICA exhibited excellent detection performance in OFCs samples with strong acidity, strong alkalinity, and deep color. Under optimal conditions, the proposed method detected B[a]P in OFCs at 0.42-300 mg/kg, and limit of detection was 0.23-1.07 mg/kg. The recovery rate was 88-106% with a coefficient of variation of 1.46-6.35%. Confirmed by natural positive OFCs samples and high-performance liquid chromatography, this GICA is accurate and reliable, with great potential for rapid and cost-effective on-site detection.

Polymer Microspheres Copolymerized with Deep Red Fluorescent Molecules as a Label for Lateral Flow Immunochromatography.

The development of fluorescently labeled microspheres is a critical aspect of advancing the technology of lateral flow immunochromatography (LFIA) for biological detection. Nevertheless, potential interference posed by the background fluorescence originating from the nitrocellulose (NC) membrane would significantly impact the sensitivity and accuracy of microsphere-based detection in LFIA. In this work, an attempt was made to extend the π-conjugated system and asymmetric structure of rhodamine fluorophore, resulting in the synthesis of dye molecules (RB2) incorporating double bonds, which can reach an absolute photoluminescence quantum yield (PLQY) of 30.01% in EtOH. Subsequently, carboxyl group functionalized fluorescent microspheres were prepared in a two-step copolymerization via soap-free emulsion polymerization. The obtained microspheres were characterized by scanning electron microscopy, transmission electron microscopy, DLS, Fourier transform infrared spectroscopy, ultraviolet spectrophotometry, and fluorescence spectrophotometry. The results showed that RB2 was successfully copolymerized into the microspheres, and the resulting microspheres had good dispersion and stability with high red fluorescence intensity (λabs ∼ 610 nm, λem ∼ 660 nm). Utilizing these microspheres, the resulting lateral flow immunoassay was successfully found to detect SARS-CoV-2 N protein with a detection limit of 2.5 pg/mL and the linear concentration spanning from 2.5 pg/mL to 10 ng/mL. The results confirm the effectiveness of the synthetic fluorescent microspheres as the label for LFIA.

Improved mRNA affinity chromatography binding capacity and throughput using an oligo-dT immobilized electrospun polymer nanofiber adsorbent.

Increased demand for mRNA-based therapeutics and improved in vitro transcription (IVT) yields have challenged the mRNA purification platform. Hybridization-affinity chromatography with an immobilized oligo-deoxythymidilic acid (oligodT) ligand is often used to capture mRNA through base pairing with the polyadenylated tail. Commercially available oligodT matrices include perfusive cross-linked poly(styrene-divinylbenzene) 50 µm POROS™ chromatography resin beads and convective polymethacrylate CIMmultus® monolithic columns consisting of 2 µm interconnected channels. POROS™ columns may be limited by poor mass transfer for larger mRNAs and slow flowrates, while monoliths can operate at higher flowrates but are limited by modest binding capacity. To enable both high flowrates and binding capacity for mRNA of all lengths, prototype chromatography media was developed by Cytiva using oligodT immobilized electrospun cellulose nanofibers (Fibro™) with a 0.3-0.4 µm pore size. In this work, four polyadenylated mRNAs ranging from ∼1900-4300 nucleotides were used to compare the dynamic binding capacity (DBC) of Fibro™, POROS® and CIMmultus® columns as a function of residence time and binding buffer compositions. Fibro™ improved the DBC ∼2-4-fold higher than CIMmultus® and ∼2-13-fold higher than POROS™ across all residence times, mRNA length, and binding matrix compositions tested. CIMmultus® DBC was least dependent on residence time and mRNA size, while both Fibro™ and POROS™ DBC increased at slower flowrates and with shorter mRNA. Surprisingly, inverse size exclusion (ISE) experiments showed that POROS™ was not limited by diffusion and POROS™ along with CIMmultus® demonstrate higher mRNA permeation however the Fibro™ prototype is not in the final configuration. Lastly, IVT reaction products were subjected to purification and oligodT elution product yield, quality, and purity were consistent across the three matrices investigated. These results highlight the benefits of high DBC and equivalent product profiles offered by the oligodT Fibro™ prototype compared to commercially available oligodT media.

Purification of pineapple bromelain by IMAC chromatography using chlorophyll-activated macroporous matrices.

This study investigated the purification of bromelain obtained from pineapple fruit using a new adsorbent for immobilized metal ion affinity chromatography (IMAC), with chlorophyll obtained from plant leaves as a chelating agent. The purification of bromelain was evaluated in batches from the crude extract of pineapple pulp (EXT), and the extract precipitated with 50 % ammonium sulfate (EXT.PR), the imidazole buffer (200 mM, pH 7.2) being analyzed and sodium acetate buffer, pH 5.0 + 1.0 NaCl as elution solutions. All methods tested could separate forms of bromelain with molecular weights between ±21 to 25 kDa. Although the technique using EXT.PR stood out in terms of purity, presenting a purification factor of around 3.09 ± 0.31 for elution with imidazole and 4.23 ± 0.12 for acetate buffer solution. In contrast, the EXT methods obtained values between 2.44 ± 0.23 and 3.21 ± 0.74 for elution with imidazole and acetate buffer, respectively, for purification from EXT.PR has lower yield values (around 5 %) than EXT (around 15 %). The number of steps tends to reduce yield and increase process costs, so the purification process in a monolithic bed coupled to the chromatographic system using the crude extract was evaluated. The final product obtained had a purification factor of 6, with a specific enzymatic activity of 59.61 ± 0.00 U·mg-1 and a yield of around 39 %, with only one band observed in the SDS-PAGE electrophoresis analysis, indicating that the matrix produced can separate specific proteins from the total fraction in the raw material. The IMAC matrix immobilized with chlorophyll proved promising and viable for application in protease purification processes.