Chromatographic Purification Research Articles

Development of novel 9H-carbazole-4H-chromene hybrids as dual cholinesterase inhibitors for the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disease affecting mental ability and neurocognitive functions. Cholinesterase enzymes affect concentration of acetylcholine in the brain, leading to dementia. Thus, there is an urgent need to develop novel dual cholinesterase inhibitors as possible anti-AD drugs. Herein, we have designed and synthesized a novel series of 9H-carbazole-4H-chromenes 4(a-l) through a one-pot three-component reaction of salicylaldehydes (1), hydroxycarbazole (2) and N-methyl-1-(methylthio)-2-nitroethenamine (3) using triethylamine as a catalyst in ethanol. Synthetic transformation involves the formation of two C-C bonds and one C-O bond in a single step to obtain desired analogs. The rapid one-pot reaction does not require chromatographic purification, proceeds under mild conditions, and exhibits good tolerance toward various functional groups with high synthetic yields. Synthesized compounds were screened for cytotoxicity using MTT assay in BV-2 microglial cells. These compounds were then in-vitro screened against acetylcholinesterase (AChE) and butyrylcholinestrase (BuChE) enzymes. Most of these ligands have shown dual cholinesterase inhibitory activity compared to the standard drug. In-vitro results showed that the compounds 4a and 4d have promising anticholinesterase response against both cholinesterase enzymes (4a, AChE IC50: 5.76µM, BuChE IC50: 48.98µM; 4d, AChE IC50: 3.58µM, BuChE IC50: 42.73µM). In-vitro results were validated by molecular docking and dynamic simulation at 100ns. Molecular docking and molecular dynamics simulation study strongly supported structural features present in these analogs. Together, these analogs could be exploited to develop dual anti-cholinesterase candidates to treat AD in combination with other drugs.

Chromatographic purification of C-phycocyanin from Spirulina platensis: assessing antioxidant activity and stability.

The efficient separation and purification of proteins like C-phycocyanin (C-PC) from Spirulina platensis are essential for their commercialization, yet they remain challenging. This study investigated three chromatographic methods for C-PC purification: weak anion exchange chromatography (DEAE), strong anion exchange chromatography (Q Sepharose), and hydrophobic interaction chromatography (HIC). Weak anion exchange chromatography achieved a recovery of 36.80 mg unit (57.08%) with a purity of 3.23, outperforming Q Sepharose (yield: 23.21 mg unit means that 46.33%, purity: 2.76) and HIC (yield: 22.95 mg unit means that 17.57%, purity: 3.02). The purified C-PC consisted of α and β subunits with molecular masses of 16 kDa and 17 kDa, respectively. Further assessment revealed its antioxidant capacity through a 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. The stability of C-phycocyanin was tested at different pH levels and temperatures. Maximum stability was observed at pH 7, and pH 4 showed the lowest stability. Glutaraldehyde-treated C-PC (GC-PC) demonstrated gradual degradation up to 50 °C, retaining 73.25% after 30 min. Notably, GC-PC exhibited stability even at higher temperatures, with degradation rates of 57.32% at 70 °C and 50.96% at 80 °C. Weak anion exchange chromatography proved superior for C-PC purification, offering higher yields and purity than Q Sepharose and HIC. The purified C-PC showed promising antioxidant capacity and stability, particularly GC-PC, which exhibited resistance to degradation, even at elevated temperatures. These findings underscore the potential of C-PC as a valuable compound for various applications, with DEAE chromatography being an efficient method for its production and commercialization. © 2024 Society of Chemical Industry.

Structural characterization of the polysaccharide from the black crystal region of Inonotus obliquus and its effect on AsPC-1 and SW1990 pancreatic cancer cell apoptosis

In this study, one water soluble polysaccharide (IOP1–1) with a weight average molecular weight of 6886 Da was obtained from the black crystal region of Inonotus obliquus by hot water extraction, DEAE-52 cellulose extraction and Sephadex-100 column chromatography purification. Structural analysis indicated that IOP1–1 was a glucan with a main chain composed of α-Glcp-(1 → 4)-α-Glcp-(1 → 4)-β-Glcp-(1 → 4)-β-Glcp-(1 → 4)-α-Glcp-(1 → 6)-β-Glcp-(1 → 4)-α-Glcp-(1 → 3)-β-Glcp-(1→. The CCK-8 assay results showed that IOP1–1 inhibited AsPC-1 and SW1990 pancreatic cancer cell proliferation in a concentration-dependent manner. Flow cytometric analysis revealed that IOP1–1 induced cell cycle arrest in AsPC-1 and SW1990 cells. Hoechst 33342 staining and Annexin V-FITC/PI double staining analysis showed that IOP1–1 could induce apoptosis in AsPC-1 and SW1990 cells. Furthermore, western blot analysis confirmed that IOP1–1 could induce apoptosis in AsPC-1 and SW1990 pancreatic cancer cells through three pathways: the mitochondrial pathway, the death receptor pathway, and endoplasmic reticulum stress. According to these research data, IOP1–1 may be utilized as an adjuvant treatment to anticancer medications, opening up new application prospects and opportunities.

Room Temperature Diels-Alder Reactions of 4-Vinylimidazoles.

In the course of studying Diels-Alder reactions of 4-vinylimidazoles with N-phenylmaleimide, it was discovered that they engage in cycloaddition at room temperature to give high yields of the initial cycloadduct as a single stereoisomer. In certain cases, the product precipitated out of the reaction mixture and could be isolated by simple filtration, thereby avoiding issues with aromatization observed during chromatographic purification. Given these results, intramolecular variants using doubly activated dienophiles were also investigated at room temperature. Amides underwent cycloaddition at room temperature in modest yields, but the initial adducts were not isolable with Nimid-benzyl-protected systems. Attempts to extend these results to the corresponding esters and hydroxamate were less successful with these substrates only undergoing cycloaddition at elevated temperatures in lower yields. Density functional theory calculations were performed to evaluate the putative transition states for both the inter- and intramolecular variants to rationalize experimental observations.

Identification of a broad-spectrum high-affinity peptide ligand for the purification of spike proteins

Since the outbreak of coronavirus disease 2019, the global demand for vaccines has increased rapidly to prevent infection and protect high-risk populations. However, identifying viral mutations poses an additional challenge for chromatographic purification of vaccines and subunit vaccines. In this study, a new affinity peptide model, X1VX2GLNX3WX4RYSK, was established, and a library of 612 peptides was generated for ligand screening. Based on a multistep strategy of ligand screening, 18 candidate peptides were obtained. The top ranking peptide, LP14 (YVYGLNIWLRYSK), and two other representative peptides, LP02 and LP06, with lower rankings were compared via molecular dynamics simulation. The results revealed that peptide binding to the receptor binding domain (RBD) was driven by hydrophobic interactions and the key residues involved in the binding were identified. Surface plasmon resonance analysis further confirmed that LP14 had the highest affinity for the wild RBD (Kd=0.520 μmol/L), and viral mutation had little influence on the affinity of LP14, demonstrating its great potential as a broad-spectrum ligand for RBD purification. Finally, chromatographic performance of LP14-coupled gel-packed column verified that both wild and omicron RBDs could be purified and were eluted by 0.1 mol/L Gly-HCl buffer (pH 3.0). This research identified a broad-spectrum peptide for RBD purification based on rational design and demonstrated its potential application in the purification of RBDs from complex feedstock.

Cost-effective in-house COVID-19 reverse transcription-polymerase chain reaction testing with yeast-derived Taq polymerase.

Despite the decline of the COVID-19 pandemic, there continues to be a persistent requirement for reliable testing methods that can be adapted to future outbreaks and areas with limited resources. While the standard approach of using reverse transcription-polymerase chain reaction (RT-PCR) with Taq polymerase is effective, it faces challenges such as limited access to high-quality enzymes and the presence of bacterial DNA contamination in commercial kits, which can impact the accuracy of test results. This study investigates the production of recombinant Taq polymerase in yeast cells and assesses its crude lysate in a multiplex RT-PCR assay for detecting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRP) and N genes, with human Ribonuclease P serving as an internal control. The unpurified yeast Taq polymerase demonstrates sensitivity comparable to commercially purified bacterial Taq polymerase and unpurified bacterial counterparts in detecting the RdRP and N genes. It exhibits the highest specificity, with 100% accuracy, for the N gene. The specificity for the RdRP gene closely aligns with that of commercially purified bacterial Taq polymerase and unpurified bacterial Taq polymerase. The use of unpurified recombinant yeast Taq polymerase shows promise as a cost-effective approach for conducting in-house COVID-19 RT-PCR testing. By eliminating the need for chromatography purification steps, the production of RT-PCR kits can be streamlined, potentially improving accessibility and scalability, especially in resource-limited settings and future pandemics.

A Practical, Scalable Synthesis of 1,18‐Octadecanedioic acid‐Paclitaxel (ODDA‐PTX)

AbstractPaclitaxel is one of the most important chemotherapy agents and it has shown activity against a variety of human cancers. In the past years several derivatives and formulations have been developed to improve its activity and reduce its toxicity. Recently, 1,18‐Octadecanedioic acid‐Paclitaxel (ODDA‐PTX), obtained by conjugation of paclitaxel with a long‐chain diacid, has emerged as a promising paclitaxel prodrug, with high efficacy and low toxicity. A novel strategy for its synthesis has been developed and is herein described. The approach is based on the mono‐allyl protection of octadecanedioic acid (ODDA) and on the Pd‐catalyzed removal of the allyl ester after coupling with paclitaxel. This strategy is superior to previously reported procedures in that it affords the ODDA‐PTX product with much higher purity, avoids chromatographic purifications and can be practically scaled‐up for the industrial production of ODDA‐PTX.

Comparative Analysis of Purification and Characterization Process of Phycocyanin Pigment from Spirulina Subsalsa with Arthrospira Platensis from Mexico

The Arthrospira maxima, Spirulina major, and Spirulina subsalsa are plenty available in Mexican lakes and ponds. This cyanobacterium is used as an important dietary supplement due to its high protein content and vitamins. However, the most commercial strain in use is A. platensis, and very few reports exist on other Spirulina species from Mexico and their potential phycocyanin production. To use at a commercial scale, it is essential to evaluate native strains in an efficient purification process. In this work, we demonstrated the potential of Spirulina subsalsa for phycocyanin pigment production and purification process, compared with native A. platensis from Mexico. The biomass yield with S. subsalsa was 4.50±0.10 g/Lt, which was higher than A. platensis, which was 1.36 g/lt. The crude phycocyanin pigment from S. subsalsa was 267±0.37 mg/g, whereas 221±0.04mg/g in the case of A. platensis. After partial purification, we found a higher purity grade in S. subsalsa, with a higher yield and a recovery percent of 39.75%. In the case of A. platensis, after partial purification, the recovery percentage was 35.37. After ionic chromatography purification, the purity of phycocyanin was 2.57±0.03 in the case of S. subsalsa, whereas 1.91±0.02 in the case of A. platensis. SDS gel electrophoresis demonstrated two phycocyanin bands showing 17 kDa and 12 kDa in the case of both species. The two-step purification process functions very well for S. subsalsa species, whereas A. platensis demonstrated efficiency with one step. The extracted C-PC from S. Sulsalsa is safe as per the cytotoxicity assay.

Cryogels and Monoliths: Promising Tools for Chromatographic Purification of Nucleic Acids.

The increasing demand for highly pure biopharmaceuticals has put significant pressure on the biotechnological industry to innovate in production and purification processes. Nucleic acid purification, crucial for gene therapy and vaccine production, presents challenges due to the unique physical and chemical properties of these molecules. Meeting regulatory standards necessitates large quantities of biotherapeutic agents of high purity. While conventional chromatography offers versatility and efficiency, it suffers from drawbacks like low flow rates and binding capacity, as well as high mass transfer resistance. Recent advancements in continuous beds, including monoliths and cryogel-based systems, have emerged as promising solutions to overcome these limitations. This review explores and evaluates the latest progress in chromatography utilizing monolithic and cryogenic supports for nucleic acid purification.

Novel purification platform based on multimodal preparative scale separation of mAb fragments and aggregates

Monoclonal antibodies (mAbs) continue to dominate the biopharmaceutical industry. Certain mAbs are prone to fragmentation and clipping and in these cases, adequate removal of these species is critical during manufacturing. Fragments can be generated during fermentation, purification, storage, formulation, and administration. Their addition to the acidic charge-variant of the purified mAb has been reported to decrease stability and potency of the final product. However, contrary to mAb aggregation, manufacturers have not given much attention to removal of fragments and clipped species and as a result most conventional mAb platforms offer at best limited capabilities for their removal. In this study, we propose a novel purification platform that uses multimodal chromatography and achieves complete removal of a range of mAb fragments and clipped products (25–120 kDa). The utility of the platform has been successfully demonstrated for 2 IgG1s and 2 IgG4s. Further, adequate removal of the various host cell impurities such as host cell proteins (<10 ppm) and host cell DNA (<5 ppb) has been achieved. Finally, the platform was able to deliver adequate removal of high molecular weight impurities (<1 %) and a 30 % clearance of the acidic charge variant. The proposed single step has been shown to deliver what the polishing chromatography and intermediate purification chromatography steps deliver in a traditional mAb platform.

Energy‐Efficient Synthesis of Haloquinazolines and Their Suzuki Cross‐Coupling Reactions in Propylene Carbonate

AbstractComparative synthesis of some new quinazoline derivatives via tetrakis(triphenylphosphine)palladium(0) catalysed Suzuki‐Miyaura cross‐coupling reaction in “green” propylene carbonate (PC) and dimethoxyethane. Three haloquinazolines were synthesised for Suzuki‐cross‐coupling from 5‐bromoanthranilic acid. Sufficient and easy method for the elimination of PC is developed, thus the optimization of the synthesis of some quinazoline derivatives was carried out. Reactions with 2‐chloro‐6‐bromo‐3‐phenylquinazolin‐4(3H)‐one proceeded faster and with a higher yield in PC than in dimethoxyethane, thus for some reactants PC is a good choice for greener synthesis. Practical considerations for using PC include shorter reaction times, improved yields, and the elimination of unnecessary solvent distillation, leading to significant energy savings. This facilitates the execution of energy‐efficient reactions in a notably environmentally friendly solvent. In contrast to DME, the use of PC does not necessitate special pre‐treatments. Moreover, less PC is required due to its high boiling point and low volatility. Considering these properties, coupled with the consistent need for chromatographic purification, the use of PC in the Suzuki‐Miyaura cross‐coupling reaction emerges as a superior and greener alternative compared to DME.

Preparation and identification of novel DPP-IV inhibitory peptides from Musculus senhousei: Peptidomic analysis, molecular simulation, and validation

Bioactive peptides have been considered effective alternatives for the treatment of type 2 diabetes targeting the dipeptidyl peptidase-IV (DPP-IV). In this study, novel DPP-IV inhibitory peptides were prepared and identified from Musculus senhousei through two-stage chromatographic purification, peptidomic analysis, in silico screening, and validation. Furthermore, molecular simulation was employed to analyze the interaction from a molecular perspective. Results showed that Musculus senhousei hydrolysate produced by 8 h Neutrase hydrolysis exhibited the significant DPP-IV inhibitory activity. Purification and identification led to the discovery of 387 peptide sequences. A total of 11 novel peptides with potential DPP-IV inhibitory activity were screened in silico. Further synthesis and validation of peptide activity showed that LTWR and DPF significantly inhibit DPP-IV in a competitive inhibitory manner, with IC50 values of 1788.67 ± 28.13 and 1399.73 ± 27.15 μM, respectively. Results from molecular docking and dynamic simulations indicated that peptides LTWR and DPF could tightly bind to the catalytic site of DPP-IV through hydrogen-bond and hydrophobic interaction. These findings suggested that Musculus senhousei could serve as a natural source of bioactive peptides for potential treatment of type 2 diabetes.

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.

Trinucleotide mRNA Cap Analogue N6-Benzylated at the Site of Posttranscriptional m6Am Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo.

Eukaryotic mRNAs undergo cotranscriptional 5'-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6Am─a common epitranscriptomic mark unique to the mRNA 5'-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N6-benzyl analogue of the m6Am-cap-m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped transcripts. The Bn6Am moiety was shown to act as a reversed-phase high-performance liquid chromatography (RP-HPLC) purification handle, allowing the separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell-line-dependent. m7GpppBn6AmpG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic settings as anticancer vaccines. The biochemical characterization suggests several phenomena potentially underlying the biological properties of AvantCap: (i) reduced propensity for unspecific interactions, (ii) involvement in alternative translation initiation, and (iii) subtle differences in mRNA impurity profiles or a combination of these effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of m6Am in mRNA.

Purification technology design, biochemical and immunological characteristics of the recombinant chimeric antigen for evaluation of T cell immunity against coronavirus infection

Diagnosis of specific T cell immunity to the antigenic determinants of SARS-CoV-2 in patients seems to be an increasingly important task due to accumulation of data about the role of T cell immune response in course of coronavirus infection and clearance of SARS-CoV-2 in case of secondary infection. Previously, we designed the recombinant CorD_PS antigen for evaluation of T cell antiviral immunity, containing conservative and immunogenic sequences of structural proteins of the SARS-CoV-2 coronavirus. E. coli CorD_PS producing strain with stable expression of the recombinant CorD_PS antigen was obtained. Aim of the present work is to design a laboratory technology for the production of recombinant antigen CorD_PS, to control the quality of the obtained chimeric protein and to study its immunological activity. Development of the cultivation conditions for E. coli CorD_PS cells was carried out in conical flasks in LB-M_Km medium at 37 °C, then scaled in a fermenter with a volume of 30 liters. Expression was induced by the addition of IPTG. Expression was controlled in culture lysates in 12% SDS-PAGE. The resulting biomass was lysed using an ultrasonic disintegrator with followed by centrifugation. The compositions of lysing and solubilizing buffers were selected, as well as conditions for refolding of the recombinant protein. Cation exchange (SP-sepharose), hydrophobic (Butylsepharose) and exclusive (Sephacryl S-200 HR) chromatography were used sequentially to purify the dissolved protein. Protein impurities in the preparation were determined by reverse-phase HPLC and electrophoresis in 12% SDS-PAGE, residual lipopolysaccharides were determined using a gel-thrombin variant of the LAL test, residual proteins of the producer strain were determined by enzyme immunoassay, residual DNA of the producer strain was determined by binding with PicoGreen dye. Specificity was controlled by indirect enzyme immunoassay. The evaluation of cytokine production by CD4+T lymphocytes in response to their stimulation by recombinant antigen ex vivo was performed on a flow cytofluorimeter. The biomass yield during cultivation of E. coli CorD_PS in a 30L fermenter was up to 20 g/L for 4 hours of 0.1 mM IPTG induction. Sequential washing of inclusion bodies from bacterial cellular components and their subsequent solubilization in a buffer containing 8 M urea allowed to obtain a solution of denatured antigen with a concentration of 10 mg/mL. The efficiency of refolding by dilution was 75%. After three stages of chromatographic purification, protein samples with a concentration of 1.2-1.4 mg/mL, HPLC purity of 98.43%, corresponding to key quality parameters according to the OFS.1.7.1.0007.15, were obtained. The recombinant antigen showed specific binding to a sample of the First WHO International Standard and sample Company Reference Standard No. 3 of anti- SARS-CoV-2 human immunoglobulins in a specified concentration range. CD4+T lymphocytes effectively responded to recombinant antigen treatment by increasing IFNγ production. The optimal concentration of recombinant coronavirus antigen was 5 μg/mL. The developed technological process makes it possible to obtain 5-7 grams of coronavirus recombinant CorD_PS antigen in one cultivation cycle in 30 liters of fermentation medium with key quality parameters according to the OFS.1.7.1.0007.15. As a result of specific immunological activity studies of the recombinant coronavirus antigen CorD_PS, the concept of its possible use as a diagnostic tool for determining the formation of a T cell immune response was confirmed.

Process Development and Scale-Up of a Novel Atypical DAT Inhibitor (S)-CE-123.

Large-scale syntheses of small molecules and kilo laboratories are crucial steps in drug development, especially in advanced stages. (S)-5-((Benzhydrylsulfinyl)methyl)thiazole, (S)-CE-123, a potent, selective, and novel atypical DAT inhibitor, has undergone iterative testing as part of the preclinical evaluation step. This required the process transfer, scale-up, and synthesis of a 1 kg preclinical batch. The Kagan protocol for asymmetric sulfide to sulfoxide oxidation was successfully applied within a four-step synthetic process for the successful upscaling of (S)-CE-123. During the scale-up of the last step, several changes were made to the original synthetic procedure, as with every increase in batch size, new problems had to be overcome. These include, among others, the workup optimization of the last step, the simplification of chromatographic purification, elution modification to improve the purity of the product and saving of workup time. Two washing steps were added to the original procedure to enhance both the yield and the enantiomeric excess value of the final product. The modifications introduced allowed access to a 1 kg (S)-CE-123 batch with a purity >99% and an enantiomeric excess value of 95%.

Abstract 2090 Bacterial expression and chromatography purification of a human period 2 domain

Abstract 2090 Bacterial expression and chromatography purification of a human period 2 domain

Novel anti-breast cancer unsaturated fatty acids isolated from Nigella sativa L.

Nigella sativa L. has been used for thousands of years as a remedy for various disorders due to its broad spectrum pharmacological action including anticancer, antifungal, anthelminthic, antidiabetic, anti-inflammatory, antioxidant, etc. A methanolic extract was prepared using the seeds of Nigella sativa L. Column chromatographic purification using silica gravity column using the methanolic extract led to the isolation of two compounds. The structures of the compounds were elucidated using NMR (1H, 13C, HSQC, HMBC) and HRMS spectral data. The isolated compounds were found to be (9Z,12Z)- 17- propyloctadeca- 9,12-diene-1,3,7,18- tetraol(I) and (9Z,12Z,15Z)-17-hydroxy- (hydroxymethyl)- 7-((Z)- 10-iododec-6-en-1-yl) heptadeca-9,12,15-trienoic acid(II). The isolated compounds were tested for anticancer properties and both of them showed promising results (4.69± 0.09 μg/ml and 9.88± 0.17 μg/ml respectively) in MDA-MB-231 cell lines.

PTSA‐Catalyzed [3+2] Cycloaddition of in Situ Generated 3‐Ylidene Oxindoles with Coumarin‐Based N‐Phenyl Enamine Derivatives

AbstractBrønsted acid mediated [3+2] cycloaddition of coumarin‐based enamines with 3‐ylidene oxindoles generated in situ from 3‐hydroxy‐3‐aroylmethyl oxindoles enabled the synthesis of polyheterocyclic compounds with a pyrrolocoumarin core structure in a highly regioselective manner. The synthetic procedure is highly efficient and metal‐free, and no chromatographic purification of the products is required. The chromenopyrrole derivatives bearing an oxindole moiety formed as rapidly interconverting rotamers.

Open-source milligram-scale, four channel, automated protein purification system.

Liquid chromatography purification of multiple recombinant proteins, in parallel, could catalyze research and discovery if the processes are fast and approach the robustness of traditional, "one-protein-at-a-time" purification. Here, we report an automated, four channel chromatography platform that we have designed and validated for parallelized protein purification at milligram scales. The device can purify up to four proteins (each with its own single column), has inputs for up to eight buffers or solvents that can be directed to any of the four columns via a network of software-driven valves, and includes an automated fraction collector with ten positions for 1.5 or 5.0 mL collection tubes and four positions for 50 mL collection tubes for each column output. The control software can be accessed either via Python scripting, giving users full access to all steps of the purification process, or via a simple-to-navigate touch screen graphical user interface that does not require knowledge of the command line or any programming language. Using our instrument, we report milligram-scale, parallelized, single-column purification of a panel of mammalian cell expressed coronavirus (SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-229E) trimeric Spike and monomeric Receptor Binding Domain (RBD) antigens, and monoclonal antibodies targeting SARS-CoV-2 Spike (S) and Influenza Hemagglutinin (HA). We include a detailed hardware build guide, and have made the controlling software open source, to allow others to build and customize their own protein purifier systems.