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

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

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

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

Comparison of separation modes for microchip electrophoresis of proteins.

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

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

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

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

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

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

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

Effect of Pressure Increase on Macromolecules' Adsorption in Ion Exchange Chromatography.

In this study a new method for evaluating the pressure effect on separations of oligonucleotides and proteins on an anion exchange column was developed. The pressure rise of up to 500 bar was attained by coupling restriction capillaries to the column outlet to minimize differences in pressure over the column. Using pH transient measurements it was demonstrated that no shift in ion exchange equilibria occurs due to a pressure increase. Results from isocratic and gradient separations of oligonucleotides (model compounds) were evaluated by stoichiometric displacement and linear gradient elution model, respectively. Both elution modes demonstrated that for smaller oligonucleotides the number of binding sites remained unchanged with pressure rise while an increase for large oligonucleotides was observed, indicating their alignment over the stationary phase. From the obtained model parameters and their pressure dependencies, a thermodynamic description was made and compared between the elution modes. A complementary pattern of a linear increase of partial molar volume change with a pressure rise was established. Furthermore, estimation of the pressure effect was performed for bovine serum albumin and thyroglobulin that required gradient separations. Again, a raise in binding site number was found with pressure increase. The partial molar volume changes of BSA and Tg at the maximal investigated pressure and minimal salt concentration were -31.6 and -34.4 cm3/mol, respectively, indicating a higher rigidity of Tg. The proposed approach provides an insight into the molecule deformation over a surface at high pressures under nondenaturing conditions. The information enables a more comprehensive UHPLC method development.

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

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

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

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

Dual metal cations coated magnetic mesoporous silica probe for highly selective capture of endogenous phosphopeptides in biological samples.

For the first time, dual metal ions (Ti4+-Zr4+) were successfully modified into the channel of magnetic mesoporous silica to obtain an affinity probe for highly selective capture of endogenous phosphopeptides in biological samples. The newly prepared Fe3O4@mSiO2@Ti4+-Zr4+ composites possessed the advantages of ordered mesoporous channels, superparamagnetism, and enhanced affinity properties of dual metal ions of Ti4+ and Zr4+. The phosphopeptide enrichment efficiency of the Fe3O4@mSiO2@Ti4+-Zr4+ composite was investigated, and the result indicated an ultrahigh size exclusive ability (weight ratio of ß-casein tryptic digests, BSA, and α-casein protein reached up to 1:1000:1000). Compared to magnetic affinity probes with single metal ions (Fe3O4@mSiO2@Ti4+, Fe3O4@mSiO2@Zr4+), the composite possessed stronger specificity, higher sensitivity, and better efficiency; and more importantly, it showed much enhanced enrichment ability towards both mono- and multi-phosphorylated peptides. Additionally, by utilizing the Fe3O4@mSiO2@Ti4+-Zr4+ affinity probe, a total number of 104 endogenous phosphopeptides including 95 mono-phosphopeptides and 9 multi-phosphopeptides were captured and identified from human saliva, indicating the great potential for the application of the novel probe for the peptidome analysis in the future. Graphic abstract.

Specific enrichment of phosphopeptides by using magnetic nanocomposites of type Fe3O4@graphene oxide and Fe3O4@C coated with self-assembled oligopeptides.

Iron(III-immobilized magnetic nano-composites (MNCs) were first fabricated using one-step aqueous self-assembly of oligopeptides (Glu-Pro-Ala-Lys-Ala-Lys-Ala-Lys; EPAK-VI) for the highly selective capture of phosphopeptides from complex biological samples. Under physiological conditions, EPAK-VI can readily self-organize into a robust and complete coating layer mainly composed of ß-sheets and ß-turns on the surface of Fe3O4@GO and Fe3O4@C MNCs. Tailored by the cyclic structure of proline, the Glu-Pro motifs of EPAK-VI are vertically erected on the surface and thus serve as an effective linker to chelate Fe3+ through carboxyl (COO-) group in the glutamic acid (E) residues. The ionic hydrogen bonds between the ε-amino groups and the surface negative charges coupled with intermolecular hydrogen bonds render the EPAK-VI coating on the MNCs insusceptible to repeated extreme washing conditions. The Fe3+-EPAK-VI coated MNCs exhibit high enrichment efficiency for ß-casein tryptic digest (0.05 fmol µL-1), excellent selectivity from mixed digests (ß-casein/bovine serum albumin, mass ratio 1:500), and high recovery rate (over 80%). Graphical abstractSchematic representation of the fabrication of Fe3+-immobilized MNCs for phosphopeptide enrichment.

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

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

Distribution of Protein Precipitation Capacity within Variable Proanthocyanidin Fingerprints.

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

QCQuan: A Web Tool for the Automated Assessment of Protein Expression and Data Quality of Labeled Mass Spectrometry Experiments.

In the context of omics disciplines and especially proteomics and biomarker discovery, the analysis of a clinical sample using label-based tandem mass spectrometry (MS) can be affected by sample preparation effects or by the measurement process itself, resulting in an incorrect outcome. Detection and correction of these mistakes using state-of-the-art methods based on mixed models can use large amounts of (computing) time. MS-based proteomics laboratories are high-throughput and need to avoid a bottleneck in their quantitative pipeline by quickly discriminating between high- and low-quality data. To this end we developed an easy-to-use web-tool called QCQuan (available at qcquan.net ) which is built around the CONSTANd normalization algorithm. It automatically provides the user with exploratory and quality control information as well as a differential expression analysis based on conservative, simple statistics. In this document we describe in detail the scientifically relevant steps that constitute the workflow and assess its qualitative and quantitative performance on three reference data sets. We find that QCQuan provides clear and accurate indications about the scientific value of both a high- and a low-quality data set. Moreover, it performed quantitatively better on a third data set than a comparable workflow assembled using established, reliable software.

An automated food protein isolation approach on preparative scale by two-dimensional liquid chromatography with active modulation interface.

In this work, an automated 2D-LC approach for protein isolation from egg samples on preparative scale is proposed. The method is based on the use of a C18 guard column installed in a switching valve to focus the proteins coming from the first dimension column, before their elution in the second column. For the first dimension separation, a size-exclusion column, packed with 3 µm ultrapure silica particles was used. An RP column based on core-shell technology was used for the second dimension separation. A standard mixture of BSA, ß-lactoglobulin, and glucose oxidase, chosen as a protein model system, was used to optimize the chromatographic separation conditions. The fully automated workflow allowed to isolate, in a single-chromatographic analysis, a protein amount of 50 µg for each peak fraction, with a total time of 15 min for the first separation and additional 30 min of the second separation for each trapped protein. The final aim was the development of proper analytical tools for protein isolation from foodstuffs to be used for the molecular identification by MS, as well as for biotherapeutic uses, allergy testing, and large-scale investigations in biological systems.

Temperature-Activated PEG Surface Segregation Controls the Protein Repellency of Polymers.

Poly(ethylene glycol) (PEG) is widely used to modulate the hydration states of biomaterials and is often applied to produce nonfouling surfaces. Here, we present X-ray scattering data, which show that it is the surface segregation of PEG, not just its presence in the bulk, that makes this happen by influencing the hydrophilicity of PEG-containing substrates. We demonstrate a temperature-dependent trigger that transforms a PEG-containing substrate from a protein-adsorbing to a protein-repelling state. On films of poly(desaminotyrosyl-tyrosine-co-PEG carbonate) with high (20 wt %) PEG content, in which very little protein adsorption is expected, quartz crystal microbalance data showed significant adsorption of fibrinogen and bovine serum albumin at 8 °C. The surface became protein-repellent at 37.5 °C. When the same polymer was iodinated, the polymer was protein-adsorbent, even when 37 wt % PEG was incorporated into the polymer backbone. This demonstrates that high PEG content by itself is not sufficient to repel proteins. By inhibiting phase separation either with iodine or by lowering the temperature, we show that PEG must phase-separate and bloom to the surface to create an antifouling surface. These results suggest an opportunity to design materials with high PEG content that can be switched from a protein-attractant to a protein-repellent state by inducing phase separation through brief exposure to temperatures above their glass transition temperature.

Preparation and evaluation of a green solvent-based molecularly imprinted monolithic column for the recognition of proteins by high-performance liquid chromatography.

A protein-based molecularly imprinted monolithic column was synthesized based on ionic liquids (ILs) and deep eutectic solvents (DESs) in a stainless steel column (50 mm × 4.6 mm id). An IL (1-allyl-3-butylimidazolium Br) and acrylamide were used as dual monomers. Another type of IL (1,2-bis [N,N'-vinylimidazolium] ethane bis Br) and N,N'-methylenebisacrylamide were used as dual cross-linking agents, and the DES (choline chloride : ethylene glycol 1 : 2) was used as a porogen in the preparation of a monolithic polymer. Bovine serum albumin (BSA) and lysozyme (Lyz), which differ greatly in molecular size, isoelectric point, and charge, were selected for imprinting templates to evaluate the recognition property of the green solvent-based MIP monolithic column. Some important factors, such as template-monomer molar ratio, total monomer concentration, and cross-linking density, were investigated systematically. Under optimal conditions, the MIP monolithic column obtained showed higher binding affinity for the templates than its corresponding non-imprinted (NIP) monolithic column.

A facile and general approach for the preparation of boronic acid-functionalized magnetic nanoparticles for the selective enrichment of glycoproteins.

Biomedical applications and biomarkers for early clinical diagnostics and the treatment of diseases demand efficient and selective enrichment platforms for glycoproteins. Thus, we herein report a facile and general approach for the preparation of boronic acid-functionalized magnetic nanoparticles for the selective enrichment of glycoproteins. More specifically, Fe3O4 magnetic nanoparticles were initially prepared via a solvothermal reaction, and core-shell-structured Fe3O4@SiO2 nanoparticles were obtained according to a sol-gel process. Subsequently, the Fe3O4@SiO2 surfaces were modified using 4-formylphenylboronic acid to allow the formation of strong yet reversible covalent bonds between boronic acid (BA) and the cis-1,2-diol groups of glycoproteins. The morphology and structure of the Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2-BA nanoparticles were characterized by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and vibrating sample magnetometry, thereby confirming their successful preparation. The obtained BA-modified Fe3O4@SiO2 magnetic nanoparticles were applied in the attempted enrichment of two glycoproteins (ovalbumin (OVA) and transferrin (TRF)) and two non-glycoproteins (bovine serum albumin (BSA) and cytochrome c (Cyt C)). The results confirmed a significant difference in affinity between glycoproteins and non-glycoproteins. In addition, the recognition capability of the Fe3O4@SiO2-BA nanoparticles was demonstrated by the selective enrichment of glycoproteins from a complex system containing both glycoproteins (i.e., TRF) and non-glycoproteins (i.e., Cyt C).

Fast reversed-phase liquid chromatographic separation of proteins by flow-through poly(styrene-co-divinylbenzene) microspheres.

With the explosive growth of the bioscience and biopharmaceuticals, the demand for high efficient analysis and separation of proteins is urgent. High-performance liquid chromatography is an appropriate technology for this purpose, and the stationary phase is the kernel to the separation efficiency. In this study, flow-through poly(styrene-co-divinylbenzene) microspheres characteristic of the binary pores, i.e. flow-through pores and mesopores, were synthesized; this special porous structure would benefit the convective mass transfer while guarantee the high specific surface area. Owing to the hydrophobic nature, poly(styrene-co-divinylbenzene) microspheres were suitable as the reversed-phase stationary phase for separation of proteins. For the high permeability of the poly(styrene-co-divinylbenzene) microspheres packed column, fast separation of the studied six proteins in ∼2 min was achieved. The recoveries of studied proteins were acceptable in the range of 79.0-99.4%. The proposed column had good pH stability of 1-13 and repeatability. Moreover, the column was applied for egg white fast separation, further demonstrating its applicability for complex bio-sample separation. The flow-through poly(styrene-co-divinylbenzene) microspheres were promising for fast separation of large molecules.

A microfluidic chip with a staircase pH gradient generator, a packed column and a fraction collector for chromatofocusing of proteins.

A microfluidic device for pH gradient chromatofocusing is presented, which performs creation of a micro-column, pH gradient generation, and fraction collection in a single device. Using a sieve micro-valve, anion exchange particles were packed into a microchannel in order to realize a solid-phase absorption column. To fractionate proteins according to their isoelectric points, elution buffer solutions with a stepwise pH gradient were prepared in 16 parallel mixing reactors and flowed through the micro-column, wherein a protein mixture was previously loaded. The volume of the column is only 20 nL, hence it allows extremely low sample consumption and fast analysis compared with a conventional system. We demonstrated separation of two proteins, albumin-fluorescein isothiocyanate conjugate (FITC-BSA) and R-Phycoerythrin (R-PE), by using a microcolumn of commercial charged polymeric particles (Source 15Q). The microfluidic device can be used as a rapid diagnostic tool to analyse crude mixtures of proteins or nucleic acids and determine adsorption/desorption characteristics of various biochemical products, which can be helpful for scientific fundamental understanding as well as instrumental in various industrial applications, especially in early stage screening and process development.