Initial Purification Step Research Articles

Fractionation and purification of a high-impurity, alkaline-pretreated, corn stover lignin with simple renewable solvents

Corn stover has been identified as a key resource in the transition from petrochemicals to sustainably sourced energy and materials; however, the as-recovered lignin from alkaline pretreatment contains unusually high levels of residual impurities, which must be removed to maximize potential application. Thus, the Aqueous Lignin Purification using Hot Agents (ALPHA) process was applied to a corn stover lignin containing ∼10 % carbohydrates and ∼3 % ash. Lignin fractions ∼100 times cleaner than the feed lignin and of controlled molecular weight (i.e., as low as one-fifth and as high as triple the absolute molecular weight (Mw) of the feed) were obtained. The ALPHA solvent was a hot (60 or 75 °C) solution of 80 % ethanol combined with the feed lignin at an unusually low, 3:1 solvent-to-feed lignin ratio – conditions that create the liquid–liquid equilibrium (LLE) phase split needed to practice ALPHA. Lignin fractionation then commenced by adding water to the original solution, decreasing the ethanol content in increments of 3–4 wt%. Surprisingly, the highest Mw fractions did not precipitate until the solution contained just below 50 % ethanol, by which point almost half of the starting lignin of near-average Mw had already precipitated. Another unexpected result was that the lignin waste stream from the initial purification step could be increased from 40 to 98 % lignin in a single equilibrium stage simply by re-processing that waste with another 80 % ethanol solution at the conditions given above. In summary, the efficacy of ALPHA for purifying and fractionating an impurities-laden corn stover lignin to ultraclean levels in a scalable manner requiring only aqueous bioethanol solutions has been demonstrated.

A simple process to purify (E)-resveratrol from grape stems with a photo-molecularly imprinted sorbent

The present work focused on the development of a process of sorption-desorption to purify (E)-resveratrol found in winemaking residues, specifically from the crude extract of grape stems. The core element of this process is a photo-molecularly imprinted sorbent (MIS) synthesized by means of the molecular imprinting technique (MIT). This sorbent is a 4-vinylpyridine rich polymer network which binds (E)-resveratrol at stereospecific sites. Comparison of sorption isotherms of the MIS and its counterpart non-imprinted sorbent (NIS) shows the higher capability of the MIS to bind (E)-resveratrol. It is noteworthy that the size of MIS particles was larger than to those of the NIS (as found by scanning electron microscopy analysis) facilitating its use in sorption-desorption processes without reaching a high backpressure. The MIS was then used to purify (E)-resveratrol from grape stems extract in a two-step purification procedure through a simple device. The initial purification step permitted to increase the purity of (E)-resveratrol from 29% to 78% (34% of recovery), whereas a further one took it to 87% (70% of recovery). The purified fractions were obtained in hydroalcoholic medium and contained other bioactive stilbenes such as (E)-Ɛ-viniferin. The results of this research provide a compelling proof of concept for room-temperature photopolymerization, enabling the development of highly efficient imprinted polymers as sorbents for purifying bioactive compounds from agricultural residues. This significant advancement paves the way for its large-scale implementation in a circular bioeconomy approach.

An efficient capture strategy for the purification of human adenovirus type 5 from cell lysates

An efficient capture step for human adenovirus type 5 from cell lysate was developed as an initial virus purification step from cell debris supernatant. Organosilane-based polymer particles were synthesized and experimental monomer screening allowed the selection of appropriate functionalities for the development of particles for virus binding. After elution, virus recoveries of 83 % were obtained with significant reduction of matrix proteins and residual host cell DNA. Therefore, the implemented capture strategy for adenovirus via polymer particles provides a scalable and reproducible approach to reduce time and cost during virus purification processes.

Enhanced Production of ECM Proteins for Pharmaceutical Applications Using Mammalian Cells and Sodium Heparin Supplementation.

The yields of soluble ECM proteins recombinantly produced with mammalian cells can be significantly enhanced by exploiting the stabilizing properties of heparin. Here, we propose a simple and straightforward scalable protocol for the mammalian cell production of ECM proteins with affinity for heparin, using heparin as a supplement. As proof of concept, we have demonstrated the high-level expression of four biomedically relevant human enzymes such as carboxypeptidase Z (CPZ), carboxypeptidase A6 (CPA6), beta-galactoside alpha-2,6-sialyltransferase 2 (ST6GAL1) and thrombin-activable fibrinolysis inhibitor (TAFI). We found a strong linear correlation between the isoelectric point (pI) of a protein and the improvement in protein expression levels upon heparin addition, providing a reference for selecting novel protein targets that would benefit from heparin supplementation. Finally, we demonstrated the compatibility of this approach with a three-step purification strategy that includes an initial heparin affinity purification step. Using CPZ as a representative example, we performed a preparative purification of this enzyme. The purified protein is enzymatically active and can be used for pharmaceutical applications as well as for high-throughput functional and structural studies.

Extraction, Purification and Characterization of Transglutaminase from Silver Beet Leaves and its Effect on Sensory, Chemical and Rheological Properties of Kareish Cheese

Strategy in transgluaminase (TGase) extracted from silver beet leaves (Beta vulgaris L.) performed by three stages. Ammonium sulfate precipitation (40-80%) is an important technique as an initial step in TGase purification, followed by an impure TGase sample is loaded into 2-(Diethylamino)ethyl ether sephadex A50. The final purification step was carried out by eluting dialyzed fractions into Sephadex G-100. The purified TGase was successfully purified by three purification steps with 224.2 U/mg of TGase specific activity, 3.4 fold increase in purification and 20.33% yields. The optimum temperature and pH of silver beet leaves TGase were 50°C and 5.0, respectively. The TGase completely lost its activity after 15 min at 80°C, however recovery of TGase after exposure to sodium chloride concentrations ranging from 2-6% does not affect its activity. The influence of different concentrations (5, 7.5 and 10U/g protein) of TGase on chemical, textural and sensorial of Kareish over storage time were detected. The yield and moisture content were reached the maximum values in cheese treated with TGase (10Ug-1protein), in addition to the same treatment received maximum average scores in sensory evaluation. The concentration of TGase is negatively proportional with the gumminess, adhesiveness and hardnesss values of resulted cheese.

Quality Screening of Incorrectly Folded Soluble Aggregates from Functional Recombinant Proteins

Solubility is the prime criterion for determining the quality of recombinant proteins, yet it often fails to represent functional activity due to the involvement of non-functional, misfolded, soluble aggregates, which compromise the quality of recombinant proteins. However, guidelines for the quality assessment of soluble proteins have neither been proposed nor rigorously validated experimentally. Using the aggregation-prone enhanced green-fluorescent protein (EGFP) folding reporter system, we evaluated the folding status of recombinant proteins by employing the commonly used sonication and mild lysis of recombinant host cells. We showed that the differential screening of solubility and folding competence is crucial for improving the quality of recombinant proteins without sacrificing their yield. These results highlight the importance of screening out incorrectly folded soluble aggregates at the initial purification step to ensure the functional quality of recombinant proteins.

Impact of temperature and pH on recombinant human IgM quality attributes and productivity

IgM antibodies are arousing considerable interest as biopharmaceuticals. Despite their immunotherapeutic potential, little is known about the impact of environmental conditions on product quantity and quality of these complex molecules. Process conditions influence the critical quality attributes (CQAs) of therapeutic proteins and thus are important parameters for biological safety and efficacy. Here, the results of a systematic study are presented that characterized the influence of temperature and pH on cell-specific productivity and IgM quality attributes. Biphasic temperature and pH shift experiments were performed as batch cultures in DASGIP® bioreactors under controlled conditions and defined by a specific design of experiment (DOE) approach. An internally-developed recombinant IgM producing CHO cell line was used. With respect to product quality, after an initial purification step efforts were focused on pentamer content, nucleic acid (NA) impurities and the glycosylation profile after an initial purification step. All quality attributes were evaluated by densitometric and chromatographic methods. The reduction of cultivation temperature severely reduced IgM titers, while pH variation had no impact. In contrast, IgM quality was not significantly influenced by bioprocessing parameters. Data revealed that an additional purification step is required to reduce the presence of NAs for in vivo applications. In conclusion, the results showed that for the chosen IgM model, IgM012_GL, variation in quality attributes is not caused by the environmental conditions of temperature and pH.

The Application of Dextran Sedimentation as an Initial Step in Neutrophil Purification Promotes Their Stimulation, due to the Presence of Monocytes.

The purification of human neutrophils for in vitro studies is challenging as they are easily activated through ex vivo manipulations. The technique of erythrocyte sedimentation combined with density gradient centrifugation remains widely practiced and was the subject of this study. Since in the sedimentation step the leukocytes are incubated with dextran, we have raised the likelihood that cellular activation would occur with mediator release leading to neutrophil activation. By comparing the activity of neutrophils purified from whole blood by the classical 2-step method of dextran sedimentation followed by low-density Ficoll-Hypaque (1.077 g/mL) medium, and the 1-step high-density Ficoll-Hypaque (1.114 g/mL) gradient centrifugation, we found that neutrophils from the 2-step method had a significant increase in cell surface CD11b expression and CD62L shedding and a marked increase in adhesion. Decreased random migration and chemotaxis and raised baseline oxidative burst activity were also observed. The effect was not specific to dextran, as using Ficoll for erythrocyte sedimentation replicated the elevated neutrophil adherence. Through the depletion of monocytes, lymphocytes, and platelets prior to sedimentation, we deduced that monocytes were responsible for the neutrophil activation. Our findings suggest that care needs to be exercised in choosing the method of neutrophil purification for functional studies.

Bio-assisted potentiometric multisensor system for purity evaluation of recombinant protein A

Recombinant proteins became essential components of drug manufacturing. Quality control of such proteins is routine task, which usually requires a lot of time, expensive reagents, specialized equipment and highly educated personnel. In this study we propose a new concept for protein purity evaluation that is based on application of bio-assisted potentiometric multisensor system. The model object for analysis was recombinant protein A from Staphylococcus aureus (SpA), which is commonly used for monoclonal antibody purification. SpA solutions with different amount of host cell related impurities (Escherichia coli, bacterial lysate) were analyzed. Two different bio-transducers were employed: proteinase K from Tritirachium album and baker's yeast Saccharomyces cerevisiae. It was shown that both bio-transducers are able to induce changes in pure and lysate-contaminated SpA samples. Different products of yeast digestion and proteolysis with proteinase of pure SpA and lysate were detected with size exclusion high-performance liquid chromatography (SE-HPLC). The induced changes of chemical composition are detectible with potentiometric multisensor system and can be related to SpA purity through projection on latent structures (PLS) regression technique. The proposed method allows for estimation of the impurity content with 12% accuracy using proteinase K and 16% accuracy using baker's yeast. The suggested approach could be useful for early contamination warning at initial protein purification steps. The analysis requires no expensive materials and equipment, no bio-material immobilization, and its duration time is comparable with other commonly used methods like chromatography or electrophoresis though the main part of this time is related to the sample preparation.

At-line mid infrared spectroscopy for monitoring downstream processing unit operations

At-line mid infrared spectroscopy (FT-MIR) was used for monitoring monoclonal antibody (mAb) titer, host cell protein levels and antibody aggregate amount in samples, originating from different downstream processing unit operations, performed during technical laboratory scale development runs. Antibody aggregate quantification was possible down to 1% [w/w], while host cell protein levels could be monitored down to 700ngml−1. Antibody titer could be determined with high accuracy for samples >0.7gL−1, with the possibility to further increase sensitivity when using samples with highly similar matrix. Comparing these results to relevant levels during downstream processing, the most promising applicability of FT-MIR is within target protein (mAb) monitoring, while HCP monitoring following intermediate and polishing chromatography steps is currently not a well-suited application. FT-MIR for aggregate monitoring is more suitable for steps resulting in higher aggregate levels such as pH stress and shear stress- related purification steps. Additionally, FT-MIR might be considered for monitoring of samples with higher HCP and aggregate amount profiles, which can occur during initial purification steps and when using alternative purification methods such as precipitation.

A cleavable ligand column for the rapid isolation of large quantities of homogeneous and functional neurotensin receptor 1 variants from E. coli

G protein-coupled receptors (GPCRs) are key players of cell signaling, thus representing important drug targets for the treatment of human diseases. Since inherent difficulties in receptor production and handling have precluded the application of many in vitro experiments, major questions about GPCR mechanisms and dynamics remain elusive to date. We recently used directed evolution in Escherichia coli on neurotensin receptor 1 (NTR1) for the generation of GPCR variants with greatly elevated functional expression levels and with excellent stability in detergent micelles. In this work we outline a highly efficient purification method for our evolved receptor variants, which is based on the application of an inexpensive, disposable high-affinity ligand column as the initial purification step. The ligand resin allows isolation of correctly folded GPCR variants directly from whole E. coli cell lysates at the scale of 10mg and it permits preparations of agonist- and antagonist-bound receptor samples. The purification principle presented here was key to the first structures of signaling-active NTR1 variants (Egloff et al., 2014). Since E. coli is uniquely suitable for the production of fully deuterated proteins, our method provides the basis for an array of NMR experiments that were not feasible for GPCRs to date, but which will shed light on novel aspects of receptor function and dynamics.

Scaled-up manufacturing of recombinant antibodies produced by plant cells in a 200-L orbitally-shaken disposable bioreactor.

Tobacco BY-2 cells have emerged as a promising platform for the manufacture of biopharmaceutical proteins, offering efficient protein secretion, favourable growth characteristics and cultivation in containment under a controlled environment. The cultivation of BY-2 cells in disposable bioreactors is a useful alternative to conventional stainless steel stirred-tank reactors, and orbitally-shaken bioreactors could provide further advantages such as simple bag geometry, scalability and predictable process settings. We carried out a scale-up study, using a 200-L orbitally-shaken bioreactor holding disposable bags, and BY-2 cells producing the human monoclonal antibody M12. We found that cell growth and recombinant protein accumulation were comparable to standard shake flask cultivation, despite a 200-fold difference in cultivation volume. Final cell fresh weights of 300-387 g/L and M12 yields of ∼20 mg/L were achieved with both cultivation methods. Furthermore, we established an efficient downstream process for the recovery of M12 from the culture broth. The viscous spent medium prevented clarification using filtration devices, but we used expanded bed adsorption (EBA) chromatography with SP Sepharose as an alternative for the efficient capture of the M12 antibody. EBA was introduced as an initial purification step prior to protein A affinity chromatography, resulting in an overall M12 recovery of 75-85% and a purity of >95%. Our results demonstrate the suitability of orbitally-shaken bioreactors for the scaled-up cultivation of plant cell suspension cultures and provide a strategy for the efficient purification of antibodies from the BY-2 culture medium.

Purification and characterization of a soluble calnexin from human placenta

Calreticulin (Crt) and calnexin (Cnx) are homologous endoplasmic reticulum (ER) chaperones involved in protein folding and quality control. Crt is a soluble ER luminal Mr 46kDa protein and Cnx is a Mr 67kDa ER membrane protein. During purification of Crt from human placenta a soluble form of Cnx (sCnx) was consistently identified in a separate ion exchange chromatography peak. The sCnx was further purified and characterised. This showed that the protein had been cleaved after residue 472 (between Gln and Met), thus liberating it from the transmembrane and cytoplasmic parts of Cnx. The extraction and initial purification steps were carried out in the presence of protease inhibitors, thus ruling out that the cleavage was an artefact of the isolation procedure. This indicates that sCnx may have a physiological chaperone function similar to that of Crt.

Stable Emulsions in Biphasic Whole‐Cell Biocatalysis: The Mechanism of scCO2‐Assisted Phase Separation

complete phase separation, enabling a further workup of the product-containing organic phase. It could be shown, that the whole cells present in the system are mainly responsible for the enhanced stability of these emulsions (over 93 % on total emulsion stability), and are stabilizing the emulsions by a Pickering type mechanism. To further investigate the underlying mechanism, different influence factors on the cell surface hydrophobicity were investigated. The results point to the fact that scCO2 triggers the surface hydrophobicity of the cells by irreversible desorption of surface associated macromolecules. The mechanism responsible for scCO2-assisted phase separation as well as the formation of these stable emulsions could be clarified for the first time ever. Understanding of the underlying mechanisms now allows for a precise definition of a process window for phase separation and thus the initial purification step. This enables the development of integrated bioprocesses by adjustment of both biotransformation and downstream processing parameters.

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 μg or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 μl of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate). Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.(1,2,3) This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.(4,5) The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD(260/280;) of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 μg or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 10(6) PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 10(8) plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.(6) For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 μl of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate). Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

Reducing Acyl Migration during Purification of 2-Arachidonoylglycerol from Biological Samples before Gas Chromatography Mass Spectrometry Analysis

Endocannabinoid 2-arachidonoylglycerol (2-AG) regulates several important physiological processes in the brain. 2-AG is commonly quantified by gas chromatography mass spectrometry after an initial purification step. The most precise and rapid purification utilizes C(18) solid-phase extraction, but quantification problems can arise with acyl migration from 2-AG to 1-arachidonoylglycerol. We found that extraction with methanol promoted this migration, but acetone and diethyl ether (Et(2)O) did not. Acetone and Et(2)O were used to develop a purification method for the direct determination of 2-AG.

Rapid method for extracting the antibiotic mutacin 1140 from complex fermentation medium yeast extract

The peptide antibiotic mutacin 1140 belongs to the group of antibiotics called lantibiotics. They are ribosomally synthesized and undergo extensive enzymatic modifications before being excreted into the culture medium. By using reverse-phase-high-performance liquid chromatography (RP-HPLC) and a semiquantitative bacteriocin bioassay to track mutacin 1140, an efficient ammonium sulfate (AS) precipitation method has been developed for removing mutacin 1140 from a complex medium containing 5% yeast extract. This method minimizes the amounts of fermentation by-products and media components that make downstream purification processes more difficult and economically infeasible. The method may be adaptable for the initial purification step of other lantibiotics. A threefold decrease in the precipitation of the medium components found in yeast extract, at pH 8.0 vs. pH 2.0, may have broad utility for the isolation of secondary metabolites produced in this complex medium. The average yield of mutacin 1140 from the fermentation medium was determined as 66%.

In this issue

AbstractAntibody humanizationRuuls et al., Biotechnol. J. 2008, 3, 1157–1171Monoclonal antibodies (mAbs) are increasingly used to treat human diseases, but as they are predominantly generated from mouse hybridomas their application has been hampered by their immunogenecity. To minimize antigenic reactions, researchers from Genmab in The Netherlands established a transgenic mouse platform for the routine production of fully humanized mAbs. In an overview of recent clinical research, the authors describe efficacy and mechanism of action of three novel humanized mAbs derived from this platform. Zanolimumab, Ofatumumab and Zalutumumab are designed to target CD4, CD20 and the epidermal growth factor receptor overexpressed on cancer cells and to attract cytotoxic reactions against them. All three humanized mAbs show improved efficacy in treatment trials of T‐cell lymphomas, B‐cell leukemias and head and neck cancer.Antibody productionZhou et al., Biotechnol. J. 2008, 3, 1185–1200Innovations in several aspects of antibody production technology have driven the commercialization of human therapeutic antibodies in recent years. As antibodies are complicated molecules that need several modes of secondary modification, like for example glycosylation and disulfide bonds, specific challenges have to be met. Moreover, one of the most important aims is to minimize production costs by increasing product throughput while maintaining product quality. Researchers from Amgen Inc (CA, USA) here review essential considerations and trends for commercial process development and optimization. One of the recent technical advances is the development and implementation of a disposable Q membrane adsorber. It is expected that production costs will be reduced dramatically at all levels of process development for largescale antibody production in the coming years.Antibody stabilityGaridel et al., Biotechnol. J. 2008, 3, 1201–1211Steady‐state intrinsic tryptophan fluorescence spectroscopy is used as a rapid, robust and economic way for screening the thermal protein conformational stability in various formulations used during the early biotechnology development phase. The most important parameters affecting protein stability in a liquid formulation, e.g. during the initial purification steps or preformulation development, are the pH of the solution, ionic strength, presence of excipients and combinations thereof. A well‐defined protocol is presented for the investigation of the thermal conformational stability of proteins. Researchers from Boehringer Ingelheim in Germany have now re‐evaluated and re‐assessed the influence of solution conditions on the emission spectra of tryptophan, phenylalanine and tyrosine. They show the advantage, challenges and applicability of using intrinsic tryptophan fluorescence spectroscopy as a routine development method in pharmaceutical biotechnology.

A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy

Steady-state intrinsic tryptophan fluorescence spectroscopy is used as a rapid, robust and economic way for screening the thermal protein conformational stability in various formulations used during the early biotechnology development phase. The most important parameters affecting protein stability in a liquid formulation, e. g. during the initial purification steps or preformulation development, are the pH of the solution, ionic strength, presence of excipients and combinations thereof. A well-defined protocol is presented for the investigation of the thermal conformational stability of proteins. This allows the determination of the denaturation temperature as a function of solution conditions. Using intrinsic tryptophan fluorescence spectroscopy for monitoring the denaturation and folding of proteins, it is crucial to understand the influence of different formulation parameters on the intrinsic fluorescence probes of proteins. Therefore, we have re-evaluated and re-assessed the influence of temperature, pH, ionic strength, buffer composition on the emission spectra of tryptophan, phenylalanine and tyrosine to correctly analyse and evaluate the data obtained from thermal-induced protein denaturation as a function of the solution parameters mentioned above. The results of this study are a prerequisite for using this method as a screening assay for analysing the conformational stability of proteins in solution. The data obtained from intrinsic protein fluorescence spectroscopy are compared to data derived from calorimetry. The advantage, challenges and applicability using intrinsic tryptophan fluorescence spectroscopy as a routine development method in pharmaceutical biotechnology are discussed.