Small and Large Extracellular Vesicles of Porcine Seminal Plasma Differ in Lipid Profile.

Seminal plasma contains a heterogeneous population of extracellular vesicles (sEVs) that remains poorly characterized. This study aimed to characterize the lipidomic profile of two subsets of differently sized sEVs, small (S-) and large (L-), isolated from porcine seminal plasma by size-exclusion chromatography and characterized by an orthogonal approach. High-performance liquid chromatography-high-resolution mass spectrometry was used for lipidomic analysis. A total of 157 lipid species from 14 lipid classes of 4 major categories (sphingolipids, glycerophospholipids, glycerolipids, and sterols) were identified. Qualitative differences were limited to two cholesteryl ester species present only in S-sEVs. L-sEVs had higher levels of all quantified lipid classes due to their larger membrane surface area. The distribution pattern was different, especially for sphingomyelins (more in S-sEVs) and ceramides (more in L-sEVs). In conclusion, this study reveals differences in the lipidomic profile of two subsets of porcine sEVs, suggesting that they differ in biogenesis and functionality.

Collaborative study for the establishment of Human immunoglobulin (molecular size) BRP replacement batches 4, 5 and 6.

Human immunoglobulin products are used for the treatment of a number of diseases, such as primary or secondary immunodeficiencies and autoimmune conditions due to the complete absence of antibodies or the production of defective immunoglobulins. Quality control of human immunoglobulin products is essential to ensure therapeutic functionality and safety. This includes testing for Fc function and anticomplementary activity (ACA), as well as verification of appropriate molecular size distribution using size-exclusion chromatography as prescribed in the European Pharmacopoeia (Ph. Eur.) monographs 0338, 0918, 2788 and 1928. To this end, specific biological reference preparations (BRPs) must be used. Stocks of the Ph. Eur. Human immunoglobulin (molecular size) BRP were running low and therefore a collaborative study was run by the European Directorate for the Quality of Medicines & HealthCare (EDQM), under the aegis of the Biological Standardisation Programme, to calibrate replacement batches. Eighteen laboratories, including manufacturers and Official Medicines Control Laboratories, took part in the study. Three batches of candidate BRPs were assessed and compared to Ph. Eur. Human immunoglobulin (molecular size) BRP 3 to ensure continuity. Based on the study results, the candidate BRPs were adopted by the Ph. Eur. Commission as Ph. Eur. Human immunoglobulin (molecular size) BRP batch 4, 5 and 6.

Role of Charge Heterogeneity on Physical Stability of Monoclonal Antibody Biotherapeutic Products.

PURPOSE: Chemical modifications in monoclonal antibodies can change hydrophobicity, charge heterogeneity as well as conformation, which eventually can impact their physical stability. In this study, the effect of the individual charge variants on physical stability and aggregation propensity in two different buffer conditions used during downstream purification was investigated. METHODS: The charge variants were separated using semi-preparative cation exchange chromatography and buffer exchanged in the two buffers with pH 6.0 and 3.8. Subsequently each variant was analysed for size heterogeneity using size exclusion chromatography and dynamic light scattering, conformational stability, colloidal stability, and aggregation behaviour under accelerated stability conditions. RESULTS: Size variants in each charge variant were similar in both pH conditions when analyzed without extended storage. However, conformational stability was lower at pH 3.8 than pH 6.0. All charge variants showed similar apparent melting temperature at pH 6.0. In contrast, at pH 3.8 variants A3, A5, B2, B3 and B4 display lower Tm, suggesting reduced conformational stability. Further, A2, A3 and A5 exhibit reduced colloidal stability at pH 3.8. In general, acidic variants are more prone to aggregation than basic variants. CONCLUSION: Typical industry practice today is to examine in-process intermediate stability with acidic species and basic species taken as a single category each. We suggest that perhaps stability evaluation needs to be performed at specie level as different acidic or basic species have different stability and this knowledge can be used for clever designing of the downstream process to achieve a stable product.

Affinity-Resolved Size Exclusion Chromatography Coupled to Mass Spectrometry: A Novel Tool to Study the Attribute-and-Function Relationship in Therapeutic Monoclonal Antibodies.

Assessment of critical quality attributes (CQAs) is an important aspect during the development of therapeutic monoclonal antibodies (mAbs). Attributes that affect either the target binding or Fc receptor engagement may have direct impacts on the drug safety and efficacy and thus are considered as CQAs. Native size exclusion chromatography (SEC)-based competitive binding assay has recently been reported and demonstrated significant benefits compared to conventional approaches for CQA identification, owing to its faster turn-around and higher multiplexity. Expanding on the similar concept, we report the development of a novel affinity-resolved size exclusion chromatography-mass spectrometry (AR-SEC-MS) method for rapid CQA evaluation in therapeutic mAbs. This method features wide applicability, fast turn-around, high multiplexity, and easy implementation. Using the well-studied Fc gamma receptor III-A (FcγRIIIa) and Fc interaction as a model system, the effectiveness of this method in studying the attribute-and-function relationship was demonstrated. Further, two case studies were detailed to showcase the application of this method in assessing CQAs related to antibody target binding, which included unusual N-linked glycosylation in a bispecific antibody and Met oxidation in a monospecific antibody, both occurring within the complementarity-determining regions (CDRs).

Rapid determination of toxic and essential metal binding proteins in biological samples by size exclusion chromatography-inductively coupled plasma tandem mass spectrometry.

Metalloproteins binding with trace elements play a crucial role in biological processes and on the contrary, those binding with exogenous heavy metals have adverse effects. However, the methods for rapid, high sensitivity and simultaneous analysis of these metalloproteins are still lacking. In this study, a fast method for simultaneously determination of both essential and toxic metal-containing proteins was developed by coupling size exclusion chromatography (SEC) with inductively coupled plasma tandem mass spectrometry (ICP-MS/MS). After optimization of the separation and detection conditions, seven metalloproteins with different molecular weight (from 16.0 to 443.0 kDa) were successfully separated within 10 min and the proteins containing iron (Fe), copper (Cu), zinc (Zn), iodine (I) and lead (Pb) elements could be simultaneously detected with the use of oxygen as the collision gas in ICP-MS/MS. Accordingly, the linear relationship between log molecular weight and retention time was established to estimate the molecular weight of unknown proteins. Thus, the trace metal and toxic metal containing proteins could be detected in a single run with high sensitivity (detection limits in the range of 0.0020-2.5 µg/mL) and good repeatability (relative standard deviations lower than 4.5 %). This method was then successfully used to analyze metal (e.g., Pb, Zn, Cu and Fe) binding proteins in the blood of Pb-intoxicated patients, and the results showed a negative correlation between the contents of zinc and lead binding proteins, which was identified to contain hemoglobin subunit. In summary, this work provided a rapid and sensitive tool for screening metal containing proteins in large number of biological samples.

Heterologous Expression and Purification of Eukaryotic ALA Synthase from E. coli.

This chapter presents a method for the heterologous expression and purification of human ALA synthase from Escherichia coli. Mature ALAS is produced with an N-terminal hexahistidine affinity tag followed by a SUMO fusion tag for solubility and ease of purification. The plasmid is introduced into competent E. coli cells, and robust protein expression is induced with IPTG. The ALAS cofactor, pyridoxal 5'-phosphate, is inserted during protein production to yield an active enzyme upon purification. After cell lysis, the tagged ALAS protein is isolated via a multistep purification that involves an initial nickel-affinity step, affinity tag cleavage and removal, and a final size exclusion chromatography polishing step. Importantly, this protocol is amenable to various ALAS truncations and mutations, opening the door to understanding ALAS biology and its intersections with iron utilization across several organisms.

Effects of Molecular Weight on the Marine Biodegradability of Poly(l-lactic acid).

Poly(l-lactic acid) (PLA) is a biodegradable bioplastic with limited marine degradation. This study examines the impact of molecular weight on PLA's marine biodegradability. We synthesized PLA with terminal hydroxyl groups (PLA-OH) with degrees of polymerization (DP) between 14 and 642 and conducted biochemical oxygen demand (BOD) tests. Samples with a DP of 422 or 642 did not degrade, like commercial PLA. However, PLA-OH with a DP below 314 showed biodegradability, with DP 14 exhibiting a higher degradability than cellulose. Size exclusion chromatography (SEC) confirmed a decrease in molecular weight for samples with DPs below 314, indicating extracellular microbial activity. These findings suggest that PLA-OH with a DP under 314 can be degraded in marine conditions, unlike high-molecular-weight PLA. If the DP of high-molecular-weight PLA can be reduced to 314 by some specific method, then it is expected that PLA can be used to create marine biodegradable materials.

Development of a Sampling and Storage Protocol of Extracellular Vesicles (EVs)-Establishment of the First EV Biobank for Polytraumatized Patients.

In the last few years, several studies have emphasized the existence of injury-specific EV "barcodes" that could have significant importance for the precise diagnosis of different organ injuries in polytrauma patients. To expand the research potential of the NTF (network trauma research) biobank of polytraumatized patients, the NTF research group decided to further establish a biobank for EVs. However, until now, the protocols for the isolation, characterization, and storage of EVs for biobank purposes have not been conceptualized. Plasma and serum samples from healthy volunteers (n = 10) were used. Three EV isolation methods of high relevance for the work with patients' samples (ultracentrifugation, size exclusion chromatography, and immune magnetic bead-based isolation) were compared. EVs were quantified using nanoparticle tracking analysis, EV proteins, and miRNAs. The effects of different isolation solutions; the long storage of samples (up to 3 years); and the sensibility of EVs to serial freezing-thawing cycles and different storage conditions (RT, 4/-20/-80 °C, dry ice) were evaluated. The SEC isolation method was considered the most suitable for EV biobanking. We did not find any difference in the quantity of EVs between serum and plasma-EVs. The importance of particle-free PBS as an isolation solution was confirmed. Plasma that has been frozen for a long time can also be used as a source of EVs. Serial freezing-thawing cycles were found to affect the mean size of EVs but not their amount. The storage of EV samples for 5 days on dry ice significantly reduced the EV protein concentration.

Label-free single-vesicle based surface enhanced Raman spectroscopy: A robust approach for investigating the biomolecular composition of small extracellular vesicles.

Small extracellular vesicles (sEVs) are cell-released vesicles ranging from 30-150nm in size. They have garnered increasing attention because of their potential for both the diagnosis and treatment of disease. The diversity of sEVs derives from their biological composition and cargo content. Currently, the isolation of sEV subpopulations is primarily based on bio-physical and affinity-based approaches. Since a standardized definition for sEV subpopulations is yet to be fully established, it is important to further investigate the correlation between the biomolecular composition of sEVs and their physical properties. In this study, we employed a platform combining single-vesicle surface-enhanced Raman spectroscopy (SERS) and machine learning to examine individual sEVs isolated by size-exclusion chromatography (SEC). The biomolecular composition of each vesicle examined was reflected by its corresponding SERS spectral features (biomolecular "fingerprints"), with their roots in the composition of their collective Raman-active bonds. Origins of the SERS spectral features were validated through a comparative analysis between SERS and mass spectrometry (MS). SERS fingerprinting of individual vesicles was effective in overcoming the challenges posed by EV population averaging, allowing for the possibility of analyzing the variations in biomolecular composition between the vesicles of similar and/or different sizes. Using this approach, we uncovered that each of the size-based fractions of sEVs contained particles with predominantly similar SERS spectral features. Indeed, more than 84% of the vesicles residing within a particular group were clearly distinguishable from that of the other EV sub-populations, despite some spectral variations within each sub-population. Our results suggest the possibility that size-based EV fractionation methods produce samples where similarly eluted sEVs are correlated with their respective biochemical contents, as reflected by their SERS spectra. Our findings therefore highlight the possibility that the biogenesis and respective biological functionalities of the various sEV fractions may be inherently different.

A native SEC-MS workflow and validation for analyzing drug-to-antibody ratio and drug load distribution in cysteine-linked antibody-drug conjugates.

The development and optimization of Antibody-Drug Conjugates (ADCs) hinge on enhanced analytical and bioanalytical characterization, particularly in assessing critical quality attributes (CQAs). The ADC's potency is largely determined by the average number of drugs attached to the monoclonal antibody (mAb), known as the drug-to-antibody ratio (DAR). Furthermore, the drug load distribution (DLD) influences the therapeutic window of the ADC, defining the range of dosages effective in treating diseases without causing toxic effects. Among CQAs, DAR and DLD are vital; their control is essential for ensuring manufacturing consistency and product quality. Typically, hydrophobic interaction chromatography (HIC) or reversed-phase liquid chromatography (RPLC) with UV detector have been used to quantitate DAR and DLD in quality control (QC) environment. Recently, Native size-exclusion chromatography-mass spectrometry (nSEC-MS) proves the potential as a platformable quantitative method for characterizing DAR and DLD across various cysteine-linked ADCs in research or early preclinical development. In this work, we established and assessed a streamlined nSEC-MS workflow with a benchtop LC-MS platform, to quantitatively monitor DAR and DLD of different chemotype and drug load level cysteine-linked ADCs. Moreover, to deploy this workflow in QC environment, complete method validation was conducted in three independent laboratories, adhering to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2(R1) guidelines. The results met the predefined analytical target profile (ATP) and performance criteria, encompassing specificity/selectivity, accuracy, precision, linearity, range, quantification/detection limit, and robustness. Finally, the method validation design offers a reference for other nSEC-MS methods that are potentially used to determine the DAR and DLD on cysteine-linker ADCs. To the best of our knowledge, this study is the first reported systematic validation of the nSEC-MS method for detecting DAR and DLD. The results indicated that the co-validated nSEC-MS workflow is suitable for DAR and DLD routine analysis in ADC quality control, release, and stability testing.

Aqueous size exclusion chromatography applied to polymer analysis: experimental conditions and molecular weight calibration curves.

Aqueous mode size exclusion chromatography (SEC) was employed for the analysis and construction of molecular weight (MW) calibration curves of three water-soluble polymers, namely, polyethylene glycol, polyethylene oxide, and polyacrylic acid sodium salt. Several calibration curves were obtained, varying chromatographic conditions such as columns arrangement, ionic strength, temperature and pH, in addition trends in polymeric chromatographic behavior were examined. The variation in SEC distribution coefficients at different temperatures was found to be below 10 %, indicating that the studied polymers follow an ideal SEC mechanism under the tested conditions. Thus, differences in chromatographic behavior were ascribed to changes in polymer configuration induced by media and/or temperature. These variations in morphology were consistent with the observed SEC behavior. Regarding MW calibration, polynomial regression models ranging from first to fifth order were applied, and the most adequate ones were selected based on their fit and prediction capabilities. Third order polynomials were the preferred models for polyethylene glycol and polyacrylic acid sodium salt, independently of chromatographic conditions. Meanwhile for polyethylene oxide, either third or fifth-order polynomial models were optimal depending on the chromatographic conditions. All the selected regression models presented coefficients of multiple determination (R2) above 0.990, while achieving relative errors of prediction (REP%) in MW ranging from 0.3 to 4 % for cross-validation.

Optimizing Messenger RNA Analysis Using Ultra-Wide Pore Size Exclusion Chromatography Columns.

Biopharmaceutical products, in particular messenger ribonucleic acid (mRNA), have the potential to dramatically improve the quality of life for patients suffering from respiratory and infectious diseases, rare genetic disorders, and cancer. However, the quality and safety of such products are particularly critical for patients and require close scrutiny. Key product-related impurities, such as fragments and aggregates, among others, can significantly reduce the efficacy of mRNA therapies. In the present work, the possibilities offered by size exclusion chromatography (SEC) for the characterization of mRNA samples were explored using state-of-the-art ultra-wide pore columns with average pore diameters of 1000 and 2500 Å. Our investigation shows that a column with 1000 Å pores proved to be optimal for the analysis of mRNA products, whatever the size between 500 and 5000 nucleotides (nt). We also studied the influence of mobile phase composition and found that the addition of 10 mM magnesium chloride (MgCl2) can be beneficial in improving the resolution and recovery of large size variants for some mRNA samples. We demonstrate that caution should be exercised when increasing column length or decreasing the flow rate. While these adjustments slightly improve resolution, they also lead to an apparent increase in the amount of low-molecular-weight species (LMWS) and monomer peak tailing, which can be attributed to the prolonged residence time inside the column. Finally, our optimal SEC method has been successfully applied to a wide range of mRNA products, ranging from 1000 to 4500 nt in length, as well as mRNA from different suppliers and stressed/unstressed samples.

Purification and Quality Control of Recombinant Proteins Expressed in Mammalian Cells: A Practical Review.

In the recent years, there has been a rapid development of new technologies and strategies when it comes to protein purification and quality control (QC), but the basic technologies for these processes go back a long way, with many improvements over the past few decades. The purpose of this chapter is to review these approaches, as well as some other topics such as the advantages and disadvantages of various purification methods for intracellular or extracellular proteins, the most effective and widely used genetically engineered affinity tags, solubility-enhancing tags, and specific proteases for removal of nontarget sequences. Affinity chromatography (AC), like Protein A or G resins for the recovery of antibodies or Fc fusion proteins or immobilized metals for the recovery of histidine-tagged proteins, will be discussed along with other conventional chromatography techniques: ion exchange (IEC), hydrophobic exchange (HEC), mixed mode (MMC), size exclusion (SEC), and ultrafiltration (UF) systems. How to select and combine these different technologies for the purification of any given protein and the minimal criteria for QC characterization of the purity, homogeneity, identity, and integrity of the final product will be presented.

Multiangle Light Scattering as a Lentivirus Purification Process Analytical Technology.

The limited biomolecular and functional stability of lentiviral vectors (LVVs) for cell therapy poses the need for analytical tools that can monitor their titers and activity throughout the various steps of expression and purification. In this study, we describe a rapid (25 min) and reproducible (coefficient of variance ∼0.5-2%) method that leverages size exclusion chromatography coupled with multiangle light scattering detection (SEC-MALS) to determine size, purity, and particle count of LVVs purified from bioreactor harvests. The SEC-MALS data were corroborated by orthogonal methods, namely, dynamic light scattering (DLS) and transmission electron microscopy. The method was also evaluated for robustness in the range of 2.78 × 105-2.67 × 107 particles per sample. Notably, MALS-based particle counts correlated with the titer of infectious LVVs measured via transduction assays (R2 = 0.77). Using a combination of SEC-MALS and DLS, we discerned the effects of purification parameters on LVV quality, such as the separation between heterogeneous LV, which can facilitate critical decision-making in the biomanufacturing of gene and cell therapies.

Not All Arms of IgM Are Equal: Following Hinge-Directed Cleavage by Online Native SEC-Orbitrap-Based CDMS.

Immunoglobulins M (IgM) are key natural antibodies produced initially in humoral immune response. Due to their large molecular weights and extensive glycosylation loads, IgMs represent a challenging target for conventional mass analysis. Charge detection mass spectrometry (CDMS) may provide a unique approach to tackle heterogeneous IgM assemblies, although this technique can be quite laborious and technically challenging. Here, we describe the use of online size exclusion chromatography (SEC) to automate buffer exchange and sample introduction, and demonstrate its adaptability with Orbitrap-based CDMS. We discuss optimal experimental parameters for online SEC-CDMS experiments, including ion activation, choice of column, and resolution. Using this approach, CDMS histograms containing hundreds of individual ion signals can be obtained in as little as 5 min from single injections of <1 µg of sample. To demonstrate the unique utility of online SEC-CDMS, we performed real-time kinetic monitoring of pentameric IgM digestion by the protease IgMBRAZOR, which cleaves specifically in the hinge region of IgM. Several digestion intermediates corresponding to processive losses of F(ab')2 subunits could be mass-resolved and identified by SEC-CDMS. Interestingly, we find that for the J-chain linked IgM pentamer, cleavage of one of the F(ab')2 subunits is much slower than the other four F(ab')2 subunits, which we attribute to the symmetry-breaking interactions of the J-chain within the pentameric IgM structure. The online SEC-CDMS methodologies described here open new avenues into the higher throughput automated analysis of heterogeneous, high-mass protein assemblies by CDMS.

Expression and Purification of Mammalian NMDA Receptor Protein for Functional Characterization.

N-methyl-D-aspartate (NMDA) receptors are critical for brain function and serve as drug targets for the treatment of neurological and psychiatric disorders. They typically form the tetrameric assembly of GluN1-GluN2 (2A to 2D) subtypes, with their diverse three-dimensional conformations linked with the physiologically relevant function in vivo. Purified proteins of tetrameric assembled NMDA receptors have broad applications in the structural elucidation, hybridoma technology for antibody production, and high-throughput drug screening. However, obtaining sufficient quantity and monodisperse NMDA receptor protein is still technically challenging. Here, we summarize a paradigm for the expression and purification of diverse NMDA receptor subtypes, with detailed descriptions on screening constructs by fluorescence size-exclusion chromatography (FSEC), generation of recombinant baculovirus, expression in the eukaryotic expression system, protein purification by affinity chromatography and size-exclusion chromatography (SEC), biochemical and functional validation assays.

Analysis of aggregation profile of glucagon using SEC-HPLC and FFF-MALS methods.

Recently, the first generic glucagon for injection was approved for the treatment of severe hypoglycemia. Unlike its brand name recombinant glucagon, the generic glucagon is synthetic. Since glucagon has a high propensity to form aggregates in solution, it is essential to assess the aggregation profile of the synthetic glucagon compared to the recombinant glucagon. In this study, two robust separation methods, size-exclusion chromatography (SEC-HPLC) and field-flow fractionation coupled with a multi-angle light scattering detector (FFF-MALS), were employed to characterize generic and brand glucagon aggregation in six lots (three newly released, three expired). The presence of aggregation in samples was determined from the generated chromatograms and analyzed. The study showed that both products have comparable aggregation profiles. The SEC-HPLC demonstrated that in both glucagon versions, the expired lots had a higher percentage of dimers than the newly released lots, but even at expiration, the amount was negligible (∼0.1%). The FFF-MALS method did not detect any dimers or higher molecular weight aggregates. Further evaluation of the detection limit found that FFF-MALS was unable to detect aggregates at amounts lower than 0.5% of total glucagon. The negligible amounts of dimer detected in the generic and brand glucagon indicate that both versions are physically stable and are not prone to aggregation under clinically relevant conditions.

Analytical Quality by Design as applied to the development of a SEC-HPLC platform procedure for the determination of monoclonal antibody purity without mobile phase additives.

This work presents the application of AQbD principles to the development of a size exclusion chromatography (SEC) HPLC procedure for the determination of monoclonal antibody (mAb) product purity using state-of-the-art column technology available via the Waters™ XBridge Premier Protein SEC column. Analytical Quality by Design (AQbD) emphasizes a systematic, risk-based lifecycle approach to analytical procedure development based on sound statistical methodologies. It has recently become increasingly recommended by regulatory agencies as a response to the need for greater efficiency, improved reliability, and increased robustness among modern analytical procedures in the pharmaceutical industry. Use of an Analytical Target Profile (ATP) and formal risk assessments informed the application of Design of Experiments (DoE) to optimize this analytical procedure, as well as assess its robustness and ruggedness. Importantly, our ruggedness results demonstrated the transferability of this procedure between two laboratories within the Catalent Biologics Global Network. Application of this analytical procedure as a platform approach for evaluating mAb purity is expected to support expedited, first-in-human timelines of mAb molecules by enabling great quantitative performance with simple mobile phase buffer compositions. Taken together, this case study demonstrates the utility of adopting AQbD principles in analytical procedure development.

Interaction of carbonic anhydrase I released from red blood cells with human plasma in vitro.

Red blood cells (RBCs) constitute ∼50% of the bloodstream and represent an important target for environmental pollutants and bacterial/viral infections, which can result in their rupture. In addition, diseases such as sickle cell anaemia and paroxysmal nocturnal haemoglobinuria can also result in the rupture of RBCs, which can be potentially life-threatening. With regard to the release of cytosolic metalloproteins from RBCs into the blood-organ system, the biochemical fate of haemoglobin is rather well understood, while comparatively little is known about another highly abundant Zn-metalloprotein, carbonic anhydrase (CA I). To gain insight into the interaction of CA I with human blood plasma constituents, we have employed a metallomics tool comprised of size-exclusion chromatography (SEC) coupled online with an inductively coupled plasma atomic emission spectrometer (ICP-AES), which allows to simultaneously observe all Cu, Fe, and Zn-metalloproteins. After the addition of CA I to human blood plasma incubated at 37°C, the SEC-ICP-AES analysis using phosphate buffered saline (pH 7.4) after 5 min, 1 h, and 2 h revealed that CA I eluted after all endogenous Zn-metalloproteins in the 30 kDa range. Matrix-assisted laser desorption-time of flight mass spectrometry analysis of the collected Zn-peak confirmed that CA I eluted from the column intact. Our in vitro results suggest that CA I released from RBCs to plasma remains free and may be actively involved in health-relevant adverse processes that unfold at the bloodstream-endothelial interface, including atherosclerosis and vision loss.

A Practical Guide for the Quality Evaluation of Fluobodies/Chromobodies.

BACKGROUND: Fluorescent proteins (FPs) are pivotal reagents for flow cytometry analysis or fluorescent microscopy. A new generation of immunoreagents (fluobodies/chromobodies) has been developed by fusing recombinant nanobodies to FPs. METHODS: We analyzed the quality of such biomolecules by a combination of gel filtration and SDS-PAGE to identify artefacts due to aggregation or material degradation. RESULTS: In the SDS-PAGE run, unexpected bands corresponding to separate fluobodies were evidenced and characterized as either degradation products or artefacts that systematically resulted in the presence of specific FPs and some experimental conditions. The elimination of N-terminal methionine from FPs did not impair the appearance of FP fragments, whereas the stability and migration characteristics of some FP constructs were strongly affected by heating in loading buffer, which is a step samples undergo before electrophoretic separation. CONCLUSIONS: In this work, we provide explanations for some odd results observed during the quality control of fluobodies and summarize practical suggestions for the choice of the most convenient FPs to fuse to antibody fragments.