Affinity Resin Research Articles

Comparison of Protein A affinity resins for twin-column continuous capture processes: Process performance and resin characteristics

Continuous chromatography is a promising technology for downstream processing of biopharmaceuticals. The operation of continuous processes is significantly different to batch-mode chromatography and needs comprehensive evaluation. In this work, the performances of four Protein A affinity resins were studied systematically for twin-column continuous capture processes. A model-based approach was used to evaluate the process performance (productivity and capacity utilization) under varying operation conditions, and the objective was to reveal the crucial resin properties for continuous capture. The trade-off between productivity and capacity utilization was found, and it is necessary to select appropriate resins for different feedstock and operation conditions. The capacity utilization heavily depends on mass transfer, and steep breakthrough curves are favorable for high capacity utilization. The productivity is determined by both equilibrium binding capacity and mass transfer, and the balance of feed amount and feed time is critical. Moreover, the influence of binding capacity and mass transfer on process productivity and parameter sensitivity with two important resin properties (equilibrium binding capacity qmax and effective pore diffusion coefficient De) were assessed by the model, and suitable resin parameter ranges for twin-column continuous capture were determined. The model-based approach is an effective and useful tool to evaluate the complex performance of different resins and guide the design of next-generation resins for continuous processes.

Purification and Characterization of Antibodies Directed against the α-Gal Epitope

The α-Gal epitope is an immunogen trisaccharide structure consisting of N-acetylglucosamine (GlcNAc)β1,4-galactose (Gal)α1,3-Gal. It is presented as part of complex-type glycans on glycoproteins or glycolipids on cell surfaces of non-primate mammalians. About 1% of all antibodies in human sera are specific toward α1,3-Gal and are therefore named as anti-α-Gal antibodies. This work comprises the purification and characterization of anti-α-Gal antibodies from human immunoglobulin G (IgG). A synthetically manufactured α Gal epitope affinity resin was used to enrich anti-α-Gal antibodies. Selectivity experiments with purified antibodies were carried out using enzyme-linked immunosorbent assays (ELISA), Western blotting, and erythrocyte agglutination. Furthermore, binding affinities toward α-Gal were determined by surface plasmon resonance (SPR) and the IgG distribution of anti α Gal antibodies (83% IgG2, 14% IgG1, 2% IgG3, 1% IgG4) was calculated applying ELISA and immunodiffusion. A range of isoelectric points from pH 6 to pH 8 was observed in 2D gel electrophoresis. Glycan profiling of anti α Gal antibodies revealed complex biantennary structures with high fucosylation grades (86%). Additionally, low amounts of bisecting GlcNAc (15%) and sialic acids (13%) were detected. The purification of anti-α-Gal antibodies from human IgG was successful, and their use as detection antibodies for α Gal-containing structures was evaluated.

A Comparative Investigation of the Bispecific Antibody: Expression in Expi293F Cells and E.coli

Aims: Dual-Character antibodies can simultaneously target two surface markers. Blinatumomab is a C19 / CD3 antibody from the BiTE family (Bispecific T cell engager antibody) and was approved by the US Food and Drug Administration for clinical use. This antibody effectively targets malignant cells in patients with acute infoblastic leukemia. In the production of large quantities of such antibodies on an industrial scale, selecting the appropriate host remains a critical issue. Mammalian cells and strains of E.coli are the most common hosts for producing antibodies and antibody components on an industrial scale, respectively. Methods & Materials: In this study, a dual-specific antibody was used in the mammalian system of plasmid pcDNA3.1 (+) and for expression in the bacterial system of plasmid pET22b. The antibody produced in both systems was purified using nickel affinity resin under similar conditions. Next, SDS-PAGE and Western blot analysis was performed on both study samples. Finally, the binding properties of the antibody secreted from both systems were assessed by the ELISA test. Findings: The present study results suggested that antibodies produced by the mammalian expression system provided better binding properties than the expression system in bacteria. Conclusion: This study indicated that in the case of antibodies to two traits of the BiTE family, like Blinatombe, mammalian cells generate a more efficient and successful expression system; although the bacterium can produce much larger amounts of the antibody.

Supporting development of mRNA-based therapies by addressing large-scale purification challenges

The field of mRNA-based therapies is a rapidly emerging area with increasing real-world applications. The potential of these therapies is being demonstrated in various fields. Although the potential of mRNA in therapies is seemingly endless, obtaining the quantities of synthetic mRNA needed for clinical treatment remains a challenging obstacle, and current methods for mRNA purification are creating a bottleneck in large-scale manufacturing. Particularly for vaccine development, obtaining the quantities of synthetic mRNA needed for clinical treatment remains an obstacle. As a result, a robust, scalable and easy-to-use platform to support all mRNA therapies is needed. To support the development of mRNA-based therapies, Thermo Fisher Scientific has developed an affinity resin for the purification and isolation of mRNA from<em>&nbsp;in vitro</em>&nbsp;transcription (IVT) manufacturing processes. The following article and case studies will highlight how the Thermo Scientific POROS™ Oligo (dT)25 affinity resin can enable efficient and simplified mRNA purification.

Abstract P-12:Three-dimensional Structure of the Yeast Transmembrane Mechanosensor Wsc1

Background: Wsc1 is the best studied of the five mechanosensors of the cell wall integrity (CWI) signal transduction pathway in Saccharomyces cerevisiae. In genetic and biophysical studies, Wsc1 functions were assessed either in living yeast cells or in crude cell extracts. So far, no attempts to purify the sensor and determine its structure have been reported. Methods: The Wsc1-green fluorescent protein (GFP) fusion was expressed in S. cerevisiae following standard protocols. For solubilization, a 5% (w/v) solution of styrene-maleic acid (SMA) copolymer was added dropwise to the membrane suspension to get a final cell-to-SMA weight ratio of 1:2.5. The suspension was incubated for 30 min at room temperature (RT) and then for 16 hours at 4 °C, followed by centrifugation for 20 min at 134000 g at 4 °C. The supernatant was subsequently purified on affinity resin. Protein samples (3 µL) were placed onto the glow-discharged grid, stained twice, using 1% aquatic uranyl acetate solution for 30 s at RT, and air-dried. Micrographs were acquired using a transmission electron microscope Jem-2100 (Jeol, Japan) equipped with a 2K x 2K CCD camera Ultrascan 1000XP (Gatan, USA). The microscope operated at 200 kV in a low dose mode, with a magnification x40000 (2.5 Å/pix) and defocus 0.5-1.9 µm. Images were acquired automatically with SerialEM software. Results: We applied the 3:1 SMA copolymer to isolate Wsc1-GFP fusions into SMA/lipid particles (SMALPs) directly from native yeast membranes. We purified Wsc1-GFP-containing SMALPs by affinity chromatography; characterization by dynamic light scattering confirmed the presence of monodisperse nano-sized particles. The intensity-weighted diameter was estimated to be in the 10-nm range. The purified Wsc1-GFP-containing SMALPs were further analyzed by transmission electron microscopy (TEM). 46500 single particles were used to generate the 3D reconstructions of Wsc1-GFP. The resulting model showed the three functional parts of the Wsc1 sensor: the extracellular part, which has a clasp-like appearance, the transmembrane domain, and the short cytosolic part of Wsc1 fused to the GFP beta-barrel. Conclusion: The 3:1 SMA copolymer enabled isolation of Wsc1-GFP fusion complexes into SMALPs directly from native yeast membranes. Image analysis of the TEM data showed the Wsc1 in its contracted conformation. We propose a model of conformational changes in Wsc1 in response to mechanical stress.

A non-affinity purification process for GMP production of prefusion-closed HIV-1 envelope trimers from clades A and C for clinical evaluation

Metastable glycosylated immunogens present challenges for GMP manufacturing. The HIV-1 envelope (Env) glycoprotein trimer is covered by N-linked glycan comprising half its mass and requires both trimer assembly and subunit cleavage to fold into a prefusion-closed conformation. This conformation, the vaccine-desired antigenic state, is both metastable to structural rearrangement and labile to subunit dissociation. Prior reported GMP manufacturing for a soluble trimer stabilized in a near-native state by disulfide (SOS) and Ile-to-Pro (IP) mutations has employed affinity methods based on antibody 2G12, which recognizes only ~30% of circulating HIV strains. Here, we develop a scalable manufacturing process based on commercially available, non-affinity resins, and we apply the process to current GMP (cGMP) production of trimers from clades A and C, which have been found to boost cross-clade neutralizing responses in vaccine-test species. The clade A trimer, which we named “BG505 DS-SOSIP.664”, contained an engineered disulfide (201C-433C; DS) within gp120, which further stabilized this trimer in a prefusion-closed conformation resistant to CD4-induced triggering. BG505 DS-SOSIP.664 was expressed in a CHO-DG44 stable cell line and purified with initial and final tangential flow filtration steps, three commercially available resin-based chromatography steps, and two orthogonal viral clearance steps. The non-affinity purification enabled efficient scale-up, with a 250 L-scale cGMP run yielding 9.6 g of purified BG505 DS-SOSIP.664. Antigenic analysis indicated retention of a prefusion-closed conformation, including recognition by apex-directed and fusion peptide-directed antibodies. The developed manufacturing process was suitable for 50 L-scale production of a second prefusion-stabilized Env trimer vaccine candidate, ConC-FP8v2 RnS-3mut-2G-SOSIP.664, yielding 7.8 g of this consensus clade C trimer. The successful process development and purification scale-up of HIV-1 Env trimers from different clades by using commercially available materials provide experimental demonstration for cGMP manufacturing of trimeric HIV-Env vaccine immunogens, in an antigenically desired conformation, without the use of costly affinity resins.

A simple protocol to purify human TFIID free of the MED26 subunit of mediator complex

The general transcription factor TFIID is a multiprotein complex that is essential for specific transcription initiation by RNA polymerase II. It is composed of the TATA box-binding protein (TBP) and ~13 different TBP-associated factors (TAFs). Purification of TFIID free of other general transcription factors and coactivators is essential to analyze the transcription regulatory mechanisms in reconstituted systems in vitro. A breakthrough in TFIID purification was the generation of HeLa cell lines that express a FLAG epitope-tagged TBP subunit and immunopurification protocols with monoclonal anti-FLAG antibodies. Purification of TFIID from HeLa nuclear extracts generally required a two-step purification procedure involving phosphocellulose P11 chromatography followed by anti-flag M2 affinity purification (Chiang et al., 1993; Ge et al., 1996) [1,2]. Here we show first that the MED26 (CRSP70) coactivator subunit of Mediator co-purifies with TFIID in the above two-step protocol and interacts strongly with TFIID under high salt conditions. We further show that a MED26-free TFIID complex can be obtained by including a simple additional DE52 chromatography step following P11 fractionation. Thus, we demonstrate that MED26 strongly interacts with TFIID and recommend the use of a P11-DE52-M2 resin affinity three-step purification procedure to obtain MED26-free TFIID for analyzing Mediator-dependent transcription regulatory mechanisms in purified transcription systems in vitro.

Tandem nanobody: A feasible way to improve the capacity of affinity chromatography

Nanobodies, referred to the binding domain of the heavy-chain-only antibodies, are the smallest antigen recognition unit. The molecular weight of monomeric nanobodies is about one-tenth of the conventional antibodies. The small size of nanobodies facilitates genetic manipulation and recombinant expression. This study aimed to investigate the effects of nanobody multivalency on the binding capacity of affinity resin. The nanobody (namely AFV), which binds to the fragment crystallizable (Fc) region of immunoglobulin G (IgG), was fused to the N-terminal of HaloTag in the form of monomeric (H-AFV), dimer (H-diAFV), trimer (H-triAFV), and tetramer (H-tetAFV). The fusion proteins were solubly expressed in Escherichia coli yielding at least 9.9 mg L−1. The biolayer interferometry confirmed an increment of avidity as the increase of AFV valences. The four recombinant proteins in crude cell lysate were site-specifically immobilized onto the Halo ligand resin via the self-labeling HaloTag, respectively. The generated affinity resins were able to isolate high purity IgG from mouse plasma. The highest improvement of the static binding capacity was achieved 73.7% by the H-diAFV resin other than the H-triAFV or H-tetAFV, as compared to the H-AFV resin.

Development of a platform process for the production and purification of single-domain antibodies.

Single‐domain antibodies (sdAbs) offer the affinity and therapeutic value of conventional antibodies, with increased stability and solubility. Unlike conventional antibodies, however, sdAbs do not benefit from a platform manufacturing process. While successful production of a variety of sdAbs has been shown in numerous hosts, purification methods are often molecule specific or require affinity tags, which generally cannot be used in clinical manufacturing due to regulatory concerns. Here, we have developed a broadly applicable production and purification process for sdAbs in Komagataella phaffii (Pichia pastoris) and demonstrated the production of eight different sdAbs at a quality appropriate for nonclinical studies. We developed a two‐step, integrated purification process without the use of affinity resins and showed that modification of a single process parameter, pH of the bridging buffer, was required for the successful purification of a variety of sdAbs. Further, we determined that this parameter can be predicted based only on the biophysical characteristics of the target molecule. Using these methods, we produced nonclinical quality sdAbs as few as 5 weeks after identifying the product sequence. Nonclinical studies of three different sdAbs showed that molecules produced using our platform process conferred protection against viral shedding of rotavirus or H1N1 influenza and were equivalent to similar molecules produced in Escherichia coli and purified using affinity tags.

Removal of Per- and Polyfluoroalkyl Substances (PFASs) in Aqueous Film-Forming Foam (AFFF) Using Ion-Exchange and Nonionic Resins.

Despite benefits to the firefighting industry, the release of per- and polyfluoroalkyl substances (PFASs) from aqueous film-forming foam (AFFF) into aquatic systems poses significant risks to human health and other organisms. While anion-exchange technologies have proven to be effective for removing perfluoroalkyl acids (PFAAs) from water, their effectiveness for removing the diverse PFAS structures discovered in AFFF remains unknown. Here, we report on the adsorption of 75 PFASs, including 63 polyfluorinated substances, in a diluted AFFF mixture using 14 commercially available ion-exchange (IX)/nonionic resins and granular activated carbon (GAC). Results showed that anion-exchange resins (AERs) exhibited significant adsorption of PFASs compared to cation-exchange resins (CERs), nonionic resins (NIRs), and GAC regardless of the PFAS's predicted charge. Isotherm data showed that macroporous AERs have a higher PFAS adsorption capacity compared to gel-type AERs. Cross-correlation comparison of PFAS/Cl- selectivity coefficients (Kex) for each PFAS-AER combination showed that the hydrophobicity of the AER functional group, and polymer matrix played a dominant role in determining resin affinity for PFASs. PFAS structural characteristics also significantly affected adsorption, with increasing chain length and a net negative charge increasing the extent of adsorption. Results from this study provide guidelines for the selection of resins to adsorb a wider range of PFASs and meaningful insights for the development of quantitative models for IX treatment of AFFF-impacted water.

Strategy for Treatment of Infected Diabetic Foot Ulcers.

Diabetic foot ulcers (DFUs) are chronic wounds that develop in 30% of diabetic patients. In DFUs, the normal wound healing process consisting of inflammation, angiogenesis, and extracellular matrix (ECM) remodeling is dysregulated and stalled. Upon injury, neutrophils and monocytes arrive at the wound and secrete matrix metalloproteinase (MMP)-8 and reactive oxygen species (ROS). ROS activates nuclear factor kappa beta (NF-κB), which upregulates MMP-9. Monocytes become macrophages, secreting tumor growth factor (TGF)-β1 and vascular endothelial growth factor (VEGF) for angiogenesis, resulting in remodeling of the ECM. MMP-9 cleaves laminin for keratinocyte migration. MMP-8 is beneficial for remodeling the ECM and healing the wound. In DFUs, the excess unregulated MMP-9 is detrimental, destroying the ECM and preventing the wound from healing. DFUs are typically infected, many with biofilm-producing bacteria that are resistant to antibiotics. Infection increases the time for wound healing and the likelihood for a lower-limb amputation. Despite the use of antibiotics, amputations occur in 24.5% of patients with DFUs. Clearly, new strategies for treatment of DFUs are needed. With the use of an affinity resin that binds exclusively to the active forms of MMPs and proteomics, we identified two proteinases, MMP-8 and MMP-9, in wounds of diabetic mice and diabetic humans. With the use of selective inhibitors, gene ablation of MMP-9, and exogenous application of MMP-8, we demonstrated that MMP-8 is beneficial to wound repair and that MMP-9 prevents the diabetic wound from healing. Our research has shown that infection increases active MMP-9, increasing inflammation and decreasing angiogenesis. As a result, infected diabetic wounds take a longer time to heal than uninfected ones. We found that active MMP-9 and NF-κB increased in human DFUs with wound severity and infection. The best strategy for treatment of DFUs is to selectively inhibit the detrimental proteinase MMP-9 without affecting the beneficial MMP-8 so that the body can repair the wound. Lead optimization of the thiirane class of inhibitors led to the discovery of (R)-ND-336, a potent (19 nM) and selective (450-fold) MMP-9 inhibitor. (R)-ND-336 accelerated wound healing in diabetic mice by decreasing ROS and NF-κB, lowering inflammation, and increasing angiogenesis. (R)-ND-336 in combination with the antibiotic linezolid improved wound healing in infected diabetic mice by inhibiting MMP-9, which mitigated macrophage infiltration and increased angiogenesis, thereby restoring the normal wound healing process.

Removing a single-arm species by Fibro PrismA in purifying an asymmetric IgG-like bispecific antibody

MabSelect PrismA is an affinity resin whose ligand is derived from the B-domain of Protein A. Compared to its predecessor resins (i.e., MabSelect SuRe LX and MabSelect SuRe), the PrismA ligand has enhanced affinity for the VH3 chain. Consistently, when being used for the purification of an asymmetric IgG-like bispecific antibody (bsAb), MabSelect PrismA resin shows certain resolution between the intact product and a single-arm byproduct, which contain the same Fc region but different numbers of VH3 domain. Fibro PrismA is a newly launched product which has the same PrismA Protein A ligand as MabSelect PrismA but uses cellulose fiber instead of resin as its matrix. It was learned that Fibro PrismA, in comparison to PrismA resin, exhibits further improved resolution, allowing efficient clearance of the single-arm byproduct as well as good recovery of the target bsAb. This finding suggests that Fibro PrismA provides a potential solution for separating antibody species containing the same Fc region but different numbers of VH3 domain, which can otherwise be difficult to separate.

Discovery and Characterization of an ALFA-Tag-Specific Affinity Resin Optimized for Protein Purification at Low Temperatures in Physiological Buffer.

Epitope tags are widely employed as tools to detect, purify and manipulate proteins in various experimental systems. We recently introduced the ALFA-tag together with two ALFA-specific single-domain antibodies (sdAbs), NbALFA and NbALFAPE, featuring high or intermediate affinity, respectively. Together, the ALFA system can be employed for a broad range of applications in microscopy, cell biology and biochemistry requiring either extraordinarily stable binding or mild competitive elution at room temperature. In order to further enhance the versatility of the ALFA system, we, here, aimed at developing an sdAb optimized for efficient elution at low temperatures. To achieve this, we followed a stringent selection scheme tailored to the specific application. We found candidates combining a fast capture of ALFA-tagged proteins with an efficient competitive elution at 4 °C in physiological buffer. Importantly, by employing a structure-guided semisynthetic library based on well-characterized NbALFA variants, the high specificity and consistent binding of proteins harboring ALFA-tags at either terminus could be maintained. ALFA SelectorCE, a resin presenting the cold-elutable NbALFACE, is an ideal tool for the one-step purification of sensitive protein complexes or temperature-labile enzymes. We believe that the general approach followed during the selection and screening can be transferred to other challenging sdAb discovery projects.

Isolation and purification of oleuropein from olive leaves using boric acid affinity resin and a novel solvent system

Oleuropein is an important natural compound with many biological activities. How to get high purity oleuropein is an urgent issue to be solved. This study provided a method for separating and purifying high purity oleuropein from olive leaves using boric acid affinity resin and a novel solvent system. And the experimental kinetic and adsorption isotherm data of boric acid affinity resin were well–fitted pseudo–first–order kinetic model and Langmuir isotherm model, respectively. The dynamic desorption experiments showed that ethanol was the most efficient eluent while had the lowest oleuropein purity. However, after the novel solvent system treated only one cycle of adsorption and desorption, the purity of oleuropein increased by 5.55 folds from 12.31 ± 0.42% – 68.30 ± 2.08 %. The results can guide the separation and purification of high purity catechol natural products using boric acid affinity resin and mixed solvent systems.

Investigation of alkaline effects on Protein A affinity ligands and resins using high resolution mass spectrometry

In this study, the enhanced alkaline stability of Protein A ligands and resins designed by protein engineering approaches is demonstrated. High throughput PreDictor™ plates were used to evaluate and compare the human Immunoglobulin G (IgG) static binding capacities (SBC) of MabSelect SuRe™ and MabSelect™ PrismA affinity chromatography (AC) resins after continuous incubation in 0.1–2.0 M NaOH for 1–72 h. The alkaline effect on the Protein A affinity ligand was studied by high resolution mass spectrometry (MS). The IgG binding capacity of the investigated AC resins show expected declining trends with increasing NaOH concentrations and incubation times. The decrease is larger for MabSelect SuRe than for MabSelect PrismA and occur at lower NaOH concentrations. MabSelect SuRe display high remaining binding capacity even after 72 h continuous incubation in 0.1 M NaOH, while higher concentrations induce an accentuated decline with incubation time. The MabSelect PrismA resin shows almost no effect on the binding capacity even after 72 h incubation in 0.5 M NaOH. Decline in capacity is first observed after 48 h incubation in 1.0 M NaOH, thus displaying the extreme alkaline stability of the PrismA affinity ligand. The MS analysis of the ligands, including a Protein A single B-domain, SuRe-domain and PrismA-domain clearly illustrate the increasing alkaline stability (B-domain < SuRe < PrismA) as the ligand has been refined using a protein engineering approach. Deamidation and ligand degradation could be monitored in relation to NaOH incubation conditions. Enzymatic digestion of MabSelect SuRe and MabSelect PrismA resins after alkaline incubation and LC-MS/MS peptide mapping facilitates identification and quantification of specific deamidation sites on the affinity ligand.

Efficient purification of selenoprotein thioredoxin reductase 1 by using chelating reagents to protect the affinity resins and rescue the enzyme activities

The application of 2’,5’-ADP Sepharose affinity chromatography is used to purify the recombinantly expressed selenoprotein thioredoxin reductases (TrxRs, TXNRDs). However, the affinity resin gradually loses its binding capacity, and the eluted fractions show low activities. The issues above might be attributed to either impairment of the ligands of the column by the released intercellular nucleases or inhibition of the enzyme by trace heavy metal impurities. In this study, chelating reagents are supplemented in crude samples and buffer, and the effects on enzyme purification are investigated. Our results show that 20 mM EDTA or EGTA fully protects/rescues TrxR1 from the impairment of heavy metal ions. More importantly, 20 mM EDTA improves the yield of TrxR1 from 50 % to >80 % after 2’,5’-ADP Sepharose affinity chromatography, therefore increasing the reproducibility of the 2’,5’-ADP Sepharose column. The purified enzyme shows an apparent 55-kDa subunit single band on a reducing SDS-PAGE gel and exhibits a specific activity >20 U/mg based on the classic DTNB reduction assay. This study would be helpful for the high-efficiency preparation of selenoproteins using NADPH as a cofactor and for the effective protection of ribonucleotide-based ligands via affinity chromatography.

Mechanistic Studies of Thrombin Generation Assay to Evaluate Procoagulant Potential of Fitusiran

Introduction: Fitusiran is a once-monthly subcutaneously administered RNA interference therapeutic targeting antithrombin III (AT) to rebalance hemostasis in patients with hemophilia A and B with or without inhibitors. As for other non-FVIII therapies in development, there is a need to better assess the procoagulant potential of fitusiran. Using thrombin generation assays (TGA), we studied the hemostatic activity in hemA plasma with low AT levels in the presence and absence of varying factor VIII (FVIII) levels to mimic conditions associated with breakthrough bleeds. To better understand the impact of AT depletion in the assay, we monitored multiple TGA parameters as well as the levels of several coagulation components potentially influencing the reactions. Methods: Pooled HemA plasma was applied to an AT affinity resin to remove AT. Residual AT activity was 5% and additional AT was spiked back to produce HemA plasmas with various amounts of AT (5, 10, 20 and 100%). FVIII was spiked into the 4 plasmas at 0, 5, 10, 20, 30, 50, and 100 IU/dL and all samples were analyzed by Calibrated Automated Thrombography (Stago). Thrombin generation parameters were calculated by thrombinoscope software.After the TGA reaction, samples were further analyzed by immunoblot for evaluation of thrombin-AT (TAT) and alpha2-macroglobulin-thrombin (a2M:T) complex formation, as well as remaining (pro)thrombin, AT and a2M levels . In parallel, a2M:T complex levels were assessed by ELISA capture of a2M followed by addition of thrombin substrate to directly measure the thrombin activity stemming from a2M-inhibited thrombin. . Results: TGA parameters for FVIII titrations in HemA plasmas with reduced AT were calculated and compared to the FVIII standard curves generated in HemA plasma with normal AT levels for each TGA parameter (lag time, time to peak, peak height, ETP and velocity index). As expected, a greater reduction of AT activity in hemA plasma correlated with increased thrombin generation compared to hemA plasma (with 100% AT). The apparent FVIII-like activity observed at low AT conditions in HemA plasma was dependent on the TGA parameter evaluated, with velocity index suggesting the lowest and ETP the highest FVIII-like activity. Furthermore, particularly at lower starting levels of AT, AT is depleted during the TGA reaction and leads to enhanced levels of a2M:T complex as observed by immunoblot or a2M:T chromocapture assays. Importantly, the a2M:T complex is capable of cleaving the assay substrate and contributing to the TGA signal. Typically, the a2M:T generated signal is calculated and substracted by the thrombinoscope software. However, under low AT conditions, increased levels of a2M-T lead to substrate consumption at later stages of the reaction, resulting in erroneous underestimation of the a2M:T activity that does not agree with direct measurement of a2M: T levels. These limitations need to be taken into consideration when interpreting TGA data under conditions of low AT activity. While this remains an area of investigation, parameters derived earlier in the reaction prior to AT depletion and significant levels of a2M:T complex formation may better correlate with the true hemostatic potential associated with AT inhibition. Conclusion: Our results show that TGA can be a valuable tool to measure the hemostatic potential associated with fitusiran therapy. We demonstrate that reduced levels of AT are associated with enhanced thrombin generation, but that activity comparison to FVIII varies significantly depending on the TGA parameter analyzed. In addition we show that under low AT conditions, AT can become depleted leading to a2M:T complex accumulation which may lead to overestimation of some TGA parameters. This overestimation is likely an artifact of the TGA method as it is a closed in vitro system that can be influenced by the depletion of components or accumulation of products. Our results suggest that TGA parameters calculated early in the reaction are less influenced by some of these limitations and may be better predictors of the true hemostatic activity of fitusiran. Furthermore, a2M may play a more active role during fitusiran treatment, ensuring free thrombin does not migrate outside of the site of injury. Disclosures Kattula: Sanofi: Current Employment, Current equity holder in publicly-traded company. Salvador:Sanofi: Current Employment, Current equity holder in publicly-traded company. Wang:Sanofi: Current Employment, Current equity holder in publicly-traded company. Ismail:Sanofi: Current Employment, Current equity holder in publicly-traded company. van der Flier:Sanofi: Current Employment, Current equity holder in publicly-traded company. Leksa:Sanofi: Current Employment, Current equity holder in publicly-traded company. Salas:Sanofi: Current Employment, Current equity holder in publicly-traded company.

Impact of salt concentration in mobile phase on antibody retention in Protein A, Protein L and KappaSelect affinity chromatography

Protein A, Protein L and KappaSelect affinity resins have been widely used for antibody purification. Elution of antibody bound to these resins is typically achieved by acidic pH. In addition, elution can be moderately adjusted by tuning the salt concentration in mobile phase as hydrophobic interactions play a major role in binding. In this study, we assessed the impact of salt concentration in mobile phase on antibody retention in these three types of affinity chromatography. The data suggest that salt concentration has a bigger impact on retention in the two light chain-binding affinity columns (i.e., Protein L and KappaSelect) than in Protein A column. In particular, lowering salt concentration in mobile phase for Protein L and KappaSelect columns allows elution become feasible at higher pH. In addition, this finding suggests that wash in these two types of column aimed at removing weakly-bound byproducts can also be performed at increased pH by lowering salt concentration in the wash buffer. Rendering wash and elution feasible at higher pH has practical value for cases where the target antibodies are sensitive to stringent conditions.

Comprehensive Analysis of Low Molecular Weight Serum Proteome Enrichment for Mass Spectrometric Studies.

Rationale: The low molecular weight (LMW) proteins present in circulating body fluids, such as serum and plasma, hold biological significance as possible biomarkers. A major obstacle in mass spectrometry-based proteomics of serum is the presence of abundant high molecular weight proteins which mask the identification and quantitation of lower molecular weight proteins. Traditional methods involve the use of affinity resins to remove high molecular weight proteins, such as albumin and immunoglobulin G, with concomitant loss of lower molecular weight proteins. Considering the importance of depleting high molecular proteins, this paper compares an affinity resin, a gel-filter, and an acetonitrile (ACN) precipitation method to achieve successful removal of high molecular weight proteins and recovery of lower molecular weight proteins. Methods: Serum enrichment was carried out by multiple methods such as with the commercially available serum protein mini kit, ACN precipitation, and a gel filter method. Mass spectrometric runs were carried out on an AB SCIEX ESI QTOF 5600 mass spectrometer. Mass spectrometry analysis of the enriched serum obtained by ACN precipitation and gel filter method was performed for global proteome profiling. Quantitative mass spectrometry using isobaric tags for relative and absolute quantitation (iTRAQ) for ACN-precipitated enriched serum was also carried out. Results: The gel filter method, though allowing for the resolution and identification of LMW proteins, was better suited for global proteome analysis and not preferred for quantitative proteomic experiments. In contrast, enrichment by the ACN precipitation method allowed for the reproducible identification and quantitation of LMW proteins having molecular weight ≥4 kDa. Conclusions: Using only chilled ACN and centrifugation, most of the highly abundant proteins were successfully removed from the serum, while recovering a significant portion of the LMW proteome. A more rapid protocol, which is compatible with iTRAQ labeling, to achieve improved results has been elucidated, thus allowing for better screening and identification of potential biomarkers.

The rapid \u201cteabag\u201d method for high-end purification of membrane proteins

Overproduction and purification of membrane proteins are generally challenging and time-consuming procedures due to low expression levels, misfolding, and low stability once extracted from the membrane. Reducing processing steps and shortening the timespan for purification represent attractive approaches to overcome some of these challenges. We have therefore compared a fast “teabag” purification method with conventional purification for five different membrane proteins (MraY, AQP10, ClC-1, PAR2 and KCC2). Notably, this new approach reduces the purification time significantly, and the quality of the purified membrane proteins is equal to or exceeds conventional methods as assessed by size exclusion chromatography, SDS-PAGE and downstream applications such as ITC, crystallization and cryo-EM. Furthermore, the method is scalable, applicable to a range of affinity resins and allows for parallelization. Consequently, the technique has the potential to substantially simplify purification efforts of membrane proteins in basic and applied sciences.