Fabrication and application of a continuously regenerated cationic impurity removal device for ion chromatography.

A continuously regenerated cationic impurity removal device (CR-CRD) has been fabricated and applied for ion chromatography (IC). The removal of cationic impurities is realized by electrodialytically replacing the cationic impurities with hydronium ions. The device is configured in a sandwich structure and the central eluent channel is respectively isolated from both electrodes by stacked cation exchange membranes and a bipolar membrane (BPM) plus stacked anion exchange membranes. The eluent channel is packed with cation exchange resins in hydronium form and their continuous regeneration can be achieved by electrodialysis. A desirable feature of the device is gas-free, and no degasser is required. It showed sufficient ability to remove cationic impurities, as indicated by > 99.9 % removal of 10 mL of 1 mM LiOH solution injected (∼10 µmol) or continuous removal of 1 mM LiOH solution at the flow rate of 1 mL/min (1 µmol/min). A useful application was for sample pretreatment in nuclear power industry, by eliminating strong matrix interference of the sample containing LiOH (1 mM) and boric acid (2000 mg/L) with trace anion analysis.

Mechanistic model-based characterization of size-exclusion-mixed-mode resins for removal of monoclonal antibody fragments.

Although antibody fragments are a critical impurity to remove from process streams, few platformable purification techniques have been developed to this end. In this work, a novel size-exclusion-mixed-mode (SEMM) resin was characterized with respect to its efficacy in mAb fragment removal. Inverse size-exclusion chromatography showed that the silica-based resin had a narrow pore size distribution and a median pore radius of roughly 6.2 nm. Model-based characterization was carried out with Chromatography Analysis and Design Toolkit (CADET), using the general rate model and the multicomponent Langmuir isotherm. Model parameters were obtained from fitting breakthrough curves, performed at multiple residence times, for a mixture of mAb, aggregates, and an array of fragments (varying in size). Accurate fits were obtained to the frontal chromatographic data across a range of residence times. Model validation was then performed with a scaled-up column, altering residence time and feed composition from the calibration run. Accurate predictions were obtained, thereby illustrating the model's interpolative and extrapolative capabilities. Additionally, the SEMM resin achieved 90% mAb yield, 37% aggregate removal, 29% [Formula: see text] removal, 54% Fab/Fc removal, 100% Fc fragments removal, and a productivity of 72.3 g mAbL×h. Model predictions for these statistics were all within 5%. Simulated batch uptake experiments showed that resin penetration depth was directly related to protein size, with the exception of the aggregate species, and that separation was governed by differential pore diffusion rates. Additional simulations were performed to characterize the dependence of fragment removal on column dimension, load density, and feed composition. Fragment removal was found to be highly dependent on column load density, where optimal purification was achieved below 100 mg protein/mL column. Furthermore, fragment removal was dependent on column volume (constant load mass), but agnostic to whether column length or diameter was changed. Lastly, the dependence on feed composition was shown to be complex. While fragment removal was inversely related to fragment mass fraction in the feed, the extent depended on fragment size. Overall, the results from this study illustrated the efficacy of the SEMM resin in fragment and aggregate removal and elucidated relationships with key operational parameters through model-based characterization.

Development of tandem cation exchange chromatography for high purity extracellular vesicle isolation: The effect of ligand steric availability.

Purification of extracellular vesicles for research and therapeutic applications requires updated methodology to address the limitations of traditional ultracentrifugation and other size-based separation techniques. Their downfalls include induced extracellular vesicle aggregation, low yields, poor scalability and one-dimensionality of the separation process, as the size or sedimentation speed of extracellular vesicles is often the only selection criterion. Ion exchange chromatography is a promising alternative or supplementary method candidate, as it offers a different approach for extracellular vesicle separation, which is surface charge. For now, mostly anion exchange chromatography has been evaluated for extracellular vesicle purification, as it successfully relies on the strongly negative surface charge of extracellular vesicles. However, as extracellular vesicles are very complex in their structure, also cation exchange chromatography could be applicable, due to individual cationic domains on the extracellular vesicle surface. Here, we compare anion exchange chromatography to different types of cation exchange chromatography for the purification of platelet extracellular vesicle samples also containing plasma-derived impurities. We found that the choice of resin structure used for cation exchange chromatography is critical for binding platelet extracellular vesicles, as a conventional-type cation exchanger was found to only capture and elute less than 20% of extracellular vesicles. With the tentacle-type resin, it was possible to obtain comparable platelet extracellular vesicle yields (over 90%) with cation exchange chromatography compared to anion exchange chromatography, as well as superior purity, especially when it was combined to conventional cation exchange resin.

Screening of six cation exchange resins for high binding capacity, monomer purity and step yield: A case study.

Cation exchange (CEX) chromatography has been widely used as a polishing step in downstream processing of therapeutic monoclonal antibodies (mAbs). It has been well documented that the performance of a particular CEX resin heavily relies on buffer type, pH and conductivity. In this work, with a case study, we screened six commercial CEX resins under different pHs and conductivities. Initial screening was conducted on Tecan to find conditions under which high binding capacity was achieved. Next, performance of each resin was further evaluated using a small column under the identified optimum binding conditions. Variations in binding capacity and purity of eluate were observed among these resins. The adopted approach allows for identification of resins exhibiting good binding capacity, aggregate clearance and recovery.

Influence of ligand density variations on the two peak elution behavior of a monoclonal antibody in cation exchange chromatography.

Cation exchange chromatography, as part of the monoclonal antibody purification train, is known as a mild polishing technique. However, in the last couple of years, more and more publications have shown unusual elution behavior, resulting from e.g. on-column (reversible) unfolding and aggregation of the predominantly mAb molecules. The stability of the investigated protein seems to play a significant role in this phenomenon. We have used a glycosylated IgG1 antibody as a model protein and investigated several influencing factors, including pH value and ligand density variations of three prototype Fractogel® cation exchange resins. Ligand density, pH and salt concentration are the main contributing factors in the Donnan effect, i.e. distribution of ions, between resin pore volume and bulk volume. This leads to a significantly lower pH value the protein is subjected to during the on-column hold time and therefore influences the conformational stability of our protein. Nano-DSF and kinetic SEC measurements show that the protein is destabilized at low pH values, but also, that the binding to the CEX resin and the elution with increasing salt concentration is responsible for the resulting two-peak elution behavior and partially reversible unfolding and aggregation.

Application of the Steric Mass Action formalism for modeling under high loading conditions: Part 2. Investigation of high loading and column overloading effects.

The application of a model-based approach for industrial chromatography development requires the capability of the model to describe protein elution under high loading and overloading conditions. In a previous work, an extensive dataset was created to model the elution behavior of a bispecific antibody (bsAb) on the strong cation exchange resin POROS™ XS. Thereby, the pH-dependence of the model parameters in the Steric Mass Action (SMA) model could be examined and described over a pH range of 4.5 to 8.9. However, discrepancies between simulated and experimental data were observed under high loading and overloading conditions, particularly in the lower pH range (pH 4.5 to 5.3) and in the higher pH range (pH 6.0 to 9.0). In this work, these discrepancies are studied by performing new experiments which show that these differences were primarily not caused by limitations of the SMA model. At lower pH values, overloading phenomena such as protein breakthrough during the loading phase, additional peaks, and peak shoulders occurred. The application of various experiments performed with different Na+ concentrations and different loading times during sample loading revealed that intraparticle diffusion effects and conformational changes of the bsAb are responsible for these overloading phenomena at low pH. The applied lumped rate mass transfer model is not adequate and should be extended to consider these effects. At higher pH, the assumption of describing the bsAb's elution behavior with only one simulated species was insufficient to predict complex peak shapes that arise because of multi-component elution of the bsAb's charge variants. The extension of the model to a simple multi-component system consisting of two variants allowed the prediction of a majority of the complex elution profiles.

Application of the Steric Mass Action formalism for modeling under high loading conditions: Part 1. Investigation of the influence of pH on the steric shielding factor.

In ion exchange chromatography, the Steric Mass Action (SMA) formalism is frequently used to simulate sorption processes at low and high column load conditions. To apply the SMA model for describing protein elution over wide ranges of pH, it is necessary to use pH-dependent model parameters. In the past, some publications have already described the pH-dependence of the characteristic protein charge and the equilibrium constant, while the influence of pH on the steric shielding factor has been mostly neglected. In this work, the pH-dependences of all relevant model parameters, including the shielding factor, were investigated, described, and implemented into the SMA model. Therefore, the elution behavior of a bispecific monoclonal antibody on the strong cation exchange resin POROS™ XS was modeled over broad ranges of pH, salt concentrations, and protein concentrations. Linear gradient elution experiments were performed to generate an extensive data set by using increasing column loadings from 0.5 up to 75.0 mgbsAb/mLresin. By using an inverse peak fitting method, shielding factors were estimated at various pH values ranging from 4.5 to 8.9. The results showed that an increasing buffer pH resulted in strongly increasing shielding factors. A semi-empirical correlation describing the shielding factor as a function of pH was established and implemented into the SMA formalism. This approach led to precise prediction of protein elution behavior using a single-component simulation. This was demonstrated by accurate simulation of linear salt, pH and dual gradient elution experiments conducted under high loading conditions.

Dissociation events during processing of monoclonal antibodies on strong cation exchange resins.

The phenomenon of pH excursion was demonstrated for pH gradient elution of monoclonal antibodies (mAbs) on strong cation exchange resins under high overloading conditions. The mAbs differed in molecular structure and isoelectric point, and the resins in matrix structure and ligand density. In all cases, elution of the proteins was accompanied with distortion of the concentration, pH and conductometric profiles. To elucidate that phenomenon, titration curves were generated for liquid solutions of the proteins as well as for suspensions of the resins with the proteins adsorbed on their surface. The course of the curves was found to be affected by the presence of the proteins both in liquid and adsorbed phases. The effect enhanced with increase in the initial pH of the binding buffer and in the protein concentration. To quantify that phenomenon, a mechanistic model was used, which accounted for the protein dissociation in both phases. The model reproduced the titration curves and the observed trends in changes of their courses. The simulation results indicated that the pattern of pH transitions recorded for different mAbs on the resins mostly depended on their adsorption affinity.

Capture and purification of an untagged nanobody by mixed weak cation chromatography and cation exchange chromatography.

Nanobodies (Nbs) are single-domain antibodies, which have potential application value in tumor-targeted therapy, immunotherapy, diagnostic probe, and molecular imaging. Typically, Nbs are captured by affinity chromatography via the addition of specific fusion tags at their N or C terminus. Nerve growth factor (NGF), which regulates the growth and development of peripheral and central neurons, maintains neuronal survival and plays a key role in both arthritis and acute and chronic pain. In this study, a method for capture and purification of an untagged Nb (anti-NGF Nb) by mixed weak cation chromatography and cation exchange chromatography was established. First, pH 4.0-5.0 was demonstrated to be the optimal loading condition for Capto MMC to capture anti-NGF by the design of experiments (DOE). Furthermore, high purity and yield products can be obtained at laboratory scale and commercial production scale by adjusting the protein pH. Additionally, direct capture of anti-NGF Nb using Capto MMC without adjusting anti-NGF Nb harvest pH was investigated. The anti-NGF Nb captured by Capto MMC was 67.2% yield, 94.5% monomer purity, and host cell protein (HCP) was reduced from 74,931 ppm to 484 ppm. The anti-NGF Nb that were further purified using Capto S ImpAct achieved 84.5% yield and 99.2% purity and 77 ppm of HCP. The scaling-up process was consistent with the results of the optimized process, further demonstrating the feasibility of this method. This outcome provides a highly promising and competitive alternative to affinity chromatography-based processing strategies for Nbs.

High throughput screening of fiber-based adsorbents for material and process development.

There has been a growing interest in fibers and fiber-based adsorbents as alternative adsorbents for preparative chromatography. While the benefits of fiber-based adsorbents in terms of productivity have been highlighted in several recent studies, microscale tools that enable a fast characterization of these novel adsorbents, and an easy integration into process development workflows, are still lacking. In the present study an automated high-throughput screening (HTS) for fiber-based adsorbents was established on a robotic liquid handling station in 96 well filter plates. Two techniques - punching and weighing - were identified as techniques that enabled accurate and reproducible portioning of short-cut fiber-based adsorbents. The impact of several screening parameters such as phase ratio, shaking frequency, and incubation time were investigated and optimized for different types of fiber-based adsorbents. The data from the developed HTS correlated with data from packed fiber columns, and binding capacities from both scales matched closely. Subsequently, the developed HTS was utilized to optimize the hydrogel architecture of anion exchange (AEX) fiber-based adsorbent prototypes. A novel AEX fiber-based adsorbent was developed that compared favorably with existing resin and membrane adsorbents in terms of productivity and DNA binding capacity. In addition, the developed HTS was also successfully employed in order to identify step elution conditions for the purification of a monoclonal antibody from product- and process-related impurities with a cation exchange (CEX) fiber-based adsorbent. Trends from the HTS were found to be in good agreement with trends from lab scale column runs. The tool developed in this paper will enable a faster and more complete characterization of fiber-based adsorbents, easier tailoring of such adsorbents towards specific process applications, and an easier integration of such materials into processes. In comparison to previous lab scale experiments, material requirements are reduced by a factor of 3-40 and time requirements are reduced by a factor of 2-5.

Exploration of fiber-based cation exchange adsorbents for the removal of monoclonal antibody aggregates.

Cation exchange chromatography (CEX) is a widely used technique for the removal of monoclonal antibody (mAb) aggregates. At present, resins are mainly used for this purpose, as convective types of adsorbents such as membrane adsorbers (MAs) have often not demonstrated overall comparable performance for this particular application. Fiber-based adsorbents can overcome the current limitations of MAs with respect to permeability, binding capacity, and adsorbent cost, and could therefore be a viable alternative to resins for the removal of mAb aggregates. It has not been evaluated, however, whether and under which conditions the use of such adsorbents is feasible for this purpose. In the present study, the use of fiber-based CEX adsorbents for mAb aggregate removal was examined. Two types of fiber-based adsorbents, an uncontrolled grafted and a controlled grafted fiber-based adsorbent, were evaluated with respect to permeability, dynamic mAb binding capacity (DBC), resolution capabilities, and the performance in bind and elute (B/E) and frontal chromatography mode with respect to typical performance indicators. The permeabilities of the fiber-based adsorbents ranged from 200 to 1700 mD, making it possible to use the fiber-based adsorbents at larger bed heights than membrane adsorbers with fast mobile phase velocities. Antibody DBCs ranged from 20 to 41 g/L at 150 cm/h, and at higher mobile phase velocities exceeded the DBC of an existing resin material, Poros 50 HS, which has frequently been used for aggregate removal. Both fiber types showed good resolution capabilities of monomer and aggregates, and provided better resolution per column length than Poros 50 HS. Typical purity and yield constraints were fulfilled for both fiber types in both B/E and frontal chromatography mode for mobile phase velocities ranging up to 480 cm/h and 1060 cm/h. The overall performance of the controlled grafted fibers was found to be superior to the performance of uncontrolled grafted fiber-based adsorbents due to higher productivity and lower buffer consumption. The overall performance of the fiber-based adsorbents was found to be comparable to the performance of Poros 50 HS at typical operating conditions. The results in this study indicate that the use of fiber-based adsorbents for mAb aggregate removal is feasible with a performance that is comparable to the performance of an existing resin material. Depending on the cost of the adsorbents and the use scenario, the usage of such adsorbents could be beneficial.

Design and synthesis of naphthalene-based chiral strong cation exchangers and their application for chiral separation of basic drugs.

In continuation of our efforts to synthesize a highly dedicated strong cation exchanger, we introduce four chiral stationary phases based on a laterally substituted naphthalene core featuring chiral 2-aminocyclohexansulfonic acid as the chiral cation-exchange site. The selectors were modified with two different terminal units, which enabled immobilization to the silica support by thiol-ene radical reaction or azide-yne click chemistry. The chromatographic parameters of these chiral stationary phases were determined using a set of chiral amines, mainly from the family of ß-blocker pharmaceuticals. The chiral stationary phases immobilized by means of click chemistry were found to be superior to those possessing the sulfide linker to the silica support. The chromatographic results and visualization of density functional theory-calculated conformations of the selectors hint at a combination of a steric and electronic effect of the triazole ring in the course of chiral resolution of the target analytes.

Novel synthesis of natural cation exchange resin by crosslinking the sodium alginate as a natural polymer with 1,6-hexamethylene diisocyanate in inert solvents: Characteristics and applications.

Novel synthesis of natural polymeric cation exchanger from sodium alginate reagent as natural polymer (CAlg-Na)n has been developed. This procedure takes place by crosslinking the solid sodium alginate with 1,6-hexamethylene diisocyanate (HDI) in dryness benzene as inert solvent (DB). The experimental results for crosslinking the alginate using different degrees of crosslinking of HDI/DB (w/w) showed that the capacity of the synthesized resin for binding the polyvalent metal ions reached its maximum at 30% of (HDI/D) ratio. This work aims to present a novel synthesis of cation exchange resin from natural polymers as an alternative promising competitor of low-cost, high performance and non-toxicity for removal of the toxic heavy metal ions in wastewater remediation and radionuclide pollutants from environmental contaminated media. The results obtained from various applied techniques for determining the bounded metal ion concentrations with (CAlg-Na)n indicated that the resin capacity was followed the order U(VI) > Cu(II) > Sr(II) > Ca(II) at 25 °C, respectively. The factors influenced the alginate affinity for chelating the metal ions were explained in some correlation terms between the alginate capacity and some physicochemical properties of chelated metal ions and its respective formed complexes. Speculated geometrical configurations for chelation were suggested and discussed.

Facile Preparation of Purified Sinapate Ethyl Ester from Rapeseed Meal Extracts Using Cation-exchange Resin in Dual Role as Adsorber and Catalyst.

In this study, cation-exchange resin was used to prepare an esterified antioxidant, sinapate ethyl ester (SE), using ethanolic extracts from rapeseed. A concentration of sinapic acid using the cation-exchange resin in 80% ethanol (aq) and subsequent interesterification of the extract in ethanol using the same resin afforded a product with a purity of 64 wt% and 100% of SE yield. Moreover, after purification using preparative thin-layer chromatography, almost 100 wt% purity was obtained. In an auto-oxidation test, purified SE conferred a much higher antioxidative effect on the bulk oil, emphasising the effectiveness of the protocol using cation-exchange resin for the purification.

Removing a light-chain missing byproduct by MMC ImpRes mixed-mode chromatography under weak partitioning mode in purifying a WuXiBody-based bispecific antibody.

WuXiBody is a bispecific antibody (bsAb) platform developed by WuXi Biologics. Its key feature is the replacement of one parental antibody's CH1/CL region with the T cell receptor (TCR) constant domain, a design that promotes cognate heavy chain (HC)-light chain (LC) pairing. BsAbs based on WuXiBody can adopt either asymmetric or symmetric format. For purifying a WuXiBody-based symmetric bsAb, we identified a LC-missing species as a major byproduct. While for bsAbs based on other platforms removal of such byproduct can pose considerable challenge to the downstream team, in this case WuXiBody's unique design makes separation relatively straightforward. We previously showed that Capto MMC ImpRes mixed-mode chromatography under bind-elute mode can effectively remove this LC-missing species. However, the dynamic binding capacity (DBC) of Capto MMC ImpRes is relatively low under the selected condition, making the process less desirable for large-scale manufacturing. In this study, we demonstrated that when Capto MMC ImpRes chromatography is conducted under weak partitioning mode, high throughput, good yield, and effective byproduct removal are simultaneously achieved.

Preparation of a polymer-based weak cation exchanger for ion chromatography via atom transfer radical polymerization.

A novel polymer-based weak cation exchanger (WCX) for ion chromatography has been described. It was prepared by grafting tert-butyl acrylate and maleic anhydride onto the surface of poly(glycidyl methacrylate-divinylbenzene) microspheres via atom transfer radical polymerization, followed by hydrolytic treatment to produce acrylic and maleic acid groups. The obtained WCX showed better separation and higher selectivity for model cations relative to solely acrylic or maleic acid. Simultaneous separation of alkali and alkaline earth cations (including NH4+) was achieved in a single isocratic run under suppressed mode, and ion exchange was found to dominate the separation process. Its utility was demonstrated for determination of several cations in a beer sample and the recovery was ranging from 98.4 to 109.9%.

Rapid and Efficient Enrichment of Snake Venoms from Human Plasma Using a Strong Cation Exchange Tip Column to Improve Snakebite Diagnosis.

Snake envenomation is a serious public health issue in many tropical and subtropical countries. Accurate diagnosis and immediate antivenom treatment are critical for effective management. However, the venom concentration in the victims' plasma is usually low, representing one of the bottlenecks in developing clinically applicable assays for venom detection and snakebite diagnosis. In this study, we attempted to develop a simple method for rapid enrichment of venom proteins from human plasma to facilitate detection. Our experiments showed that several major protein components of both Naja atra (N. atra) and Bungarus multicinctus (B. multicinctus) venoms have higher isoelectric point (pI) values relative to high-abundance human plasma proteins and could be separated via strong cation exchange-high-performance liquid chromatography (SCX-HPLC). Based on this principle, we developed an SCX tip column-based protocol for rapid enrichment of N. atra and B. multicinctus venom proteins from human plasma. Application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) led to the identification of cytotoxin and beta-bungarotoxin as the major proteins enriched by the SCX tip column in each venom sample. The entire process of venom enrichment could be completed within 10-15 min. Combination of this method with our previously developed lateral flow strip assays (rapid test) significantly enhanced the sensitivity of the rapid test, mainly via depletion of the plasma protein background, as well as increase in venom protein concentration. Notably, the SCX tip column-based enrichment method has the potential to efficiently enrich other Elapidae snake venoms containing proteins with higher pI values, thereby facilitating venom detection with other assays. This simple and rapid sample preparation method should aid in improving the clinical utility of diagnostic assays for snakebite.

Preparation of palm (Elaeis oleifera) pressed fibre cellulose nanocrystals via cation exchange resin: characterisation and evaluation as Pickering emulsifier.

BACKGROUND: Palm pressed fibre (PPF) is a cellulose-rich biomass residue produced during palm oil extraction. Its high cellulose content allows the isolation of cellulose nanocrystal (CNC). CNC has attracted scientific interest due to its biodegradability, biocompatibility and low cost. The present study isolated CNC from PPF using a cation exchange resin, which is an environmentally friendly and less harsh hydrolysis method than conventional mineral acid hydrolysis. Isolated CNC was used to stabilise an oil-in-water emulsion and the emulsion stability was evaluated in terms of droplet size, morphology and physical stability. RESULTS: PPF was subjected to alkali and bleach treatment prior to hydrolysis, which successfully removed 54% and 75% of non-cellulosic components (hemicellulose and lignin, respectively). Hydrolysis conditions of 5 h, 15:1 (w/w) resin-to-pulp ratio and 50 °C produced CNC particles of 50-100 nm in length. CNC had a crystallinity index of 42% and appeared rod-like morphologically. CNC-stabilised emulsion had better stability when used in combination with soy lecithin (SL), a well-established, commonly used food stabiliser. Emulsion stabilised by the binary mixture of CNC and SL had droplet size, morphology and physical stability comparable to those of emulsion stabilised using SL. CONCLUSIONS: CNC was successfully isolated from PPF through a cation exchange resin. This offers an alternative usage for the underutilised PPF to be converted into value-added products. Isolated CNC was also found to have promising potential in the stabilisation of Pickering emulsions. These results provide useful information indicating CNC as a natural and sustainable stabiliser for food, cosmeceutical and pharmaceutical applications. © 2021 Society of Chemical Industry.

Cross-scale quality assessment of a mechanistic cation exchange chromatography model.

Cation exchange chromatography (CEX) is an essential part of most monoclonal antibody (mAb) purification platforms. Process characterization and root cause investigation of chromatographic unit operations are performed using scale down models (SDM). SDM chromatography columns typically have the identical bed height as the respective manufacturing-scale, but a significantly reduced inner diameter. While SDMs enable process development demanding less material and time, their comparability to manufacturing-scale can be affected by variability in feed composition, mobile phase and resin properties, or dispersion effects depending on the chromatography system at hand. Mechanistic models can help to close gaps between scales and reduce experimental efforts compared to experimental SDM applications. In this study, a multicomponent steric mass-action (SMA) adsorption model was applied to the scale-up of a CEX polishing step. Based on chromatograms and elution pool data ranging from laboratory- to manufacturing-scale, the proposed modeling workflow enabled early identification of differences between scales, for example, system dispersion effects or ionic capacity variability. A multistage model qualification approach was introduced to measure the model quality and to understand the model's limitations across scales. The experimental SDM and the in silico model were qualified against large-scale data using the identical state of the art equivalence testing procedure. The mechanistic chromatography model avoided limitations of the SDM by capturing effects of bed height, loading density, feed composition, and mobile phase properties. The results demonstrate the applicability of mechanistic chromatography models as a possible alternative to conventional SDM approaches.

Optimisation of the use of sliding window deconvolution for comprehensive characterisation of trastuzumab and adalimumab charge variants by native high resolution mass spectrometry.

The biopharmaceutical industry continues to develop mAb-based biotherapeutics in increasing numbers. Due to their complexity, there are several critical quality attributes (CQAs) that need to be measured and controlled to guarantee product safety and efficacy. Charge variant analysis is a widely used method to monitor changes in product quality during the manufacturing process of monoclonal antibodies (mAbs) and, together with a bottom-up peptide centred approach, acts as a key analytical platform to fulfil regulatory requirements. Native MS measures biomolecules under conditions that preserve most aspects of protein tertiary and quaternary structure, enabling direct characterization of large intact proteins such as mAbs. The resulting native mass spectrum of a mAb is characterized by a narrower charge-state envelope that simplifies the spectra and also condenses the ion signals into fewer peaks, increasing the signal-to-noise ratio. Algorithmic spectral deconvolution is needed for routine accurate and rapid molecular weight determination, and consequently, multiple deconvolution algorithms have evolved over the past decade. Here, we demonstrate the utility of the sliding window algorithm as a robust and powerful deconvolution tool for comprehensive characterisation of charge variant analysis data for mAbs. Optimum performance is evaluated by studying the impact of critical software parameters on detection, identification and relative quantitation of protein isoforms. By combining molecular mass and retention time information, it was possible to identify multiple modifications on adalimumab and trastuzumab, both IgG1 mAbs, including lysine truncation, deamidation and succinimide formation, along with the N-glycan distribution of each of the identified charge variants. Sliding window deconvolution also provides a key benefit of low abundant variant detection in a single analysis and the ability to detect co-eluting components with different relative abundances. The studied mAbs demonstrate the algoritms applicability for efficient data processing of both simple and complex mAbs analysed using pH gradient cation exchange chromatography coupled to native mass spectrometry.