Thermostability optimization of the aspartate/alanine exchange transporter from Tetragenococcus halophilus.

Aspartate/alanine exchange transporter (AspT) is a secondary transporter isolated from the lactic acid bacterium Tetragenococcus halophilus D10 strain. This transporter cooperates with aspartate decarboxylase to produce proton-motive force through decarboxylative phosphorylation. A method that successfully analyzes the AspT mechanism could serve as a prototype for elucidating the substrate transport mechanism of other exchange transporters; therefore, the purpose of this study was to search for conditions that improve the thermal stability of AspT for 3D structure analysis. We used the fluorescence size-exclusion chromatography-based thermostability assay to evaluate conditions that contribute to AspT stability. We found that the AspT thermostability was enhanced at pH 5.0 to 6.0 and in the presence of Na+ and Li+. Pyridoxal phosphate, a coenzyme of aspartate decarboxylase, also had a thermostabilizing effect on AspT. Under the conditions obtained from these results, it was possible to increase the temperature at which 50% of dimer AspT remained by 14°C. We expect these conditions to provide useful information for future structural analysis of AspT.

Exploring the Combined Action of Adding Pertuzumab to Branded Trastuzumab versus Trastuzumab Biosimilars for Treating HER2+ Breast Cancer.

The binding activity of various trastuzumab biosimilars versus the branded trastuzumab towards the glycosylated extracellular domain of the human epidermal growth factor receptor 2 (HER2) target in the presence of pertuzumab was investigated. We employed size exclusion chromatography with tetra-detection methodology to simultaneously determine absolute molecular weight, concentration, molecular size, and intrinsic viscosity. All trastuzumab molecules in solution exhibit analogous behavior in their binary action towards HER2 regardless of the order of addition of trastuzumab/pertuzumab. This analogous behavior of all trastuzumab molecules, including biosimilars, highlights the robustness and consistency of their binding activity towards HER2. Furthermore, the addition of HER2 to a mixture of trastuzumab and pertuzumab leads to increased formation of high-order HER2 complexes, up to concentrations of one order of magnitude higher than in the case of sequential addition. The observed increase suggests a potential synergistic effect between these antibodies, which could enhance their therapeutic efficacy in HER2-positive cancers. These findings underscore the importance of understanding the complex interplay between therapeutic antibodies and their target antigens, providing valuable insights for the development of more effective treatment strategies.

Identification of heavily glycated proteoforms by hydrophilic-interaction liquid chromatography and native size-exclusion chromatography - High-resolution mass spectrometry.

BACKGROUND: The non-enzymatic glycation of proteins and their advanced glycation end products (AGEs) are associated with protein transformations such as in the development of diseases and biopharmaceutical storage. The characterization of heavily glycated proteins at the intact level is of high interest as it allows to describe co-occurring protein modifications. However, the high heterogeneity of glycated protein makes this process challenging, and novel methods are required to accomplish this. RESULTS: In this study, we investigated two novel LC-HRMS methods to study glycated reference proteins at the intact protein level: low-flow hydrophilic-interaction liquid chromatography (HILIC) and native size-exclusion chromatography (SEC). Model proteins were exposed to conditions that favored extensive glycation and the formation of AGEs. After glycation, complicated MS spectra were observed, along with a sharply reduced signal response, possibly due to protein denaturation and the formation of aggregates. When using HILIC-MS, the glycated forms of the proteins could be resolved based on the number of reducing monosaccharides. Moreover, some positional glycated isomers were separated. The SEC-MS method under non-denaturing conditions provided insights into glycated aggregates but offered only a limited separation of glycated species based on molar mass. Overall, more than 25 different types of species were observed in both methods, differing in molar mass by 14-162 Da. 19 of these species have not been previously reported. SIGNIFICANCE: The proposed strategies show great potential to characterize highly glycated intact proteins from native and denaturing perspectives and provide new opportunities for fast clinical diagnoses and investigating glycation-related diseases.

Characterization of Palm Fatty Acid Distillate, Diacylglycerol Regioisomers, and Esterification Products Using High-Performance Size Exclusion Chromatography.

High-performance size exclusion chromatography (HPSEC) equipped with an evaporative light scattering detector (ELSD) was utilized for characterization of palm fatty acid distillate (PFAD) and its esterified products, with a particular focus on lipid profiles and diacylglycerol (DAG) regioisomers. The separation of triacylglycerol (TAG), DAG, monoacylglycerol (MAG), and free fatty acid (FFA) was achieved through a single 100-Å Phenogel column, coupled with a 2-cm C18 guard, utilizing toluene/acetic acid (100:0.25, v/v) as the mobile phase. This separation was based on size sieving principles and the interactions between the hydroxyl group(s) and the Phenogel matrix. The limit of detection (LOD) and limit of quantification (LOQ) for the esterified PFAD products analyzed by this method fell within the range of 4.8-5.5 µg/mL and 14.7-16.7 µg/mL, respectively. Additionally, the same column, paired with a 2-cm silica guard and a mobile phase comprised of toluene/isooctane/acetic acid (35:65:0.15, v/v/v), was used for the characterization of DAG regioisomers within the esterified PFAD. LODs and LOQs for sn-1,3-DAG and sn- 1,2-DAG were determined to be 39.2 and 118.7 µg/mL, and 32.8 and 99.5 µg/mL, respectively. Investigation of esterified PFAD products prepared using 4% H2SO4 at 120°C. After 2 h, the analysis revealed the highest MAG content at 31.85%, accompanied by 51.54% DAG, 2.35% TAG, and a residual 14.27% FFA. Notably, as the reaction time extended, the MAG content decreased, while both DAG and TAG levels exhibited an increasing trend. Further examination of DAG regioisomers during PFAD esterification, under varying catalyst concentrations (2-10%) and reaction temperatures (80-140°C), demonstrated a significant increase in the percentage of sn-1,3-DAG, inversely correlated with the reduction in FFA from 2% H 2 SO 4 and 80°C onwards. Remarkably, the percentage of sn-1,2-DAG remained relatively stable regardless of changes in catalyst concentrations or temperatures, confirming its susceptibility to isomerization into the thermodynamically more stable sn-1,3-DAG form. This study provides valuable insights into the composition and behavior of esterified PFAD products.

Solution Structures of Two Different FRP-OCP Complexes as Revealed via SEC-SANS.

Photosynthetic organisms have established photoprotective mechanisms in order to dissipate excess light energy into heat, which is commonly known as non-photochemical quenching. Cyanobacteria utilize the orange carotenoid protein (OCP) as a high-light sensor and quencher to regulate the energy flow in the photosynthetic apparatus. Triggered by strong light, OCP undergoes conformational changes to form the active red state (OCPR). In many cyanobacteria, the back conversion of OCP to the dark-adapted state is assisted by the fluorescence recovery protein (FRP). However, the exact molecular events involving OCP and its interaction with FRP remain largely unraveled so far due to their metastability. Here, we use small-angle neutron scattering combined with size exclusion chromatography (SEC-SANS) to unravel the solution structures of FRP-OCP complexes using a compact mutant of OCP lacking the N-terminal extension (∆NTEOCPO) and wild-type FRP. The results are consistent with the simultaneous presence of stable 2:2 and 2:1 FRP-∆NTEOCPO complexes in solution, where the former complex type is observed for the first time. For both complex types, we provide ab initio low-resolution shape reconstructions and compare them to homology models based on available crystal structures. It is likely that both complexes represent intermediate states of the back conversion of OCP to its dark-adapted state in the presence of FRP, which are of transient nature in the photocycle of wild-type OCP. This study demonstrates the large potential of SEC-SANS in revealing the solution structures of protein complexes in polydisperse solutions that would otherwise be averaged, leading to unspecific results.

Quantitation of Active Pharmaceutical Ingredients in Polymeric Drug Products Using Elemental Analysis.

Polymeric prodrugs have gained significant popularity as a strategy to enhance the bioavailability and improve the pharmacokinetic properties of active pharmaceutical ingredients (API). Since the amount of the API in a polymeric prodrug product directly impacts both safety and efficacy, there is a pressing need for robust and accurate analytical methods to quantify the API in these formulations. Presently, drug quantification methods include reversed-phase high-performance liquid chromatography (RP-HPLC) and size exclusion chromatography (SEC)-based molecular weight determination. Even though these methods are highly precise and reproducible, a deep understanding of chromatography is required for complex method development, including optimization of the elution profile and selecting the appropriate column and mobile phase. In this chapter, we introduce the automated elemental analyzer for drug quantification, which is simple to use and does not require special method development.

Adaptable SEC-SAXS data collection for higher quality structure analysis in solution.

The two major challenges in synchrotron size-exclusion chromatography coupled in-line with small-angle x-ray scattering (SEC-SAXS) experiments are the overlapping peaks in the elution profile and the fouling of radiation-damaged materials on the walls of the sample cell. In recent years, many post-experimental analyses techniques have been developed and applied to extract scattering profiles from these problematic SEC-SAXS data. Here, we present three modes of data collection at the BioSAXS Beamline 4-2 of the Stanford Synchrotron Radiation Lightsource (SSRL BL4-2). The first mode, the High-Resolution mode, enables SEC-SAXS data collection with excellent sample separation and virtually no additional peak broadening from the UHPLC UV detector to the x-ray position by taking advantage of the low system dispersion of the UHPLC. The small bed volume of the analytical SEC column minimizes sample dilution in the column and facilitates data collection at higher sample concentrations with excellent sample economy equal to or even less than that of the conventional equilibrium SAXS method. Radiation damage problems during SEC-SAXS data collection are evaded by additional cleaning of the sample cell after buffer data collection and avoidance of unnecessary exposures through the use of the x-ray shutter control options, allowing sample data collection with a clean sample cell. Therefore, accurate background subtraction can be performed at a level equivalent to the conventional equilibrium SAXS method without requiring baseline correction, thereby leading to more reliable downstream structural analysis and quicker access to new science. The two other data collection modes, the High-Throughput mode and the Co-Flow mode, add agility to the planning and execution of experiments to efficiently achieve the user's scientific objectives at the SSRL BL4-2.

Molecular size distribution in pentavalent (A, C, Y, W, X) meningococcal polysaccharide conjugate vaccine by HPSEC-UV-MALS-RI method- a conceivable stability indicating parameter.

Molecular size distribution (MSD) of polysaccharides serves as a key parameter that directly correlates to the immunogenicity of vaccine. MSD at meningococcal polysaccharide (A, C, Y and W) or conjugate bulk level is well established under detailed pharmacopeial and WHO guidelines. We report here, a newly developed method for determination of molecular size distribution of pentavalent Meningococcal conjugate vaccine comprising of A, C, Y, W and X (MenFive). Although serogroup specific molecular size could not be estimated here; lot to lot consistency monitoring, molecular aggregates distribution in final lot, are key takeaways of this method. Determination of MSD in pentavalent fill finished product was quite challenging. Various columns/detectors combination, buffers, physico-chemical conditions (temperature, 2-8 °C, 25 °C, 40 °C and 60 °C; flow rate, 0.3 mL to 0.8 mL), liquid/lyophilized formulations, were explored. Polymer-based packed columns were explored for estimation for MSD by aqueous size exclusion chromatography, using combinations of- Shodex OHPAK SB 807 HQ, Shodex OHPAK SB 806 HQ, G6000 PWXL, coupled with guard Shodex OHPAK SB-G-6B. MenFive showed heterogenous distribution of molecules ranging from 200 to 19000 kDa, indicating its complex nature. However, 1000-8000 kDa was dominant range, comprising of ≥ 50 % distribution of molecules, in both liquid as well as lyophilized formulations, with average molecular weight around 6000-6500 kDa. The molar mass distribution after slicing would provide an insight to the conformation of molecules through its presentation as HMW, LMW, aggregates and subsequently, the presence of dominant population of molecules of a particular molecular weight and its total contribution in the sample.

Two-Dimensional SEC-SEC-UV-MALS-dRI Workflow for Streamlined Analysis and Characterization of Biopharmaceuticals.

The emergence of complex biological modalities in the biopharmaceutical industry entails a significant expansion of the current analytical toolbox to address the need to deploy meaningful and reliable assays at an unprecedented pace. Size exclusion chromatography (SEC) is an industry standard technique for protein separation and analysis. Some constraints of traditional SEC stem from its restricted ability to resolve complex mixtures and notoriously long run times while also requiring multiple offline separation conditions on different pore size columns to cover a wider molecular size distribution. Two-dimensional liquid chromatography (2D-LC) is becoming an important tool not only to increase peak capacity but also to tune selectivity in a single online method. Herein, an online 2D-LC framework in which both dimensions utilize SEC columns with different pore sizes is introduced with a goal to increase throughput for biomolecule separation and characterization. In addition to improving the separation of closely related species, this online 2D SEC-SEC approach also facilitated the rapid analysis of protein-based mixtures of a wide molecular size range in a single online experimental run bypassing time-consuming deployment of different offline SEC methods. By coupling the second dimension with multiangle light scattering (MALS) and differential refractive index (dRI) detectors, absolute molecular weights of the separated species were obtained without the use of calibration curves. As illustrated in this report for protein mixtures and vaccine processes, this workflow can be used in scenarios where rapid development and deployment of SEC assays are warranted, enabling bioprocess monitoring, purity assessment, and characterization.

Evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometry.

Aggregates are recognized as one of the most critical product-related impurities in monoclonal antibody (mAb)-based therapeutics due to their negative impact on the stability and safety of the drugs. So far, investigational efforts have primarily focused on understanding the causes and effects of mAb self-aggregation, including both internal and external factors. In this study, we focused on understanding mAb stability in the presence of its monovalent fragment, formed through hinge cleavage and loss of one Fab unit (referred to as "Fab/c"), a commonly observed impurity during manufacturing and stability. The Fab/c fragments were generated using a limited IgdE digestion that specifically cleaves above the IgG1 mAb hinge region, followed by hydrophobic interaction chromatographic (HIC) enrichment. Two IgG1 mAbs containing different levels of Fab/c fragments were incubated under thermally accelerated conditions. A method based on size exclusion chromatography coupled with native mass spectrometry (SEC-UV-native MS) was developed and used to characterize the stability samples and identified the formation of heterogeneous dimers, including intact dimer, mAb-Fab/c dimer, Fab/c-Fab/c dimer, and mAb-Fab dimer. Quantitative analyses on the aggregation kinetics suggested that the impact of Fab/c fragment on the aggregation rate of individual dimer differs between a glycosylated mAb (mAb1) and a non-glycosylated mAb (mAb2). An additional study of deglycosylated mAb1 under 25°C accelerated stability conditions suggests no significant impact of the N-glycan on mAb1 total aggregation rate. This study also highlighted the power of SEC-UV-native MS method in the characterization of mAb samples with regard to separating, identifying, and quantifying mAb aggregates and fragments.

Effect of Sample Preprocessing and Size-Based Extraction Methods on the Physical and Molecular Profiles of Extracellular Vesicles.

Extracellular vesicles (EVs) are nanometric lipid vesicles that shuttle cargo between cells. Their analysis could shed light on health and disease conditions, but EVs must first be preserved, extracted, and often preconcentrated. Here we first compare plasma preservation agents, and second, using both plasma and cell supernatant, four EV extraction methods, including (i) ultracentrifugation (UC), (ii) size-exclusion chromatography (SEC), (iii) centrifugal filtration (LoDF), and (iv) accousto-sorting (AcS). We benchmarked them by characterizing the integrity, size distribution, concentration, purity, and expression profiles for nine proteins of EVs, as well as the overall throughput, time-to-result, and cost. We found that the difference between ethylenediaminetetraacetic acid (EDTA) and citrate anticoagulants varies with the extraction method. In our hands, ultracentrifugation produced a high yield of EVs with low contamination; SEC is low-cost, fast, and easy to implement, but the purity of EVs is lower; LoDF and AcS are both compatible with process automation, small volume requirement, and rapid processing times. When using plasma, LoDF was susceptible to clogging and sample contamination, while AcS featured high purity but a lower yield of extraction. Analysis of protein profiles suggests that the extraction methods extract different subpopulations of EVs. Our study highlights the strengths and weaknesses of sample preprocessing methods, and the variability in concentration, purity, and EV expression profiles of the extracted EVs. Preanalytical parameters such as collection or preprocessing protocols must be considered as part of the entire process in order to address EV diversity and their use as clinically actionable indicators.

Performance and mechanism of amphiphilic polymeric chelator for enhanced removal of high concentrations of Cu(II) from wastewater.

An amphiphilic polymeric chelator (APC16-g-SX) grafted with sodium xanthate (SX) groups was successfully prepared for the efficient removal of high concentrations of Cu(II) from wastewater. The ordinary polymeric chelator (PAM-g-SX) based on linear polyacrylamide (PAM) was also prepared for comparative studies. The polymeric chelators were characterized by Fourier transform infrared spectroscopy (FT-IR), solid-state nuclear magnetic resonance (13C-NMR), gel permeation chromatography (GPC), elemental analyzer, and scanning electron microscope (SEM). The chelating performance of these polymeric chelators was investigated, and the mechanism of APC16-g-SX for enhanced removal of Cu(II) from wastewater was proposed based on fluorescence spectroscopy, cryo-scanning electron microscope (Cryo-SEM), energy-dispersive spectrometer (EDS), and X-ray photoelectron spectroscopy (XPS) tests. The results show that as the initial Cu(II) concentration in the wastewater increases, APC16-g-SX shows more excellent chelating performance than ordinary PAM-g-SX. For the wastewater with an initial Cu(II) concentration of 200 mg/L, the removal rate of Cu(II) was 99.82% and 89.34% for both 500 mg/L APC16-g-SX and PAM-g-SX, respectively. The pH of the system has a very great influence on the chelating performance of the polymeric chelators, and the increase in pH of the system helps to improve the chelating performance. The results of EDS and XPS tests also show that N, O, and S atoms in APC16-g-SX were involved in the chelation of Cu(II). The mechanism of enhanced removal of Cu(II) by APC16-g-SX can be attributed to the spatial network structure constructed by the self-association of hydrophobic groups that enhances the utilization of chelation sites.

Size-exclusion chromatography combined with DIA-MS enables deep proteome profiling of extracellular vesicles from melanoma plasma and serum.

Extracellular vesicles (EVs) are important players in melanoma progression, but their use as clinical biomarkers has been limited by the difficulty of profiling blood-derived EV proteins with high depth of coverage, the requirement for large input amounts, and complex protocols. Here, we provide a streamlined and reproducible experimental workflow to identify plasma- and serum- derived EV proteins of healthy donors and melanoma patients using minimal amounts of sample input. SEC-DIA-MS couples size-exclusion chromatography to EV concentration and deep-proteomic profiling using data-independent acquisition. From as little as 200 µL of plasma per patient in a cohort of three healthy donors and six melanoma patients, we identified and quantified 2896 EV-associated proteins, achieving a 3.5-fold increase in depth compared to previously published melanoma studies. To compare the EV-proteome to unenriched blood, we employed an automated workflow to deplete the 14 most abundant proteins from plasma and serum and thereby approximately doubled protein group identifications versus native blood. The EV proteome diverged from corresponding unenriched plasma and serum, and unlike the latter, separated healthy donor and melanoma patient samples. Furthermore, known melanoma markers, such as MCAM, TNC, and TGFBI, were upregulated in melanoma EVs but not in depleted melanoma plasma, highlighting the specific information contained in EVs. Overall, EVs were significantly enriched in intact membrane proteins and proteins related to SNARE protein interactions and T-cell biology. Taken together, we demonstrated the increased sensitivity of an EV-based proteomic workflow that can be easily applied to larger melanoma cohorts and other indications.

Evaluation of size-exclusion chromatography, multi-angle light scattering detection and mass photometry for the characterization of mRNA.

The recent approval of messenger ribonucleic acid (mRNA) as vaccine to combat the COVID-19 pandemic has been a scientific turning point. Today, the applicability of mRNA is being demonstrated beyond infectious diseases, for example in cancer immunotherapy, protein replacement therapy and gene editing. mRNA is produced by in vitro transcription (IVT) from a linear DNA template and modified at the 3' and 5' ends to improve translational efficiency and stability. Co-existing impurities such as RNA fragments and double-stranded RNA (dsRNA), amongst others, can drastically impact mRNA quality and efficacy. In this study, size-exclusion chromatography (SEC) is evaluated for the characterization of IVT-mRNA. The effect of mobile phase composition (ionic strength and organic modifier), pH, column temperature and pore size (300 Å, 1000 Å, and 2000 Å) on the separation performance and structural integrity of IVT-mRNA varying in size is described. Non-replicating, self-amplifying (saRNA), temperature degraded, and ribonuclease (RNase) digested mRNA, the latter to characterize the 3' poly(A) tail, were included in the study. Beyond ultraviolet (UV) detection, refractive index (RI) and multi-angle light scattering (MALS) detection were implemented to accurately determine molecular weight (MW) of mRNA. Finally, mass photometry is introduced as a complementary methodology to study mRNA under native conditions.

Accelerated development of a SEC-HPLC procedure for purity analysis of monoclonal antibodies using design of experiments.

The complex structure of biopharmaceutical products poses an inherent need for their thorough characterization to ensure product quality, safety, and efficacy. Analytical size exclusion chromatography (SEC) is a widely used technique throughout the development and manufacturing of monoclonal antibodies (mAbs) which quantifies product size variants such as aggregates and fragments. Aggregate and fragment content are critical quality attributes (CQAs) in mAb products, as higher contents of such size heterogeneities impact product quality. Historically, SEC methods have achieved sufficient separation between the high molecular weight (HMW) species and the main product. In contrast, some low molecular weight (LMW) species are often not sufficiently different in molecular mass from the main product, making it difficult to achieve appropriate resolutions between the two species. This lack of resolution makes it difficult to consistently quantify the LMW species in mAb-based therapeutics. The following work uses a design of experiments (DoE) approach to establish a robust analytical SEC procedure by evaluating SEC column types and mobile phase compositions using two mAb products with different physiochemical properties. The resulting optimized procedure using a Waters™ BioResolve column exhibits an improved ability to resolve and quantify mAb size variants, highlighting improvement in the resolution of the LMW species. Additionally, the addition of L-arginine as a mobile phase additive showed to reduce secondary interactions and was beneficial in increasing the recoveries of the HMW species.

Fast insights into chitosan-cleaving enzymes by simultaneous analysis of polymers and oligomers through size exclusion chromatography.

The thorough characterization of chitosan-cleaving enzymes is crucial to unveil structure-function relationships of this promising class of biomolecules for both, enzymatic fingerprinting analyses and to use the enzymes as biotechnological tools to produce tailor-made chitosans for diverse applications. Analyzing polymeric substrates as well as oligomeric products has been established as an effective way to understand the actions of enzymes, but it currently requires separate, rather laborious methods to obtain the full picture. Here, we present ultra high performance size exclusion chromatography coupled to refractive index and mass spectrometry detection (UHPSEC-RI-MS) as a straightforward method for the semi-quantitative analysis of chitosan oligomers of up to ten monomers in length. Additionally, the method allows to determine the average molecular weight of the remaining polymers and its distribution. By sampling live from an ongoing enzymatic reaction, UHPSEC-RI-MS offers the unique opportunity to analyze polymers and oligomers simultaneously-i.e., to monitor the molecular weight reduction of the polymeric substrate over the course of the digestion, while at the same time analyzing the emerging oligomeric products in a semi-quantitative manner. In this way, a single simple analysis yields detailed insights into an enzyme's action on a given substrate.

Adeno-associated virus analysis by size exclusion chromatography within 3 minutes using short bio-inert columns made with 3 µm particles operated at high flowrates.

Adeno-associated virus (AAV) analytical characterization is crucial to the well-defined and reproducible production of human gene therapies utilizing the AAV vector modality. The establishment of analytical methods based upon technology platforms currently widely used by bio-therapeutic manufacturers, namely HPLC, will assist efforts to produce high quality AAV reproducibly and decrease chemical manufacturing and control challenges in method portability and reliability. AAV analysis by size exclusion chromatography (SEC) is currently practiced with columns and mobile phase conditions traditional to SEC of proteins. Here, an improved method to measure multiple AVV critical quality attributes (CQA) rapidly by SEC is explored. The use of short columns made with small particles at high flow rates resulted in up to 80 % reduction in analysis time and 66 % in sample consumption while maintaining reliable quantitation of AAV aggregate or high molecular weight (HMW) content. These results were demonstrated across four different AAV serotypes. Furthermore, critical AAV sample handling learnings are shared.

Considering the selectivity of pore size gradient size exclusion chromatography columns.

One of the most significant performance determining variables of a size exclusion column is the pore size of its packing material. This is most definitely the case for assigning the suitability of a given column for differently sized analytes. As technologies for particle and column manufacturing continue to advance, it is worth contemplating the value of more finely controlled manipulation of this parameter. The change in a packing material's pores across the length of a size exclusion column was thus explored. A change in average pore diameter and pore size distribution was studied by means of theoretical modeling. These parameters were investigated for independent and combinatorial effects. From our predictions, versus tandem column chromatography, a gradient column apparatus does not yield sizable increases in monomer to dimer selectivity of any given critical pair. Instead, our modeling suggests it can yield more universally effective separations of multiple pairs of species at once, as is sometimes necessary when analyzing the high molecular weight components of highly aggregated drug substances.

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.

Characterization of antibody surface properties and selection of a diverse subset for development of a generic size-exclusion chromatography method.

Aggregates are an important quality attribute for biotherapeutics because they can affect the safety and efficacy of the drug and they are therefore routinely monitored, mainly with size-exclusion chromatography (SEC). However, there is often a need to tailor a SEC method to a specific molecule. This study describes the development of a generic SEC method tested on 138 antibodies with the variable domains taken from clinical stage antibodies. We report on the discovery of a subset of 12 antibodies that represents the full range of physiochemical properties found in the 138 antibodies. This subset is shown to be an efficient and reliable test set when developing chromatographic methods for antibodies. An understanding of the nature of the analyte-stationary phase interactions was gained when using this set with its wide range of physiochemical properties. Highly hydrophobic antibodies interact strongly with some modern silica hybrid materials causing the elution time to increase significantly, while a hydrophilically modified hybrid surface showed highly reduced interactions for the hydrophobic antibodies. Highly hydrophilic antibodies, on the other hand, exhibited asymmetric peaks to a certain extent on all stationary phases, while the elution time was not affected. The developed SEC method was shown to have satisfactory performance in terms of linearity, repeatability, range, and accuracy and exhibit very narrow distributions of elution time and peak symmetry when testing the 138 antibodies indicating its generic performance.