Dynamic Binding Capacity Research Articles

Different VH3-Binding Protein A Resins Show Comparable VH3-Binding Mediated By product Separation Capabilities Despite Having Varied Dynamic Binding Capacities Towards A VH3 Fab.

Protein A resins have been widely used for product capture during mAb, bispecific antibody (bsAb), and Fc-fusion protein purification. While Protein A ligands mainly bind the Fc region, many of them can also bind the VH3 domain. During mAb/bsAb purification, certain truncated byproducts may contain the same Fc region as the product but fewer numbers of the VH3 domain. In such a scenario, VH3-binding Protein A resins provide a potential means for byproduct separation based on the difference in VH3-binding valency. As the ligands of different VH3-binding Protein A resins are derived from distinct domains of the native Protein A, it would be interesting to know whether they possess comparable capabilities for separating species with the same Fc region but different numbers of VH3 domain. This study aims to explore the potential of different VH3-binding Protein A resins for separating antibody species with the same Fc region but different numbers of VH3 domain. The VH3 Fab was released from a VH3-containing mAb by papain digestion. Post digestion, the released VH3 Fab was purified sequentially using CaptureSelect CH1-XL and MabSelect SuRe affinity chromatography. The purified VH3 Fab was used as the load material to assess the dynamic binding capacity (DBC) of five VH3-binding Protein A resins (i.e., Amshpere A3, Jetted A50, MabCapture C, MabSelect and MabSelect PrismA). The potential of VH3-binding Protein A resins for separating species having the same Fc region but different numbers of VH3 domain was evaluated using an artificial mixture composed of the product and a truncated byproduct, which contained one and zero VH3 domain, respectively (both species contained the same Fc region). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to monitor Fab purification and separation of species containing the same Fc region but different numbers of VH3 domain. When loaded with an isolated VH3 Fab, different VH3-binding Protein A resins showed varied DBCs. Nevertheless, when these Protein A resins were used to separate a truncated byproduct, which contained the Fc region only without any VH3 domain, from the product, which included one VH3 domain in addition to the Fc region, they showed comparable capabilities for separating these two species. Although different VH3-binding Protein A resins showed varied DBCs towards a VH3 Fab, they exhibited comparable capabilities for separating species with the same Fc region but different numbers of VH3 domain.

Purification of his-tagged proteins using printed monolith adsorption columns

Bio-separation is a crucial process in biotechnology and biochemical engineering for separating biological macromolecules, and the field has long relied on bead-based and expanded bed chromatography. Printed monolith adsorption (PMA) is a new alternative to which uses a 3D-printed monolithic structure containing self-supporting, ordered flow channels. PMA allows for direct purification of biological molecules from crude cell lysates and cell cultures, and like the other technologies, can functionalized to specifically target a molecule and enable affinity chromatography. Here we have combined PMA technology with an immobilized metal affinity ligand (iminodiacetic acid) to provide selectivity of binding to polyhistidine-tagged proteins during PMA chromatography. Two different PMA structures were created and tested for both static and dynamic protein-binding capacity. At comparative linear flow rates, the dynamic binding capacity of both columns was ≈3 mg/mL, while static capacity was shown to differentiate based on column voidage. We show that a polyhistidine-tagged protein can be directly purified from crude lysate with comparable results to the available commercial providers of IMAC, and with a substantially reduced purification time.

Biolayer interferometry for adeno-associated virus capsid titer measurement and applications to upstream and downstream process development

Faster and more accurate analytical methods are needed to support the advancement of recombinant adeno-associated virus (rAAV) production systems. Recently, bio-layer interferometry (BLI) has been developed for high-throughput AAV capsid titer measurement by functionalization of the AAVX ligand onto biosensor probes (AAVX-BLI). In this work, an AAVX-BLI method was evaluated using Octet® AAVX biosensors across four rAAV serotypes (rAAV2, 5, 8, and 9) and applied in an upstream and downstream processing context. AAVX-BLI measured capsid titer across a wide concentration range (1×1010 – 1×1012 capsids/mL) for different rAAV serotypes and sample backgrounds with reduced measurement variance and error compared to an enzyme-linked immunosorbent assay (ELISA) method. Biosensors were regenerated for repeated use, with lysate samples showing reduced regeneration capacity compared to purified and supernatant samples. The AAVX-BLI method was applied in a transfection optimization study where direct capsid titer measurement of culture supernatants generated a representative response surface for total vector genome (VG) titer. For rAAV purification, AAVX-BLI was used to measure dynamic binding capacity with POROS™ CaptureSelect™ AAVX affinity chromatography, showing resin breakthrough dependence on operating flow rate. Measurement accuracy, serotype and sample background flexibility, and high sample throughput make AAVX-BLI an attractive alternative to other capsid titer measurement techniques.

Biocatalytic Method for Producing an Affinity Resin for the Isolation of Immunoglobulins.

Affinity chromatography is a widely used technique for antibody isolation. This article presents the successful synthesis of a novel affinity resin with a mutant form of protein A (BsrtA) immobilized on it as a ligand. The key aspect of the described process is the biocatalytic immobilization of the ligand onto the matrix using the sortase A enzyme. Moreover, we used a matrix with primary amino groups without modification, which greatly simplifies the synthesis process. The resulting resin shows a high dynamic binding capacity (up to 50 mg IgG per 1 mL of sorbent). It also demonstrates high tolerance to 0.1 M NaOH treatment and maintains its effectiveness even after 100 binding, elution, and sanitization cycles.

Comparison of batch and continuous multi-column capture of monoclonal antibodies with convective diffusive membrane adsorbers

Protein A affinity membrane adsorbers are a promising alternative to resins to intensify the manufacturing of monoclonal antibodies. This study examined the process performance of convective diffusive membrane adsorbers operated in batch and continuous multi-column mode. Therefore, three different processes were compared regarding membrane utilization, productivity, and buffer consumption: the batch process, the rapid cycling parallel multi-column chromatography process, and the rapid cycling simulated moving bed process. The influence of the monoclonal antibody loading concentration (between 0.5 g L-1 and 5.2 g L-1) and the loading flow rate (between 1.25 MV min-1 and 10 MV min-1) on the monoclonal antibody binding behavior of the membrane adsorber were studied with breakthrough curve experiments. The determined breakthrough curves were used to calculate the monoclonal antibody dynamic binding capacity, the duration of the loading steps for each process, and the number of required membrane adsorbers for the continuous processes rapid cycling parallel multi-column chromatography and rapid cycling simulated moving bed. The highest productivity for the batch (176 g L-1 h-1) and rapid cycling parallel multi-column chromatography process (176 g L-1 h-1) was calculated for high monoclonal antibody loading concentrations and low loading flow rates. In contrast, the rapid cycling simulated moving bed process achieved the highest productivity (217 g L-1 h-1) for high monoclonal antibody loading concentrations and loading flow rates. Furthermore, due to the higher membrane utilization, the buffer consumption of the rapid cycling simulated moving bed process (1.1 L g-1) was up to 1.9 times lower than that of the batch or rapid cycling parallel multi-column chromatography operation (2.1 L g-1).

Development of temperature-controlled batch and 3-column counter-current protein A system for improved therapeutic purification

Maximizing product quality attributes by optimizing process parameters and performance attributes is a crucial aspect of bioprocess chromatography process design. Process parameters include but are not limited to bed height, eluate cut points, and elution pH. An under-characterized chromatography process parameter for protein A chromatography is process temperature. Here, we present a mechanistic understanding of the effects of temperature on the protein A purification of a monoclonal antibody (mAb) using a commercial chromatography resin for batch and continuous counter-current systems. A self-designed 3D-printed heating jacket controlled the 1 mL chromatography process temperature during the loading, wash, elution, and cleaning-in-place (CIP) steps. Batch loading experiments at 10, 20, and 30 °C demonstrated increased dynamic binding capacity (DBC) with temperature. The experimental data were fit to mechanistic and correlation-based models that predicted the optimal operating conditions over a range of temperatures. These model-based predictions optimized the development of a 3-column temperature-controlled periodic counter-current chromatography (TCPCC) and were validated experimentally. Operating a 3-column TCPCC at 30 °C led to a 47% increase in DBC relative to 20 °C batch chromatography. The DBC increase resulted in a two-fold increase in productivity relative to 20 °C batch. Increasing the number of columns to the TCPCC to optimize for increasing feed concentration resulted in further improvements to productivity. The feed-optimized TCPCC showed a respective two, three, and four-fold increase in productivity at feed concentrations of 1, 5, and 15 mg/mL mAb, respectively. The derived and experimentally validated temperature-dependent models offer a valuable tool for optimizing both batch and continuous chromatography systems under various operating conditions.

Grafting chromatographic monoliths with charged linear polymers for highly productive and selective protein or plasmid DNA purification

Increased requirements for plasmid DNA (pDNA) in gene therapy and vaccination efforts bring the need for efficient large-scale production processes with high purity and productivity. Chromatographic purification of pDNA is often a bottleneck due to low dynamic binding capacities (DBC), pDNA recovery and/or low selectivity of columns. Ion exchangers (IEX) with charged groups on surface extenders (polyions formed by a grafting reaction and subsequents modification) have attracted attention due to their greatly enhanced productivity compared with traditional IEX. The ultimate goal of our investigation was to increase the DBC for pDNA on a monolith stationary phase by grafting the surface with linear polymethacrylate brushes (grafted chains), while retaining high pDNA recovery and chromatographic selectivity between pDNA and RNA impurities. The inner walls of the Convective Interaction Media® monolith channels were successfully coated with polyglycidyl methacrylate linear chains of varying densities and lengths by employing a controlled radical polymerization reaction. Glycidyl groups throughout the length of the formed grafted chains were converted to cation- (CEX) or anion-exchanging (AEX) moieties. Preliminary chromatographic evaluation of novel grafted CEX columns for different model proteins exhibited 10-times higher DBC with high elution recovery (99 %). Similar DBC improvement was proven for 7.3 kilobase pair pDNA, where the DBC10 increased from 1.8 mg pDNA per mL of non-grafted chromatographic support up to 15.2 mg per the most densely distributed and the longest grafted chains, modified with weak AEX groups. However, the DBC increase was partially at the expense of pDNA elution recovery, which presumably decreased due to entanglement of pDNA molecules inside the dense grafted layer. To confirm the hypothesis, we have prepared a series of columns differing in length and density of grafted chains and tested them for pDNA capture. A satisfactory compromise between high pDNA DBC and elution recovery was found with relatively long and low density grafted chains. The optimal grafted AEX column with 1 mL bed volume was evaluated for pDNA capture from neutralized E. coli lysate. With a capacity of 13.5 mg pDNA per mL support, ≥95 % elution recovery, and complete RNA clearance, the pDNA was successfully purified at loading flow rate of at least 15 column volumes per min. According to our knowledge and literature search, these process characteristics would enable one of the highest pDNA isolation productivities to be achieved with currently tested IEX.

Exploring the effects of resin particle sizes on enhancing antibody binding capacity of a hybrid biomimetic ligand

Particle size is a critical parameter of chromatographic resins that significantly affects protein separation. In this study, effects of resin particle sizes (31.26 μm, 59.85 μm and 85.22 μm named Aga-31, Aga-60 and Aga-85, respectively) on antibody adsorption capacity and separation performance of a hybrid biomimetic ligand were evaluated. Their performance was investigated through static adsorption and breakthrough assays to quantify static and dynamic binding capacity (Qmax and DBC). The static adsorption results revealed that the Qmax for hIgG was 152 mg/g resin with Aga-31, 151 mg/g resin with Aga-60, and 125 mg/g resin with Aga-85. Moreover, the DBC at 10% breakthrough for hIgG with a residence time of 2 min was determined to be 49.4 mg/mL for Aga-31, 45.9 mg/mL for Aga-60, and 38.9 mg/mL for Aga-85. The resins with smaller particle sizes exhibited significantly higher capacity compared to typical commercial agarose resins and a Protein A resin (MabSelect SuRe). Furthermore, the Aga-31 resin with the hybrid biomimetic ligand demonstrated exceptional performance in terms of IgG purity (>98%) and recovery (>96%) after undergoing 20 separation cycles from CHO cell supernatant. These findings are helpful in further chromatographic resin design for the industrial application of antibody separation and purification.

Preparation technology comparison and performance evaluation of different protein A affinity chromatographic materials

Protein A affinity chromatographic materials are widely used in clinical medicine and biomedicine because of their specific interactions with immunoglobulin G (IgG). Both the characteristics of the matrix, such as its structure and morphology, and the surface modification method contribute to the affinity properties of the packing materials. The specific, orderly, and oriented immobilization of protein A can reduce its steric hindrance with the matrix and preserve its bioactive sites. In this study, four types of affinity chromatographic materials were obtained using agarose and polyglycidyl methacrylate (PGMA) spheres as substrates, and multifunctional epoxy and maleimide groups were used to fix protein A. The effects of the ethylenediamine concentration, reaction pH, buffer concentration, and other conditions on the coupling efficiency of protein A and adsorption performance of IgG were evaluated. Multifunctional epoxy materials were prepared by converting part of the epoxy groups of the agarose and PGMA matrices into amino groups using 0.2 and 1.6 mol/L ethylenediamine, respectively. Protein A was coupled to the multifunctional epoxy materials using 5 mmol/L borate buffer (pH 8) as the reaction solution. When protein A was immobilized on the substrates by maleimide groups, the agarose and PGMA substrates were activated with 25% (v/v) ethylenediamine for 16 h to convert all epoxy groups into amino groups. The maleimide materials were then converted into amino-modified materials by adding 3 mg/mL 3-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) dissolved in dimethyl sulfoxide (DMSO) and then suspended in 5 mmol/L borate buffer (pH 8). The maleimide groups reacted specifically with the C-terminal of the sulfhydryl group of recombinant protein A to achieve highly selective fixation on both the agarose and PGMA substrates. The adsorption performance of the affinity materials for IgG was improved by optimizing the bonding conditions of protein A, such as the matrix type, matrix particle size, and protein A content, and the adsorption properties of each affinity material for IgG were determined. The column pressure of the protein A affinity materials prepared using agarose or PGMA as the matrix via the maleimide method was subsequently evaluated at different flow rates. The affinity materials prepared with PGMA as the matrix exhibited superior mechanical strength compared with the materials prepared with agarose. Moreover, an excellent linear relationship between the flow rate and column pressure of 80 mL/min was observed for this affinity material. Subsequently, the effect of the particle size of the PGMA matrix on the binding capacity of IgG was investigated. Under the same protein A content, the dynamic binding capacity of the affinity materials on the PGMA matrix was higher when the particle size was 44-88 μm than when other particle sizes were used. The properties of the affinity materials prepared using the multifunctional epoxy and maleimide-modified materials were compared by synthesizing affinity materials with different protein A coupling amounts of 1, 2, 4, 6, 8, and 10 mg/mL. The dynamic and static binding capacities of each material for bovine IgG were then determined. The prepared affinity material was packed into a chromatographic column to purify IgG from bovine colostrum. Although all materials showed specific adsorption selectivity for IgG, the affinity material prepared by immobilizing protein A on the PGMA matrix with maleimide showed significantly better performance and achieved a higher dynamic binding capacity at a lower protein grafting amount. When the protein grafting amount was 15.71 mg/mL, the dynamic binding capacity of bovine IgG was 32.23 mg/mL, and the dynamic binding capacity of human IgG reached 54.41 mg/mL. After 160 cycles of alkali treatment, the dynamic binding capacity of the material reached 94.6% of the initial value, indicating its good stability. The developed method is appropriate for the production of protein A affinity chromatographic materials and shows great potential in the fields of protein immobilization and immunoadsorption material synthesis.

Preparation of a macroporous anion exchange chromatographic medium by polyamine grafting and evaluation of its protein-adsorption behavior

The macroporous anion exchange chromatographic medium (FastSep-PAA) was prepared through grafting polyallylamine (PAA) onto polyacrylate macroporous microspheres (FastSep-epoxy). The effects of the synthesis conditions, including the PAA concentration, reaction time, and reaction solution pH, on the ion exchange (IC) of the medium were investigated in detail. When the PAA concentration, reaction time, and reaction solution pH were increased, the IC of the medium increased, and optimal synthesis conditions were then selected in combination with changes of protein binding capacity. A scanning electron microscope was used to examine the surface morphology of the medium. The medium possessed high pore connectivity. Furthermore, the pore structure of the medium was preserved after the grafting of PAA onto the macroporous microspheres. This finding demonstrates that the density of the PAA ligands does not appear to have any discernible impact on the structure of the medium; that is, no difference in the structure of the medium is observed before and after the grafting of PAA onto the microspheres. The pore size and pore-size distribution of the medium before and after grafting were determined by mercury intrusion porosimetry and the nitrogen adsorption method to investigate the relationship between pore size (measured in the range of 300-1000 nm) and protein adsorption. When the pore size of the medium was increased, its protein binding capacity did not exhibit any substantial decrease. An increase in pore size may hasten the mass transfer of proteins within the medium. Among the media prepared, that with a pore size of 400 nm exhibited the highest dynamic-binding capacity (DBC: 70.3 g/L at 126 cm/h). The large specific surface area of the medium and its increased number of protein adsorption sites appeared to positively influence its DBC. When the flow rate was increased, the protein DBC decreased in media with original pore sizes of less than 700 nm. In the case of the medium with an original pore size of 1000 nm, the protein DBC was independent of the flow rate. The protein DBC decreased by 3.5% when the flow rate was increased from 126 to 628 cm/h. In addition, the protein DBC was maintained at 57.7 g/L even when the flow velocity was 628 cm/h. This finding reveals that the diffusion rate of protein molecules at this pore size is less restricted and that the prepared medium has excellent mass-transfer performance. These results confirm that the macroporous polymer anion exchange chromatographic medium developed in this study has great potential for the high-throughput separation of proteins.

Continuous multi-column capture of monoclonal antibodies with convective diffusive membrane adsorbers.

Downstream processing is the bottleneck in the continuous manufacturing of monoclonal antibodies (mAbs). To overcome throughput limitations, two different continuous processes with a novel convective diffusive protein A membrane adsorber (MA) were investigated: the rapid cycling parallel multi-column chromatography (RC-PMCC) process and the rapid cycling simulated moving bed (RC-BioSMB) process. First, breakthrough curve experiments were performed to investigate the influence of the flow rate on the mAb dynamic binding capacity and to calculate the duration of the loading steps. In addition, customized control software was developed for an automated MA exchange in case of pressure increase due to membrane fouling to enable robust, uninterrupted, and continuous processing. Both processes were performed for 4 days with 0.61 g L-1 mAb-containing filtrate and process performance, product purity, productivity, and buffer consumption were compared. The mAb was recovered with a yield of approximately 90% and productivities of 1010 g L-1 d-1 (RC-PMCC) and 574 g L-1 d-1 (RC-BioSMB). At the same time, high removal of process-related impurities was achieved with both processes, whereas the buffer consumption was lower for the RC-BioSMB process. Finally, the attainable productivity for perfusion bioreactors of different sizes with suitable MA sizes was calculated to demonstrate the potential to operate both processes on a manufacturing scale with bioreactor volumes of up to 2000 L.

Capture of fully assembled secretory immunoglobulin A by affinity chromatography with nanobodies as ligands

Secretory immunoglobulin A (sIgA) has strongpotential for passive immunization and other therapeutic applications. This complex molecule with a molecular mass of 410 kDa consists of two IgA monomers linked by a connecting chain (J) and a secretory component (SC). Fully assembled sIgA was overexpressed in CHO cells. In order to exploit the therapeutic potential of recombinant sIgA expressed in CHO cells, a platform process for capture of the antibody directly from the clarified culture supernatant using available affinity chromatography material was investigated. The hydrodynamic radius was 10.2 ± 0.9 nm determined by dynamic light scattering and the isoelectric point 5.5 by electrophoretic mobility using the Malvern Zetasizer. Capture Select IgA and Capture Select IgA-CH1 chromatography media with camelid-derived single-domain antibody fragments comprising the 3 CDR domain antibody fragment with a 13 kD or 14 kDa as ligand were used to purify the antibody. sIgA was directly captured from the clarified culture supernatant and a highly purified, full-length, correctly assembled sIgA was obtained. The residence time must be in the range of 5–10 min to achieve a high binding capacity. The effective diffusivity of sIgA was in the range of 10−9 cm2 s−1 and the dynamic binding capacity was in the range of 10 mg/ml packed bed. We conclude that the current version of the resin has a pore size that is too small for this type of molecule to achieve substantial resin utilization and productivity, although affinity ligand-based purification led to high degree of purity suited for an industrial capture process.

Mixed-mode size-exclusion silica resin for polishing human antibodies in flow-through mode

The polishing step in the downstream processing of therapeutic antibodies removes residual impurities from Protein A eluates. Among the various classes of impurities, antibody fragments are especially challenging to remove due to the broad biomolecular diversity generated by a multitude of fragmentation patterns. The current approach to fragment removal relies on ion exchange or mixed-mode adsorbents operated in bind-and-gradient-elution mode. However, fragments that bear strong similarity to the intact product or whose biophysical features deviate from the ensemble average can elude these adsorbents, and the lack of a chromatographic technology enabling robust antibody polishing is recognized as a major gap in downstream bioprocessing. Responding to this challenge, this study introduces size-exclusion mixed-mode (SEMM) silica resins as a novel chromatographic adsorbent for the capture of antibody fragments irrespective of their biomolecular features. The pore diameter of the silica beads features a narrow distribution and is selected to exclude monomeric antibodies, while allowing their fragments to access the pores where they are captured by the mixed-mode ligands. The static and dynamic binding capacity of the adsorbent ranged respectively between 30-45 and 25–33 gs of antibody fragments per liter of resin. Selected SEMM-silica resins also demonstrated the ability to capture antibody aggregates, which adsorb on the outer layer of the beads. Optimization of the SEMM-silica design and operation conditions – namely, pore size (10 nm) and ligand composition (quaternary amine and alkyl chain) as well as the linear velocity (100 cm/h), ionic strength (5.7 mS/cm), and pH (7) of the mobile phase – afforded a significant reduction of both fragments and aggregates, resulting into a final antibody yield up to 80% and monomeric purity above 97%.

Improved mRNA affinity chromatography binding capacity and throughput using an oligo-dT immobilized electrospun polymer nanofiber adsorbent

Increased demand for mRNA-based therapeutics and improved in vitro transcription (IVT) yields have challenged the mRNA purification platform. Hybridization-affinity chromatography with an immobilized oligo-deoxythymidilic acid (oligodT) ligand is often used to capture mRNA through base pairing with the polyadenylated tail. Commercially available oligodT matrices include perfusive cross-linked poly(styrene-divinylbenzene) 50 µm POROS™ chromatography resin beads and convective polymethacrylate CIMmultus® monolithic columns consisting of 2 µm interconnected channels. POROS™ columns may be limited by poor mass transfer for larger mRNAs and slow flowrates, while monoliths can operate at higher flowrates but are limited by modest binding capacity. To enable both high flowrates and binding capacity for mRNA of all lengths, prototype chromatography media was developed by Cytiva using oligodT immobilized electrospun cellulose nanofibers (Fibro™) with a 0.3–0.4 µm pore size. In this work, four polyadenylated mRNAs ranging from ∼1900–4300 nucleotides were used to compare the dynamic binding capacity (DBC) of Fibro™, POROS® and CIMmultus® columns as a function of residence time and binding buffer compositions. Fibro™ improved the DBC ∼2–4-fold higher than CIMmultus® and ∼2–13-fold higher than POROS™ across all residence times, mRNA length, and binding matrix compositions tested. CIMmultus® DBC was least dependent on residence time and mRNA size, while both Fibro™ and POROS™ DBC increased at slower flowrates and with shorter mRNA. Surprisingly, inverse size exclusion (ISE) experiments showed that POROS™ was not limited by diffusion and POROS™ along with CIMmultus® demonstrate higher mRNA permeation however the Fibro™ prototype is not in the final configuration. Lastly, IVT reaction products were subjected to purification and oligodT elution product yield, quality, and purity were consistent across the three matrices investigated. These results highlight the benefits of high DBC and equivalent product profiles offered by the oligodT Fibro™ prototype compared to commercially available oligodT media.

Affinity Resins for the Isolation of Immunoglobulins G Obtained Using Biocatalytic Technology.

Affinity chromatography resins that are obtained by conjugation of matrices with proteins of bacterial origin, like protein A, are frequently used for the purification of numerous therapeutic monoclonal antibodies. This article presents the development of a biocatalytic method for the production of novel affinity resins with an immobilized mutant form of protein A via sortase A mediated reaction. The conditions for activation of the agarose Seplife 6FF matrix, selection of different types of linkers with free amino groups and conditions for immobilization of recombinant protein A on the surface of the activated matrix were studied. Finally, the basic operational properties, like dynamic binding capacity (DBC), temperature dependance of DBC and stability during the cleaning-in-place process of the affinity resin with the Gly-Gly-EDA-Gly-Gly linker, were assessed using recombinant hyperchimeric monoclonal antibodies. The main characteristics show comparable results with the widely used commercial samples.

On-line PAT based monitoring and control of resin aging in protein A chromatography for COGs reduction

Resin aging is a common occurrence in chromatographic processes and generally influenced by factors such as cleaning procedure and composition of the feed stream. Two major events occur along with protein fouling, one is the loss of protein A ligand and the other is non-specific, irreversible interactions of foulants with resin particles. Both these are responsible for resin aging. As a result, the performance of the resin suffers a fall, and this can be quantified through indicators like reduction in dynamic binding capacity, increased column pressure, or peak broadening. The number of reuse cycles of a resin has a major influence on the cost per batch. This is even more significant in the case of protein A resin, which is the primary cost driver for downstream processing. In this work, we first identify chromatogram characteristics that correlate to resin aging. Next, we propose a data monitoring-based tool for prediction of resin aging. Principal component analysis of the UV data of Mab 1 showed a deviation at 120th cycle and an out of specification at around 149th cycle, corroborating with yield decline. Batch level modelling could deliver a predictable trend for resin aging and was demonstrated for two different Mabs (Mab1 and Mab2). The results demonstrate that significant resin aging can be detected 20–25 cycles prior to observable yield decline. A control strategy has been suggested such that once the deviation has been detected, additional resin cleaning is triggered. Overall, a 50–100 Protein A cycle enhancement in resin lifespan could be achieved.

Preparation of Protein A Membranes Using Propargyl Methacrylate-Based Copolymers and Copper-Catalyzed Alkyne-Azide Click Chemistry.

The development of convective technologies for antibody purification is of interest to the bioprocessing industries. This study developed a Protein A membrane using a combination of graft polymerization and copper(I)-catalyzed alkyne-azide click chemistry. Regenerated cellulose supports were functionalized via surface-initiated copolymerization of propargyl methacrylate (PgMA) and poly(ethylene glycol) methyl ether methacrylate (PEGMEMA300), followed by a reaction with azide-functionalized Protein A ligand. The polymer-modified membranes were characterized using attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR), gravimetric analysis, and permeability measurements. Copolymer composition was determined using the Mayo-Lewis equation. Membranes clicked with azide-conjugated Protein A were evaluated by measuring static and dynamic binding (DBC10) capacities for human immunoglobulin G (hIgG). Copolymer composition and degree of grafting were found to affect maximum static binding capacities, with values ranging from 5 to 16 mg/mL. DBC10 values did not vary with flow rate, as expected of membrane adsorbers.

Evaluation of novel chromatographic prototypes for supercoiled plasmid DNA polishing.

Since the world first approved gene therapeutics, nucleic acid-based therapies have gained prominence. Several strategies for DNA-based therapy have been approved, and numerous clinical trials for plasmid DNA (pDNA)-based vaccines are currently in development. Due to the rising interest in pDNA for vaccination and gene therapy, plasmid manufacturing must become more effective. One of the most critical steps is downstream processing, involving isolation and purification procedures. To comply with the regulatory guidelines, pDNA must be available as a highly purified, homogeneous preparation of supercoiled pDNA (sc pDNA). This process undertakes several challenges, primarily due to the diversity of molecules derived from the producer organism. In this study, different resins were tested for the adsorption and selective polishing of sc pDNA. To identify optimal pDNA adsorption conditions, batch and column assays were performed with different resins while promoting electrostatic and hydrophobic interactions. The effect of ionic strength, pH, and contact time were evaluated and optimized. Additionally, static and dynamic binding capacities were determined for the selected resins. Analytical chromatography and agarose gel electrophoresis were used to assess the selectivity of the most promising resins toward sc pDNA isoform. Also, genomic DNA, endotoxins, and proteins were quantified to characterize the final sc pDNA quality. At the same time, the recovery and purity yields were evaluated by quantification of sc pDNA after the purification procedure. Overall, the results of the chromatographic assays using agmatine- and arginine-based resins have shown promising potential for sc pDNA polishing. Both resins demonstrated excellent binding capacity for pDNA, with agmatine outperforming arginine-based resin in terms of capacity. However, arginine-based resin exhibited a superior pDNA recovery yield, reaching a notable 52.2% recovery compared to 10.09% from agmatine. Furthermore, both resins exhibited high relative purity levels above 90% for the sc pDNA. The comprehensive characterization of the recovered sc pDNA also revealed a significant reduction in gDNA levels, reinforcing the potential of these prototypes for obtaining high-quality and pure sc pDNA. These findings highlight the promising applications of both resins in scalable pDNA purification processes for gene therapy and biopharmaceutical applications.

Enhanced adsorption performance of varying-length mRNA on Oligo dT affinity resins through optimal pore size and grafting

The enormous potential of mRNA-based therapeutics in vaccines and medicine has led to the consideration of various mRNA sizes, ranging from ∼1000 to 10,000 nucleotides (nt). However, the commonly used Oligo-deoxythymidine (Oligo dT) affinity resins are not initially designed for such a wide range of sizes, and present challenges in achieving optimal performance for the separation of a given mRNA. In this paper, two factors of the resin beads, pore size and grafting were focused. The effects of varying pore sizes, ranging from 150 to 850 nm, on both static and dynamic adsorption for varying-length mRNA (1000–8500 nt) were investigated. It was found that static adsorption capacity and uptake kinetics were correlated with the mRNA length. Interestingly, dynamic binding capacities (DBCs) showed an increase-then-decrease trend with increasing pore size. An optimal pore size of about 350 nm could lead to the highest DBCs for all tested mRNA. Additionally, the effects of polymer grafting on the adsorption performance were studied. The results revealed that polymer-grafted resin exhibited 24–55% higher DBCs than non-grafted resin. The enhanced DBCs could be attributed to the improved uptake rate facilitated by the polymer grafted. These investigations emphasize the importance of optimizing pore size and grafting for Oligo dT affinity resins, providing valuable guidance for selecting and designing new resins for different mRNA products.

Evaluation of mild pH elution protein A resins for antibodies and Fc-fusion proteins

Protein A affinity chromatography is widely used as a capture step for monoclonal antibodies (mAb) and molecules that possess an Fc-domain, such as fusion proteins and bispecific antibodies. However, the use of low pH (3.0–4.0) to elute the molecule and achieve acceptable yield (>85 %) can lead to product degradation (e.g. fragmentation, aggregation) for molecules sensitive to low pH. In this paper, we describe a comprehensive evaluation of two protein A resins with ligands designed to elute at a milder pH as a result of modified sequences in their Fc and VH3 binding regions. One of the evaluated resins has been made commercially available by Purolite and named Praesto Jetted A50 HipH. Results demonstrated that Jetted A50 HipH could elute the Fc-fusion protein and most mAbs evaluated with an elution pH at or above 4.6. Elution and wash optimization determined run conditions for high recovery (>90 % monomer yield), reduction of high molecular weight (HMW) species (>50 %), and significant host cell protein (HCP) clearance at the mildest elution pH possible. For a pH-stable mAb and a pH-sensitive fusion protein, cell culture material was purified with optimized conditions and demonstrated the mild elution pH resins' ability to purify product with acceptable yield, comparable or better impurity clearance, and significantly milder native eluate pH compared to traditional resins. The benefits of the mild elution pH resins were clearly exemplified for the pH-sensitive protein, where a milder elution buffer and native eluate pH resulted in only 2 % HMW in the eluate that remained stable over 48 h. In contrast, a traditional protein A resin requiring low pH elution led to eluate HMW levels of 8 %, which increased to 16 % over the same hold time. Additionally, these resins have high dynamic binding capacity and allow the use of traditional HCP washes. Therefore, Jetted A50 HipH is an ideal candidate for a platform protein A resin and provides flexibility for pH-sensitive proteins and stable mAbs, while preserving product quality, recovery, and seamless integration into a downstream process.