•12 Protein A stationary phases were compared using nine Fc-containing proteins.•Morphological properties were investigated using four orthogonal techniques.•Product quality and yield were evaluated ...under realistic bioprocess conditions.•Mass transport was quantified using the chromatography general rate model.•Dynamic binding capacities for mAbs ranged from 37–72 mg/mL at 4 min residence time.
We describe a comprehensive evaluation of 12 Protein A stationary phases for capture of biotherapeutics. We first examine the morphological properties of the stationary phases using a variety of orthogonal techniques including electron microscopy, particle sizing, pressure-flow behavior, and isocratic pulse response. A panel of nine proteins spanning a wide range of structures and biochemical properties was then used to assess equilibrium uptake, mass transport, dynamic binding capacity, and elution pH. Process performance and product quality were also examined under realistic bioprocess conditions using clarified mammalian cell culture broth. Equilibrium isotherms were found to be highly favorable, with equilibrium binding capacity for monoclonal and bispecific antibodies ranging from 47–100 mg/mL packed bed across all stationary phases tested. Effective pore diffusivities, De, were obtained by fitting the chromatography general rate model to breakthrough data. The fitted De values for monoclonal antibodies ranged from 1.1–5.7 × 10−8 cm2/s. The stationary phases had high dynamic binding capacities for the model proteins. The highest dynamic capacities for monoclonal and bispecific antibodies were seen with MabSelect SuRe pcc and MabSelect PrismA, which ranged from 58–74 mg/mL packed bed at 4 min residence times. Product capture using clarified cell culture broth as a feedstock showed high yields and elution pool volumes that ranged from 2–3 column volumes in most cases. Host cell protein, DNA, and aggregate levels in the elution pool were dependent on the specific nature of protein being purified, and levels were consistent between stationary phases. Lastly, we perform an analysis of bivariate correlations and discuss considerations for process design and optimization.
•Structure and function are compared for core shell resins Capto Core 400 and 700.•Capto Core 400 has higher binding capacity for small proteins but slower kinetics.•Capto Core 700 allows large ...proteins up to 660 kDa to diffuse and bind.•The pore diffusion model describes transport through the shell and binding in the core.•Large bound proteins hinder diffusion of smaller proteins in the core in both resins.
Structural and functional characteristics of the two core-shell resins Capto™ Core 400 and 700, which are useful for the flow-through purification of bioparticles such as viruses, viral vectors, and vaccines, are compared using bovine serum albumin (BSA) and thyroglobulin (Tg) as models for small and large protein contaminants. Both resins are agarose-based and contain an adsorbing core surrounded by an inert shell. Although shell thicknesses are comparable (3.6 and 4.2 µm for Capto Core 400 and 700, respectively), the two resins differ substantially in pore size (pore radii of 19 and 50 nm, respectively). Because of the smaller pores and higher surface area, the BSA binding capacity of Capto Core 400 is approximately double that of Capto Core 700. However, for the much larger Tg, the attainable capacity is substantially larger for Capto Core 700. Mass transfer in both resins is affected by diffusional resistances through the shell and within the adsorbing core. For BSA, core and shell effective pore diffusivities are about 0.25 × 10−7 and 0.6 × 10−7 cm2/s, respectively, for Capto Core 400, and about 1.6 × 10−7 and 2.6 × 10−7 cm2/s, respectively, for Capto Core 700. These values decrease dramatically for Tg to 0.022 × 10−7 and 0.088 × 10−7 cm2/s and to 0.13 × 10−7 and 0.59 × 10−7 cm2/s for Capto Core 400 and 700, respectively. Adsorbed Tg further hinders diffusion of BSA in both resins. Column measurements show that, despite the higher static capacity of Capto Core 400 for BSA, the dynamic binding capacity is greater for Capto Core 700 as a result of its faster kinetics. However, some of this advantage is lost if the feed is a mixture of BSA and Tg since, in this case, Tg binding leads to greater diffusional hindrance for BSA.
•Development of reproducible and scalable grafting procedure for chromatographic monoliths.•Up to 72 mg lysozyme /mL dynamic binding capacity in CEX mode using grafted monoliths.•Grafted monoliths ...result in up to 15 mg pDNA /mL dynamic binding capacity in AEX mode.•Long and dense grafted layer is optimal for pDNA capture in terms of elution recovery.•pDNA isolation on 1 mL scale with 3.4 g/L·h productivity and complete RNA removal.
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.
•Weak ion-exchange nanofiber exhibits an effective capture for lysozyme.•At pH 9, the EBC value was approximately 698.91 mg/g.•A comparison was made between linear and nonlinear analyses on the ...breakthrough curves.•Breakthrough curves were well-fitted by the Yoon-Nelson model.
Nanofibers prepared by electrospinning technology are widely used as an adsorber in protein purification processes. However, the evaluation of dynamic adsorption performance has not been reported yet.
Lysozyme was subjected to assess dynamic adsorption of weak ion exchange nanofiber membranes by applying a continuous flow system. Different operating conditions were selected to study the lysozyme adsorption breakthrough curve, which included four factors: pH, feedstock lysozyme concentration, feed flow rate, and stacked membrane layers. The breakthrough curve was applied to analyze the performance for dynamic binding with lysozyme as a model protein. In addition, the adsorption models (Bohart-Adams, Thomas, Yoon-Nelson, and BDST) were fitted to the breakthrough curves using linear and nonlinear regression analysis.
It was found that the four operating parameters had considerable influences on the dynamic adsorption performance. Among these models, the breakthrough curve was well-fitted by using linear regression analysis of the Yoon-Nelson model. The breakthrough curve characteristics could be used to describe the dynamic binding performance of interested membranes to lysozyme molecules.
Protein A affinity chromatography is a key step in isolation of biotherapeutics (BTs) containing fragment crystallizable regions, including monoclonal and bispecific antibodies. Dynamic binding ...capacity (DBC) analysis assesses how much BT will bind to a protein A column. DBC reduces with column usage, effectively reducing the amount of recovered product over time. Drug regulatory bodies mandate chromatography resin lifetime for BT isolation, through measurement of parameters including DBC, so this feature is carefully monitored in industrial purification pipelines. High-performance affinity chromatography (HPAC) is typically used to assess the concentration of BT, which when loaded to the column results in significant breakthrough of BT in the flowthrough. HPAC gives an accurate assessment of DBC and how this changes over time but only reports on protein concentration, requires calibration for each new BT analyzed, and can only be used offline. Here we utilized Raman spectroscopy and revealed that this approach is at least as effective as both HPAC and ultraviolet chromatogram methods at monitoring DBC of protein A resins. In addition to reporting on protein concentration, the chemical information in the Raman spectra provides information on aggregation status and protein structure, providing extra quality controls to industrial bioprocessing pipelines. In combination with partial least square (PLS) analysis, Raman spectroscopy can be used to determine the DBC of a BT without prior calibration. Here we performed Raman analysis offline in a 96-well plate format, however, it is feasible to perform this inline. This study demonstrates the power of Raman spectroscopy as a significantly improved approach to DBC monitoring in industrial pipelines.
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•Structural and adsorptive properties of multimodal anion exchangers are characterized•Adsorption rates are controlled by pore diffusion•Mass transfer of large proteins is highly restricted in ...agarose-based resins•Polymer-based resins provide higher dynamic binding capacity at short residence times
The effect of bead and ligand structure on protein adsorption was investigated for multimodal anion exchangers combining a quaternary ammonium ion group with hydrophobic moieties: Nuvia aPrime 1 and aPrime 2, based on a 54 μm diameter polymeric bead, and Capto Adhere ImpRes and Capto Adhere, based on agarose beads 51 and 78 μm diameter, respectively. Bovine serum albumin (BSA) monomer, BSA dimer, and thyroglobulin (Tg) were used as model proteins. Based on TEM imaging and iSEC, the Nuvia resins have a microgranular structure and large pores (110 nm radius), while the Capto resins have a fibrous structure and smaller pores (32-36 nm radius). Comparable binding capacities (80-110 mg/mL), decreasing as salt is added, are observed for all three proteins on the Nuvia resins. Higher capacities (110-130 mg/mL), also decreasing as salt is added, are observed for BSA monomer and dimer on the Capto resins. However, the Tg binding capacity is very low in this case and increases as salt is added. Confocal laser scanning microscopy show that the kinetics are controlled by pore diffusion for all four resins, but with diffusivities that decrease as the protein size increases especially for the Capto resins. For Tg at low salt, binding is restricted to a thin shell close to the bead surface for both Capto resins. The ratio of effective and free diffusivity is about 0.30, 0.18, and 0.08 for BSA monomer, BSA dimer, and Tg, respectively, on the Nuvia resin. These values decrease to about 0.11, 0.04, and 0.01, respectively, for the Capto resins as a result of diffusional hindrance. Dynamic binding capacities are consistent with the equilibrium and rate behaviors.
•Macroporous epoxy-based resins have been characterized.•Equilibrium binding capacity for therapeutic proteins was reached in a short time.•The large pore of resins improved the penetration of ...therapeutic proteins.•Efficient separation of therapeutic proteins was achieved at high linear velocity.•Resolution of proteins mixture was successful at high linear velocity.
This study investigated the adsorption capacity and mass transfer properties of a novel macroporous epoxy-polymer-based anion-exchanger, MPR Q, for the efficient separation of therapeutic proteins. MPR Q resin was prepared by phase separation based on spinodal decomposition followed by dextran grafting and ligand conjugation. Under static conditions, MPR Q exhibited a binding capacity of 49.8 mg-IgG/cm3-resin at pH 10, whereas the fastest adsorption was observed among the anion-exchanger resins tested. Inverse size-exclusion chromatography (iSEC) experiments revealed that the apparent pore diameter of MPR Q was approximately 90 nm, which was sufficiently large for the penetration of human IgG and bovine IgM. Moreover, the reduced height equivalent to a theoretical plate, h, of human IgG, determined using the linear gradient elution method was 65.8 and was not significantly changed in the range of linear velocities from 20.37 to 50.93 cm/min. The dynamic binding capacity at 10% breakthrough of MPR Q, determined by frontal analysis, exhibited a capacity of 43.8 mg/cm3 at 5.09 cm/min and 58% of DBC10% was maintained even though the linear velocity was increased to 50.93 cm/min. Furthermore, a resolution for separation of IgG and BSA by MPR Q was 1.06 at 5.09 cm/min, while it was higher than that for the conventional resin at all linear velocities from 5.09 cm/min to 50.93 cm/min. Thus, it was suggested that the MPR Q developed in this study is a promising resin that can efficiently separate large biomacromolecules such as human IgG at higher velocities.
•Grafted anion exchangers were synthesized by “grafting from” method with DMAPAA.•Protein binding capacity reached over 360 mg/mL at an ionic capacity of 458 mmol/L.•Very high uptake rate was ...maintained below 100 mmol/L NaCl.•Dynamic binding capacity kept over 180 mg/mL at flow velocity up to 1350 cm/h.•Dynamic binding capacity kept over 120 mg/mL below 100 mmol/L NaCl.
Ion exchangers of high binding capacity and uptake rate are desired for high performance purification of proteins by ion exchange chromatography. For this purpose, we have herein developed a new anion exchanger by grafting N,N-Dimethylaminopropyl acrylamide (DMAPAA) onto Sepharose FF by atom transfer radical polymerization. Six DMAPAA-grafted Sepharose FF resins of the same grafting density but different chain lengths (ionic capacities, ICs) were prepared, and named as FF-pDMAPAAn (n denotes IC value, 65–831 mmol/L). Bovine serum albumin (BSA) adsorption and its chromatography were studied to evaluate the anion exchange resins. It was found that the equilibrium protein adsorption capacity (qm) increased rapidly from 91 mg/mL at IC = 65 mmol/L till reaching a peak value (368 mg/mL) at IC = 458 mmol/L and then decreased slightly with further increasing IC (chain length) to 831 mmol/L. The uptake rate, De/D0, defined as the ratio of effective diffusion coefficient (De) to free diffusivity of the protein (D0), also presented similar changes as a function of IC. Namely, De/D0 increased from 0.25 to 2.45 in the IC range of 65 to 458 mmol/L, kept almost unchanged from 458 to 573 mmol/L and then decreased slightly with further increase of IC to 831 mmol/L. Therefore, FF-pDMAPAA458 presented the best performance of the six resins for BSA adsorption, possessing very high qm and De/D0 values (368 mg/mL and 2.45, respectively). The resin was thus extensively characterized in the effect of ionic strength and column chromatography. The qm and De/D0 of FF-pDMAPAA458 were much higher than other anion exchangers reported in literatures and kept over 230 mg/mL and 2.3, respectively, at salt concentrations up to 100 mmol/L. Therefore, the dynamic binding capacity of the FF-pDMAPAA458 column was remarkably higher than Q Sepharose FF and other two typical polymer-grafted anion exchangers reported in literatures in the salt concentration range, and kept as high as 180–220 mg/mL in the superficial flow velocity rang of 150–1350 cm/h. Taken together, the results demonstrated that FF-pDMAPAA resins of appropriate ionic capacities are promising for high-performance protein chromatography.
Increased need for plasmid DNA (pDNA) with sizes above 10 kbp (large pDNA) in gene therapy and vaccination brings the need for its large‐scale production with high purity. Chromatographic ...purification of large pDNA is often challenging due to low process yields and column clogging, especially using anion‐exchanging columns. The goal of our investigation was to evaluate the mass balance and pDNA isoform composition at column outlet for plasmids of different sizes in combination with weak anion exchange (AEX) monolith columns of varying channel size (2, 3 and 6 µm channel size). We have proven that open circular pDNA (OC pDNA) isoform is an important driver of reduced chromatographic performance in AEX chromatography. The main reason for the behaviour is the entrapment of OC pDNA in chromatographic supports with smaller channel sizes. Entrapment of individual isoforms was characterised for porous beads and convective monolithic columns. Convective entrapment of OC pDNA isoform was confirmed on both types of stationary phases. Porous beads in addition showed a reduced recovery of supercoiled pDNA (on an 11.6 kbp plasmid) caused by diffusional entrapment within the porous structure. Use of convective AEX monoliths or membranes with channel diameter >3.5 µm has been shown to increase yields and prevent irreversible pressure build‐up and column clogging during purification of plasmids at least up to 16 kbp in size.
Protein A chromatography is one of the most widely used purification steps in the manufacturing of the various classes of recombinant and non-recombinant antibodies. Due to the higher cost, lower ...binding capacity, and limited life cycle of Protein A ligand, this affinity-based purification step is often one of the most significant contributors to the cost of manufacturing of monoclonal antibody (mAb) products. In the last decade, there has been significant progress in improving the Protein A chromatography throughput by designing new engineered Staphylococcal Protein A (SPA) variants with higher dynamic binding capacity, considerable alkaline tolerance, and mild acidic elution pH. This review aims at summarizing the various protein engineering approaches used for improving the throughput of the Protein A-based affinity purification of various immunoglobulins. With biopharmaceutical producers operating under ever-increasing pressure towards reducing the cost of manufacturing, these advances in engineered protein A variants will help in processing larger cell culture volumes with high throughput and thereby significantly lower the cost of raw materials.
•Protein A engineering is essential for high-throughput affinity capture of mAbs•DBC, alkaline tolerance and elution pH critically affect the Protein A performance•Homo/hetero-oligomerization of engineered protein A domains enhances DBC•Alkaline tolerance can be improved by replacing asparagine residues•Improving specificity of IgG-Fc interaction increases elution pH
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