Search

Advances in cell culture technology have enabled the production of antibody titers upwards of 30 g/L. These highly productive cell culture systems can potentially lead to productivity bottlenecks in downstream purification due to lower column loadings, especially in the primary capture chromatography step. Alternative chromatography solutions to help remedy this bottleneck include the utilization of continuous processing systems such as periodic counter-current chromatography (PCC). Recent studies have provided methods to optimize and improve the design of PCC for cell culture titers up to about 3 g/L. This paper defines a continuous loading strategy for PCC that is independent of cell culture background and encompasses cell culture titers up to about 31 g/L. Initial experimentation showed a challenge with determining a difference in change in UV280 nm signal (ie. ΔUV) between cell culture feed and monoclonal antibody (mAb) concentration. Further investigation revealed UV280 nm absorbance of the cell culture feedstock without antibody was outside of the linear range of detection for a given cell pathlength. Additional experimentation showed the difference in ΔUV for various cell culture feeds can be either theoretically predicted by Beer’s Law given a known absorbance of the media background and impurities or experimentally determined using various UV280 nm cell pathlengths. Based on these results, a 0.35 mm pathlength at UV280 nm was chosen for dynamic control to overcome the background signal. The pore diffusion model showed good agreement with the experimental frontal analysis data, which resulted in definition of a ΔUV setpoint range between 20 and 70% for 3C-PCC experiments. Product quality of the elution pools was acceptable between various cell culture feeds and titers up to about 41 g/L. Results indicated the following ΔUV setpoints to achieve robust dynamic control and maintain 3C-PCC yield: ∼20-45% for titers greater than 10 g/L depending on UV absorbance of the HCCF and ∼45-70% for titers of up to 10 g/L independent of UV absorbance of the HCCF. The strategy and results presented in this paper show column loading in a continuous chromatography step can be dynamically controlled independent of the cell culture feedstock and titer, and allow for enhanced process control built into the downstream continuous operations. [ABSTRACT FROM AUTHOR]

• Modifications in Fc and VH3 binding region of ProA ligand can allow mild elution pH. • Jetted A50 HipH can elute Fc-containing proteins at or above pH 4.6. • Mild elution pH can be achieved with high yield and >50 % HMW reduction. • pH-sensitive proteins can elute within stable pH region and stay stable over time. • Jetted A50 HipH is a flexible platform ProA resin for stable and labile 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. [ABSTRACT FROM AUTHOR]

• Model-assisted approaches for continuous chromatograph were reviewed. • Model-assisted methods for process development were focused and discussed. • Strategies for model-assisted process characterization was proposed. • Current model-assisted process design approaches were reviewed. • Perspectives of model-assisted tools for the future digitalized manufacturing were discussed. Continuous manufacturing in monoclonal antibody production has generated increased interest due to its consistent quality, high productivity, high equipment utilization, and low cost. One of the major challenges in realizing continuous biological manufacturing lies in implementing continuous chromatography. Given the complex operation mode and various operation parameters, it is challenging to develop a continuous process. Due to the process parameters being mainly determined by the breakthrough curves and elution behaviors, chromatographic modeling has gradually been used to assist in process development and characterization. Model-assisted approaches could realize multi-parameter interaction investigation and multi-objective optimization by integrating continuous process models. These approaches could reduce time and resource consumption while achieving a comprehensive and systematic understanding of the process. This paper reviews the application of modeling tools in continuous chromatography process development, characterization and design. Model-assisted process development approaches for continuous capture and polishing steps are introduced and summarized. The challenges and potential of model-assisted process characterization are discussed, emphasizing the need for further research on the design space determination strategy and parameter robustness analysis method. Additionally, some model applications for process design were highlighted to promote the establishment of the process optimization and process simulation platform. [ABSTRACT FROM AUTHOR]

Antibodies and other protein products such as interferons and cytokines are biopharmaceuticals of critical importance which, in order to be safely administered, have to be thoroughly purified in a cost effective and efficient manner. The use of aqueous two-phase extraction (ATPE) is a viable option for this purification, but these systems are difficult to model and optimization procedures require lengthy and expensive screening processes. Here, a methodology for the rapid screening of antibody extraction conditions using a microfluidic channel-based toolbox is presented. A first microfluidic structure allows a simple negative-pressure driven rapid screening of up to 8 extraction conditions simultaneously, using less than 20 μL of each phase-forming solution per experiment, while a second microfluidic structure allows the integration of multi-step extraction protocols based on the results obtained with the first device. In this paper, this microfluidic toolbox was used to demonstrate the potential of LYTAG fusion proteins used as affinity tags to optimize the partitioning of antibodies in ATPE processes, where a maximum partition coefficient ( K ) of 9.2 in a PEG 3350/phosphate system was obtained for the antibody extraction in the presence of the LYTAG-Z dual ligand. This represents an increase of approx. 3.7 fold when compared with the same conditions without the affinity molecule ( K = 2.5). Overall, this miniaturized and versatile approach allowed the rapid optimization of molecule partition followed by a proof-of-concept demonstration of an integrated back extraction procedure, both of which are critical procedures towards obtaining high purity biopharmaceuticals using ATPE. [ABSTRACT FROM AUTHOR]

In this paper, we discuss the optimization and implementation of a high throughput process development (HTPD) tool that utilizes commercially available micro-liter sized column technology for the purification of multiple clinically significant monoclonal antibodies. Chromatographic profiles generated using this optimized tool are shown to overlay with comparable profiles from the conventional bench-scale and clinical manufacturing scale. Further, all product quality attributes measured are comparable across scales for the mAb purifications. In addition to supporting chromatography process development efforts ( e.g. , optimization screening), comparable product quality results at all scales makes this tool is an appropriate scale model to enable purification and product quality comparisons of HTPD bioreactors conditions. The ability to perform up to 8 chromatography purifications in parallel with reduced material requirements per run creates opportunities for gathering more process knowledge in less time. [ABSTRACT FROM AUTHOR]

Chemical or enzymatic modifications of therapeutic monoclonal antibodies (mAbs) having high risk towards safety and efficacy are defined as critical quality attributes (CQAs). During therapeutic mAbs process development, a variety of analytical techniques have to be used for the thorough characterization and quantitative monitoring of CQAs. This paper describes the development of a rapid analytical platform to assess and rank charge variants of mAbs. The workflow is first based on a cation exchange chromatography (CEX) comparative analysis of intact IgGs versus F(ab)’2 and Fc sub-domains generated by IdeS digestion. This analytical procedure was validated with FDA and EMA approved mAbs. Then, functional assays and peptide mapping can be performed in a second instance. This approach can be used during the early stage of drug research and development to screen lead molecules and select optimized candidates (best clone, best formulation) which could be “easily” developed (OptimAbs). [ABSTRACT FROM AUTHOR]

This paper deals with the theoretical design and experimental validation of an affinity-based continuous multi-column chromatography process for the purification of single-chain Fragment variable (scFv) antibodies. An open-loop 3-zone pH-gradient simulated moving bed (SMB) process was investigated exploiting the highly specific affinity of metal ions toward histidine-tagged recombinant proteins. The separation problem was simplified by considering the cell culture supernatant as a pseudo-binary mixture. The influence of mobile phase pH on the adsorption isotherm parameters was estimated by the inverse method using recorded pH-gradient batch elution profiles. Suitable operating parameters for the SMB process were identified using an equilibrium stage model and subsequently validated in a lab-scale SMB unit. Finally, the performance of the pH-gradient SMB process was compared against a non-optimized batch process. Biologically active single-chain Fragment variable antibody formats were purified continuously with 9% more recovery, 11 times more productivity (576 mg of purified scFv per day and liter stationary phase in SMB) and enriched by a factor of 2.5 compared to those obtained in the non-optimized batch process. [ABSTRACT FROM AUTHOR]

Abstract: This paper describes the application of sol–gel immunoaffinity columns for clean up of ochratoxin A contaminated cereal crops. Monoclonal antibodies selective for OTA have been entrapped into the pores of a sol–gel matrix in order to prepare immunoaffinity columns. Different parameters such as amount of entrapped antibodies and loading conditions were optimized to obtain highest possible recoveries of OTA. The method has been found to be a suitable tool in sample preparation prior to HPLC-FLD determination and as selective as conventional commercially available immunoaffinity columns. In the clean up of different cereals mean recoveries of 82±5%, 90±6% and 91±3%, were obtained for wheat, barley and rye, respectively, with sol–gel columns containing 1mg of anti-OTA antibodies. The detection limit (signal-to-noise ratio, 3) was 0.5μg/kg and the limit of quantification (signal-to-noise ratio, 10) determined to be 1μg/kg. Sol–gel columns can be reused 7 times without significant loss of recovery. After 10 applications the recovery decreased to approx. 50%. [Copyright &y& Elsevier]

Abstract: Protein glycosylation has significant effects on the structure and function of proteins. The efficient separation and enrichment of glycoproteins from complex biological samples is one key aspect and represents a major bottleneck of glycoproteome research. In this paper, we have explored pH multimodal hydrophobic interaction chromatography to separate glycosylated from non-glycosylated forms of proteins. Three different proteins, ribonuclease, invertase and IgG, have been examined and different glycoforms have been identified. The media itself shows strong responsiveness to small variations in pH, which makes it possible to fine-tune the chromatographic conditions according to the properties of the protein isolated. Optimal glycoprotein separation has been obtained at pH 4. The pH responsive multimodal HIC medium in contrast to conventional HIC media is able to resolve contaminating DNA. [ABSTRACT FROM AUTHOR]

Abstract: This paper describes a novel mixed-bed immunoaffinity column (IAC) method. The IAC was produced by coupling anti-quinolone and anti-sulfonamide broad-specificity monoclonal antibodies to Sepharose 4B for simultaneously isolating 13 quinolones (QNs) and 6 sulfonamides (SAs) from swine and chicken muscle tissues, followed by antibiotic determination using liquid chromatography–tandem mass spectrometry (LC–MS/MS). A new broad-specificity Mab (B1A4E8) toward sulfonamides was produced using sulfamethoxazole as hapten that demonstrated cross-reactivities to 6 SAs in the range of 31–112%. IAC optimized conditions were found that allowed the IAC to be reused for selective binding of both SAs and QNs. Recoveries of all 19 antibiotics from animal muscle ranged from 72.6 to 107.6%, with RSDs below 11.3% and 15.4% for intra-day and inter-day experiments, respectively. The limit of quantification ranged from 0.5 to 3.0ng/g. The strategy used here for a mixed-bed IAC may be used to study other compounds and more than two classes of analytes simultaneously. [Copyright &y& Elsevier]

Abstract: Efficient purification of protein biopharmaceuticals from transgenic plants is a major challenge, primarily due to low target protein expression levels, and high impurity content in the feed streams. These challenges may be addressed by using membrane chromatography. This paper discusses the use of cation-exchange and Protein A affinity-based membrane chromatographic techniques, singly and in combination for the purification of an anti-Pseudomonas aerugenosa O6ad human IgG1 monoclonal antibody from transgenic tobacco. Protein A membrane chromatography on its own was unable to provide a pure product, mainly due to extensive non-specific binding of impurities. Moreover, the Protein A membrane showed severe fouling tendency and generated high back-pressure. With cation-exchange membrane chromatography, minimal membrane fouling and high permeability were observed but high purity could not be achieved using one-step. Therefore, by using a combination of the cation-exchange and Protein A membrane chromatography, in that order, both high purity and recovery were achieved with high permeability. The antibody purification method was first systematically optimized using a simulated feed solution. Anti-P. aeruginosa human IgG1 type monoclonal antibody was then purified from transgenic tobacco juice using this optimized method. [Copyright &y& Elsevier]

Abstract: For accelerating the purification process development of human monoclonal antibodies (hmAbs) for pharmaceutical drugs, we designed a standardized method for setting the conditions of the purification process, which could be applied to hmAbs for the early phase of pharmaceutical development. The process includes three sequential chromatography steps: Protein A affinity chromatography (AFC), anion-exchange chromatography (AIEC) and cation-exchange chromatography (CIEC), and also includes a low pH virus inactivation step after the AFC step. We predicted the elution pH in the AFC and elution salt concentration in the CIEC from the amino acid sequences of hmAbs, as described in our previous paper. The mobile phase pH in AIEC and the pH for virus inactivation were also predicted based on the amino acid sequence of hmAb. As a case study, six hmAbs (two of IgG1, two of IgG2 and two of IgG4) were purified with the standardized method. The recovery, purity and clearance of impurities (DNA, host cell proteins (HCP), and Protein A) were examined. All the six hmAbs were purified with high recovery and high clearance of the impurities. Factors affecting the impurities level in the purified products are also discussed. [Copyright &y& Elsevier]

Abstract: Protein A chromatography has come to be widely adopted for large-scale purification of monoclonal antibodies and Fc fusion proteins. The low pH conditions required for Protein A elution can often lead to aggregation issues for these products. A concerted study of the kinetics of aggregate formation and their relation to chromatography on Protein A media has been lacking. This paper provides a framework to describe aggregation kinetics for an Fc fusion protein that was highly susceptible to aggregate formation under low pH conditions. In contrast to what is usually expected to be a higher order reaction, first order aggregation kinetics were observed for this protein over a wide range of conditions. A comparison of the rate constants of aggregation forms an effective means of comparing various stabilizing additives to the elution buffer with one another. Inclusion of urea in the elution buffer at moderate concentrations (<2M) and low temperature operation of the Protein A column were both found to be effective solutions to the aggregation issue. Elution from the Protein A resin was found to increase the aggregation rate constants over and above what would be expected from exposure to low pH conditions in solution alone. This demonstrates that Protein A–Fc interactions can destabilize product structure and increase the tendency to aggregate. The results presented here are anticipated to assist the development of Protein A process conditions for products that are prone to form high molecular weight aggregates during column elution. [Copyright &y& Elsevier]

Abstract: In the production of monoclonal antibodies, separate chains of the antibody are often present in the product mixture as well as other contaminating proteins. These fragments should be removed from the whole antibodies. This paper shows the purification of monoclonal immunoglobulin G (IgG) from its heavy chain contaminant. The heavy chain fragment is simulated experimentally using bovine serum albumin (BSA), which has approximately the same molecular weight. The purification is performed using traditional size-exclusion chromatography (SEC) and using surfactant-aided SEC (SASEC), testing two different surfactants (C12E23 and Tween20) and two different gels (Sephacryl S200HR and Sephacryl S300 HR). Pulse experiments show that with SASEC both BSA and IgG are more distributed towards the solid phase than compared to using SEC. This effect is larger on IgG, the largest component than on BSA. As a consequence, azeotropes will be formed at a specific surfactant concentration. Above this concentration the selectivity is reversed and increased to values higher than obtained with conventional SEC. At 7.5% (w/w) of C12E23, BSA actually elutes before IgG. These experiments further show that when using SASEC larger productivity, higher yields and lower solvent consumption can be achieved without loss of purity of IgG when compared to conventional SEC. Mathematical simulation of the separation of BSA and IgG using simulated moving bed (SMB) chromatography indicates a large increase in productivity when applying a surfactant gradient in SASEC SMB compared to conventional isocratic SEC-SMB. Furthermore, solvent consumption reductions with a factor 15 prove possible as well as concentrating the IgG by a factor 2. [Copyright &y& Elsevier]

Abstract: Although lectin selection is gaining increasing acceptance as a tool for targeting glycosylation in glycoproteomics, most of the work has been directed at N-glycosylation. The work reported here focuses on the use of lectins in the study of O-glycosylation. The problem with using lectins for studying O-glycosylation is that they are not sufficiently specific. This paper reports that through the use of serial lectin affinity chromatography (SLAC) it is possible to select predominantly O-glycosylated peptides from tryptic digests of human serum. Jacalin is relatively specific for O-glycosylation but has the problem that it also selects high mannose N-type glycans. This problem was addressed by using a concanavalin A affinity column to first remove high mannose, hybrid-type and biantennary complex-type N-type glycans before application of the Jacalin columns. When used in a serial format, concanavalin A and Jacalin together provide essentially O-glycosylated peptides. The glycoprotein parents of glycopeptides were identified by deglycosylating the selected O-glycopeptides by oxidative elimination. These peptides were then separated by RPC and further analyzed using ESI-MS/MS and MALDI-MS/MS. Using this approach all the O-glycosylated sites in a model protein (fetuin) and over thirty glycoprotein parents from human serum were identified. It is concluded that a serial combination of Con A and Jacalin can be of utility in the study of O-glycosylation in glycoproteomics. [Copyright &y& Elsevier]

Abstract: Humanized monoclonal antibodies (mAbs) hold significant promise as biopharmaceuticals. One of the main challenges faced in the purification of mAbs is their separation from bovine serum albumin, which is the main protein present in most mammalian cell culture media. This paper discusses the purification of humanized mAb hIgG1-CD4 from CHO cell culture media by hydrophobic interaction membrane chromatography using a stack of microporous synthetic membranes. The effects of solution conditions on mAb solubility and binding on the membrane were first studied. The separation of a simulated mixture of bovine albumin and the mAb was then carried out to examine the feasibility of mAb purification. Separation experiments carried out under optimized conditions demonstrated that this membrane-based technique could be used for mAb purification from cell culture media. High purity (97%) and recovery (in excess of 97%) were obtained. [Copyright &y& Elsevier]

The present paper describes a chromatographic capture/purification step for the recovery of proteins directly from undiluted and unclarified cell culture broths using supermacroporous dimethylacrylamide (DMAA) cryogel. The interconnected character and the size (10–100 μm) of the pores of the adsorbent make it possible to process whole cell fermentation broths without blocking the column. Cu2+–iminodiacetic acid (IDA) DMAA cryogel has been used for the isolation and purification of excreted (His)6-tagged single chain (sc) Fv antibody fragments, (His)6-scFv, from E. coli cell culture. Bound protein was recovered with 0.2 M imidazole or with 20 mM EDTA and was practically cell-free. Chromatographic capture using Cu2+–IDA cryogel column was performed at flow rates of 300 and 600 cm/h, respectively and resulted in 84–96% recovery of (His)6-scFv fragments with a purification factor of 13–15. The DMAA cryogel adsorbent is mechanically stable, can withstand harsh cleaning-in-place procedure and is relatively inexpensive. Chromatographic isolation of proteins using cryogels allows efficient removal of cells and can be operated at a flow rate as high as 600 cm/h. This novel technique has proven to be a scalable process, does not require special equipment and can be a good alternative to expanded bed adsorption and other integrated isolation techniques. [Copyright &y& Elsevier]

In process-scale antibody purification, protein-A affinity chromatography is commonly used as the initial purification step. In this paper, two different protein-A media were evaluated. These adsorbents have a porous glass backbone with different pore sizes: 700 A˚ and 1000 A˚. Adsorption equilibrium data of human immunoglobulins on these media were measured via a batch technique and correlated using the Langmuir isotherm model. A larger static capacity was found for the smaller pore size material, which is probably a result of the larger specific surface area and associated higher ligand concentration. The protein uptake kinetics were also obtained via a stirred tank experiment using different initial protein concentrations. A surface layer model was used to represent the protein uptake by the media and to estimate values of a concentration-independent effective diffusivity within the particle. Experimental breakthrough curves were also obtained from packed beds operated under different conditions. Calculated breakthrough profiles were found to be in good agreement with the experimental results. Experimental breakthrough data were used to determine the dependence of the dynamic capacity of the media as a function of the fluid residence time. A larger dynamic capacity was also found for the smaller pore size media. The permeability of large scale packed beds was also reported and used in conjunction with the dynamic capacity to calculate the process production rate. [Copyright &y& Elsevier]

• Evaluated mAb retention, domain contributions, and surface properties from pH 5–7. • One mAb showed a pH-dependent spectrum of domain contributions. • pH can tune the relative importance of Fab vs. Fc binding sites for some mAbs. • The titration of Histidine residues plays an important role in this pH range. The present paper builds upon previous work on mAb domain contributions to multimodal (MM) chromatography by examining how pH can impact mAb surface properties and retention in these systems. Linear salt gradient experiments were carried out between pH 5–7 for several mAbs with different pI and surface hydrophobicities in four different MM CEX resins at two ligand densities. mAb retention showed an inverse, non-linear correlation with pH. Changing pH affected the elution order, creating unique windows of selectivity in each of the MM CEX resins. One mAb showed a pH-dependent spectrum of domain contributions, demonstrating that pH can be used to tune the relative importance of the (Fab) 2 and Fc domains for some mAbs in MM systems. Positive, negative, and hydrophobic patches were calculated between pH 5–7 for the mAbs. Visualizing these patches on the protein surface demonstrated that each mAb showed a unique distribution of surface charge and hydrophobicity that changed with pH. The sum of patch areas was tracked across this pH range to quantitatively understand how pH impacted these important surface properties. The quantitative analysis then was narrowed to consider only patches in the CDR loops, which were hypothesized to be an important interaction site for some mAbs in these systems. Interestingly, differences in the titration of CDR loop patches for each mAb were shown to be a result of Histidine titrations and patches in this region were qualitatively correlated with experimental trends including the observed elution order reversals. These results indicate that pH potentially can be employed as a lever for the strategic design of multimodal steps to create flow through, bind and elute, or weak partitioning operations with important implications for the design of integrated and/or continuous downstream purification processes. Furthermore, the ability to tune domain contributions in MM separations using pH creates intriguing possibilities for current downstream challenges such as the removal of product-related impurities, as well as the purification of bispecific mAbs. [ABSTRACT FROM AUTHOR]