Polishing Chromatography Research Articles

Novel purification platform based on multimodal preparative scale separation of mAb fragments and aggregates

Monoclonal antibodies (mAbs) continue to dominate the biopharmaceutical industry. Certain mAbs are prone to fragmentation and clipping and in these cases, adequate removal of these species is critical during manufacturing. Fragments can be generated during fermentation, purification, storage, formulation, and administration. Their addition to the acidic charge-variant of the purified mAb has been reported to decrease stability and potency of the final product. However, contrary to mAb aggregation, manufacturers have not given much attention to removal of fragments and clipped species and as a result most conventional mAb platforms offer at best limited capabilities for their removal. In this study, we propose a novel purification platform that uses multimodal chromatography and achieves complete removal of a range of mAb fragments and clipped products (25–120 kDa). The utility of the platform has been successfully demonstrated for 2 IgG1s and 2 IgG4s. Further, adequate removal of the various host cell impurities such as host cell proteins (<10 ppm) and host cell DNA (<5 ppb) has been achieved. Finally, the platform was able to deliver adequate removal of high molecular weight impurities (<1 %) and a 30 % clearance of the acidic charge variant. The proposed single step has been shown to deliver what the polishing chromatography and intermediate purification chromatography steps deliver in a traditional mAb platform.

A model-based performance analysis of integrated chromatography-ultrafilter separation systems for monoclonal antibody (mAb) manufacturing

Robust and accurate modelling of bioprocess systems offers the opportunity to visualise strategies for rapid-scale up of production platforms from benchtop to industrial scale, but few studies investigate how mechanistic and hybrid models can be used to design integrated bio-manufacturing. This is especially true for downstream purification technologies used to separate mAbs from culture by-products, e.g. host cell proteins. Polishing chromatography (cation-exchange mode) is commonly used to remove these by-products, but optimal bind and elution operation of these columns is complex. Ultrafiltration is typically applied in conjunction with diafiltration post-polishing chromatography in order to obtain suitable final product titers and excipient concentrations. This study presents a combination of dynamic models to probe batch polishing chromatography and ultrafiltration operating strategies achieving industrial specifications for recovery yield and mAb titer. A series of elution and filtration strategies are implemented, resulting in a total of 40 flowsheets explored. A steric mass action model with 7 pH-dependent parameters is employed to simulate scale-up polishing chromatography column behaviour. Moreover, a published gel layer ultrafiltration model based on Darcy’s law is implemented to purify the outlet of the polishing chromatography column. Strategies that are successful in achieving the target 90% recovery yield and 100 g L−1 concentration specifications are compared via a comprehensive analysis, to deduce the operational protocols which offer minimised salt consumption and pressure drop whilst respecting design criteria.

Behavior of host-cell-protein-rich aggregates in antibody capture and polishing chromatography

Recent work has shown that aggregates in monoclonal antibody (mAb) solutions may be made up not just of mAb oligomers but can also harbor hundreds of host-cell proteins (HCPs), suggesting that aggregate persistence through downstream purification operations may be related to HCP clearance. We have examined this in a primary analysis of aggregate persistence through processing steps that are typically implemented for HCP reduction, demonstrating that the phenomenon is relevant to depth filtration, protein A chromatography and flow-through anion-exchange (AEX) polishing. Confocal laser scanning microscopy observations show that aggregates compete with the mAb to adsorb specifically in protein A chromatography and that this competitive interaction is integral to the efficacy of protein A washes. Column chromatography reveals that the protein A elution tail can have a relatively high concentration of aggregates, which corroborates analogous observations from recent HCP studies. Similar measurements in flow-through AEX chromatography show that relatively large aggregates that harbor HCPs and that persist into the protein A eluate can be retained to an extent that appears to depend primarily on the resin surface chemistry. The total aggregate mass fraction of both protein A eluate pools (∼ 2.4 – 3.6%) and AEX flow-through fractions (∼ 1.5 – 3.2%) correlates generally with HCP concentrations measured using enzyme-linked immunosorbent assay (ELISA) as well as the number of HCPs that may be identified in proteomic analysis. This suggests that quantification of the aggregate mass fraction may serve as a convenient albeit imperfect surrogate for informing early process development decisions regarding HCP clearance strategies.

Continuous manufacturing of monoclonal antibodies: Dynamic control of multiple integrated polishing chromatography steps using BioSMB

We propose a strategy for automation and control of multi-step polishing chromatography in integrated continuous manufacturing of monoclonal antibodies. The strategy is demonstrated for a multi-step polishing process consisting of cation exchange chromatography in bind-and-elute mode followed by mixed-mode chromatography in flowthrough mode. A BioSMB system with a customized Python control layer is used for automation and scheduling of both the chromatography steps. Further, the BioSMB valve manifold is leveraged for in-line conditioning between the two steps, as tight control of pH and conductivity is essential when operating with multimodal resins because even slight fluctuations in load conditions adversely affect the chromatography performance. The pH and conductivity of the load to the multimodal chromatography columns is consistent, despite the elution gradient of the preceding cation exchange chromatography step. Inputs from the BioSMB pH and conductivity sensors are used for real-time control of the 7 pumps and 240 valves to achieve in-line conditioning inside the BioSMB manifold in a fully automated manner. This is confirmed by showcasing different elution strategies in cation exchange chromatography, including linear gradient, step gradient and process deviations like tubing leakage. In all the above cases, the model was able to maintain the pH and conductivity of multimodal chromatography load within the range of 6 ± 0.1 pH and 7 ± 0.3 mS/cm conductivity. The strategy eliminates the need for using multiple BioSMB units or integrating external pumps, valves, mixers, surge tanks, or sensors between the two steps as is currently the standard approach, thus offering a simple and robust structure for integrating multiple polishing chromatography steps in continuous downstream monoclonal antibody purification trains.

Continuous manufacturing of monoclonal antibodies: Automated downstream control strategy for dynamic handling of titer variations

Handling long-term dynamic variability in harvest titer is a critical challenge in continuous downstream manufacturing. This challenge is becoming increasingly important with the advent of high-titer clones and modern upstream perfusion processes where the titer can vary significantly across the course of a campaign. In this paper, we present a strategy for real-time, dynamic adjustment of the entire downstream train, including capture chromatography, viral inactivation, depth filtration, polishing chromatography, and single-pass formulation, to accommodate variations in titer from 1–7 g/L. The strategy was tested in real time in a continuous downstream purification process of 36 h duration with induced titer variations. The dynamic control strategy leverages real-time NIR-based concentration sensors in the harvest material to continuously track the titer, integrated with an in-house Python-based control system that operates a BioSMB for carrying out capture and polishing chromatography, as well as a series of pumps and solenoid valves for carrying out viral inactivation and formulation. A set of 9 different methods, corresponding to the different harvest titers have been coded onto the Python controller. The methods have a varying number of chromatography columns (3–6 for Protein A and 2–10 for CEX), designed to ensure proper scheduling and optimize productivity across the entire titer variation space. The approach allows for a wide range of titers to be processed on a single integrated setup without having to change equipment or to re-design each time. The strategy also overcomes a key unexplored challenge in continuous processing, namely hand-shaking the downstream train to upstream conditions with long-term titer variability while maintaining automated operation with high productivity and robustness.

AI-ML applications in bioprocessing: ML as an enabler of real time quality prediction in continuous manufacturing of mAbs

As continuous manufacturing of biotherapeutics gains steam, there is an increasing interest in using machine learning (ML) techniques for real time prediction of product quality and for process control. This paper focuses on application of different ML techniques for predicting critical process attributes that are pertinent to capture and polishing chromatography. Data from pH, UV, and conductivity sensors are acquired and pre-processed. For the present case study, tree-based regression techniques (decision tree and random forest) outperformed in all cases. The final model, random forest regression model, resulted in prediction errors of <5% for all predicted attributes. The results showed that random forest models exhibit optimal performance for smaller process-scale datasets with low chances of overfitting, are computationally inexpensive and do not require a graphics processing unit. The proposed approach is well suited for implementation in a continuous mAb train for real time prediction of chromatography process attributes.

Proceedings of the 2019 Viral Clearance Symposium, Session 2: New Modalities in Chromatography and Adsorptive Filters.

In Session 2 of the 2019 Viral Clearance Symposium, new outcomes of viral clearance performance of polishing chromatography and depth filters utilized in the purification processes of biotechnologically produced recombinant proteins such as monoclonal antibodies were presented. It was shown that flow through both multimodal anion-exchange and anion-exchange chromatography are effective tools for removal of retroviruses and, under defined conditions, also for parvoviruses. Cation-exchange chromatography, when operated in bind/elute mode, hydrophobic interaction chromatography, and depth filtration can also be potent for virus removal, but are less robust for parvoviruses. Despite these data provide further understanding of virus removal mechanisms by chromatography and depth filtration operations, more investigation is required for a clear understanding of the molecular mechanisms of virus removal and the relevance of different moleculés interactions including resin, virus, and product.

Control of surge tanks for continuous manufacturing of monoclonal antibodies.

Surge tanks are critical but often overlooked enablers of continuous bioprocessing. They provide multiple benefits including dampening of concentration gradients and allowing process resumption efforts in case of equipment failure or unexpected deviations, which can occur during a continuous campaign of weeks or months. They are also useful in enabling steady-state operation across a continuous train by facilitating mass balance between unit operations such as chromatography which have periodic loading and elution cycles. In this paper, we propose a design of a system of surge tanks for a monoclonal antibody (mAb) production process consisting of cell culture, clarification, capture chromatography, viral inactivation, polishing chromatography, and single-pass ultrafiltration and diafiltration. A Python controller has been developed for robust control of the continuous train. The controller has four layers, namely data acquisition, process scheduling, deviation handling, and real-time execution. A set of general guidelines for surge tank placement and sizing have been proposed together with process control strategies based on the design space of the individual unit operations, failure modes analysis of the different equipment, and expected variability in the process feed streams for both fed-batch and perfusion bioreactors. The control system has been successfully demonstrated for several continuous runs of up to 36 h in duration and is able to leverage surge tanks for robust control of the continuous train in the face of product variability as well as process errors while maintaining critical quality attributes. The proposed set of strategies for surge tank control are adaptable to most continuous processing setups for mAbs, and together form the first framework that can fully realize the benefits of surge tanks in continuous bioprocessing.

Comparative evaluation of integrated purification pathways for bacterial modular polyomavirus major capsid protein VP1 to produce virus-like particles using high throughput process technologies

Modular virus-like particles and capsomeres are potential vaccine candidates that can induce strong immune responses. There are many described protocols for the purification of microbially-produced viral protein in the literature, however, they suffer from inherent limitations in efficiency, scalability and overall process costs. In this study, we investigated alternative purification pathways to identify and optimise a suitable purification pathway to overcome some of the current challenges. Among the methods, the optimised purification strategy consists of an anion exchange step in flow through mode followed by a multi modal cation exchange step in bind and elute mode. This approach allows an integrated process without any buffer adjustment between the purification steps. The major contaminants like host cell proteins, DNA and aggregates can be efficiently removed by the optimised strategy, without the need for a size exclusion polishing chromatography step, which otherwise could complicate the process scalability and increase overall cost. High throughput process technology studies were conducted to optimise binding and elution conditions for multi modal cation exchanger, Capto™ MMC and strong anion exchanger Capto™ Q. A dynamic binding capacity of 14 mg ml−1 was achieved for Capto™ MMC resin. Samples derived from each purification process were thoroughly characterized by RP-HPLC, SEC-HPLC, SDS-PAGE and LC-ESI-MS/MS Mass Spectrometry analytical methods. Modular polyomavirus major capsid protein could be purified within hours using the optimised process achieving purities above 87% and above 96% with inclusion of an initial precipitation step. Purified capsid protein could be easily assembled in-vitro into well-defined virus-like particles by lowering pH with addition of calcium chloride to the eluate. High throughout studies allowed the screening of a vast design space within weeks, rather than months, and unveiled complicated binding behaviour for CaptoTM MMC.

Antibody Aggregate Removal Using a Mixed-Mode Chromatography Resin.

In monoclonal antibody (mAb) production, aggregates represent a major class of product-related impurities that needs to be removed by the downstream process. Protein A chromatography is generally less effective at removing antibody aggregates under typical conditions, and in most cases aggregate removal relies on a subsequent polishing chromatography. Here we describe a procedure for effective removal of antibody aggregates using the mixed-mode chromatography resin Capto MMC ImpRes. Clearance of aggregates was confirmed by analytical size-exclusion chromatography (SEC) and native gel electrophoresis.

Control of leached beta-glucan levels from depth filters by an improved depth filtration flush strategy.

Beta-glucans are polysaccharides of D-glucose monomers linked by (1-3) beta-glycosidic bonds, are found to have a potential immunogenicity risk in biotherapeutic products, and are labeled as process contaminants. A common source of beta-glucans is from the cellulose found in traditional depth filter media. Typically, beta-glucan impurities that leach into the product from the primary clarification depth filters can be removed by the subsequent bind-and-elute affinity chromatography capture step. Beta-glucans can also be removed by a bind-and-elute cation exchange chromatography step, which is useful for removing beta-glucans introduced by a post-Protein A depth filtration step. However, the increasing prevalence of flowthrough polishing chromatography poses a challenge for beta-glucan removal due to the lack of any bind-and-elute chromatography steps after the post-Protein A depth filter. In this work, a depth filter flush strategy was developed to control beta-glucan leaching into the product pool. Different loading conditions for the depth filtration and subsequent chromatography steps were evaluated to determine the robustness of the optimized flush strategy. Carry through runs demonstrated greater than two-fold reduction in beta-glucan levels using the optimized wash as compared to standard filter flush conditions.

Good modeling practice for industrial chromatography: Mechanistic modeling of ion exchange chromatography of a bispecific antibody

In the biopharmaceutical industry, development and characterization of chromatography processes is typically based on statistical models. Although these approaches are easy to apply, the resulting models may fail to predict non-linear behavior in preparative chromatography with complex protein feed streams. An alternative to empirical methods are mechanistic models. In chemical engineering, mechanistic modeling has been a standard method for decades. As mechanistic models continue their advance in the biopharmaceutical industry, this study underlines the need of a standardized methodology for mechanistic model calibration.A lumped rate model was applied to the polishing chromatography of a bispecific antibody. Following guidelines for good modeling practice, the model was thoroughly analyzed. Potential limitations such as over-parameterization, parameter correlations, imprecise parameter estimates or systematic errors were considered by evaluation of parameter confidence intervals, visual sensitivity analysis and model validation across different scales. Application of simulations for identification of critical process parameters will be discussed.

Hybrid optimization of preparative chromatography for a ternary monoclonal antibody mixture.

In the purification of monoclonal antibodies, ion-exchange chromatography is typically used among the polishing steps to reduce the amount of product-related impurities such as aggregates and fragments, whilst simultaneously reducing HCP, residual Protein A and potential toxins and viruses. When the product-related impurities are difficult to separate from the products, the optimization of these chromatographic steps can be complex and laborious. In this paper, we optimize the polishing chromatography of a monoclonal antibody from a challenging ternary feed mixture by introducing a hybrid approach of the simplex method and a form of local optimization. To maximize the productivity of this preparative bind-and-elute cation-exchange chromatography, wide ranges of the three critical operational parameters-column loading, the initial salt concentration, and gradient slope-had to be considered. The hybrid optimization approach is shown to be extremely effective in dealing with this complex separation that was subject to multiple constraints based on yield, purity, and product breakthrough. Furthermore, it enabled the generation of a large knowledge space that was subsequently used to study the sensitivity of the objective function. Increased design space understanding was gained through the application of Monte Carlo simulations. Hence, this work proposes a powerful hybrid optimization method, applied to an industrially relevant process development challenge. The properties of this approach and the results and insights gained, make it perfectly suited for the rapid development of biotechnological unit operations during early-stage bioprocess development.

Investigating the combination of single‐pass tangential flow filtration and anion exchange chromatography for intensified mAb polishing

There is growing interest within the biopharmaceutical industry to improve manufacturing efficiency through process intensification, with the goal of generating more product in less time with smaller equipment. In monoclonal antibody (mAb) purification, a unit operation that can benefit from intensification is anion exchange (AEX) polishing chromatography. Single-pass tangential flow filtration (SPTFF) technology offers an opportunity for process intensification by reducing intermediate pool volumes and increasing product concentration without recirculation. This study evaluated the performance of an AEX resin, both in terms of host cell protein (HCP) purification and viral clearance, following concentration of a mAb feed using SPTFF. Results show that preconcentration of AEX feed material improved isotherm conditions for HCP binding, resulting in a fourfold increase in resin mAb loading at the target HCP clearance level. Excellent clearance of minute virus of mouse and xenotropic murine virus was maintained at this higher load level. The increased mAb loading enabled by SPTFF preconcentration effectively reduced AEX column volume and buffer requirements, shrinking the overall size of the polishing step. In addition, the suitability of SPTFF for extended processing time operation was demonstrated, indicating that this approach can be implemented for continuous biomanufacturing. The combination of SPTFF concentration and AEX chromatography for an intensified mAb polishing step which improves both manufacturing flexibility and process productivity is supported.

Integrated continuous bioprocessing: Economic, operational, and environmental feasibility for clinical and commercial antibody manufacture.

This paper presents a systems approach to evaluating the potential of integrated continuous bioprocessing for monoclonal antibody (mAb) manufacture across a product's lifecycle from preclinical to commercial manufacture. The economic, operational, and environmental feasibility of alternative continuous manufacturing strategies were evaluated holistically using a prototype UCL decisional tool that integrated process economics, discrete‐event simulation, environmental impact analysis, operational risk analysis, and multiattribute decision‐making. The case study focused on comparing whole bioprocesses that used either batch, continuous or a hybrid combination of batch and continuous technologies for cell culture, capture chromatography, and polishing chromatography steps. The cost of goods per gram (COG/g), E‐factor, and operational risk scores of each strategy were established across a matrix of scenarios with differing combinations of clinical development phase and company portfolio size. The tool outputs predict that the optimal strategy for early phase production and small/medium‐sized companies is the integrated continuous strategy (alternating tangential flow filtration (ATF) perfusion, continuous capture, continuous polishing). However, the top ranking strategy changes for commercial production and companies with large portfolios to the hybrid strategy with fed‐batch culture, continuous capture and batch polishing from a COG/g perspective. The multiattribute decision‐making analysis highlighted that if the operational feasibility was considered more important than the economic benefits, the hybrid strategy would be preferred for all company scales. Further considerations outside the scope of this work include the process development costs required to adopt continuous processing. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:854–866, 2017

Application of simplex-based experimental optimization to challenging bioprocess development problems: Case studies in downstream processing.

The identification of feasible operating conditions during the early stages of bioprocess development is implemented frequently through High Throughput (HT) studies. These typically employ techniques based on regression analysis, such as Design of Experiments. In this work, an alternative approach, based on a previously developed variant of the Simplex algorithm, is compared to the conventional regression-based method for three experimental systems involving polishing chromatography and protein refolding. This Simplex algorithm variant was found to be more effective in identifying superior operating conditions, and in fact it reached the global optimum in most cases involving multiple optima. By contrast, the regression-based method often failed to reach the global optimum, and in many cases reached poor operating conditions. The Simplex-based method is further shown to be robust in dealing with noisy experimental data, and requires fewer experiments than regression-based methods to reach favorable operating conditions. The Simplex-variant also lends itself to the use of HT analytical methods, when they are available, which can assist in avoiding analytical bottlenecks. It is suggested that this Simplex-variant is ideally suited to rapid optimization in early-phase process development. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:404-419, 2016.

Caprylic acid-induced impurity precipitation from protein A capture column elution pool to enable a two-chromatography-step process for monoclonal antibody purification.

This article presents the use of caprylic acid (CA) to precipitate impurities from the protein A capture column elution pool for the purification of monoclonal antibodies (mAbs) with the objective of developing a two chromatography step antibody purification process. A CA-induced impurity precipitation in the protein A column elution pool was evaluated as an alternative method to polishing chromatography techniques for use in the purification of mAbs. Parameters including pH, CA concentrations, mixing time, mAb concentrations, buffer systems, and incubation temperatures were evaluated on their impacts on the impurity removal, high-molecular weight (HMW) formation and precipitation step yield. Both pH and CA concentration, but not mAb concentrations and buffer systems, are key parameters that can affect host-cell proteins (HCPs) clearance, HMW species, and yield. CA precipitation removes HCPs and some HMW species to the acceptable levels under the optimal conditions. The CA precipitation process is robust at 15-25°C. For all five mAbs tested in this study, the optimal CA concentration range is 0.5-1.0%, while the pH range is from 5.0 to 6.0. A purification process using two chromatography steps (protein A capture column and ion exchange polishing column) in combination with CA-based impurity precipitation step can be used as a robust downstream process for mAb molecules with a broad range of isoelectric points. Residual CA can be effectively removed by the subsequent polishing cation exchange chromatography.

Domain antibody downstream process optimization: High‐throughput strategy and analytical methods

Application of scale‐down high‐throughput screening has become integral for process development of antibody therapeutic products. In this work, methods are described for using high‐throughput techniques to develop a multicolumn chromatography purification protocol for a small domain antibody with very limited material (&lt;200 mg). Screenings utilized resin slurry plates to explore and narrow potential operating space, and miniature columns were used to either confirm operating spaces or further explore impurity separations. Lab scale column confirmations were performed when appropriate. Affinity capture chromatography as well as ion exchange and multimodal polishing chromatography steps were explored. Feedstreams were pooled and recycled to preserve material for the different chromatography steps. Precise high‐throughput analytical assays were developed to fully characterize the domain antibody to a similar extent as a typical commercial therapeutic protein program. Optimized two‐column and three‐column processes provided overall chromatography yields of 66 and 58%, respectively, and were able to meet typical early phase requirements for removal of impurities such as aggregates, host cell protein, endotoxin, and other product‐related impurities. This study provides a comprehensive example of how a thorough biologics downstream process can be developed with a minimum of material.

Development of advanced host cell protein enrichment and detection strategies to enable process relevant spike challenge studies.

An orthogonal chromatography methodology for the enrichment of host cell protein (HCP) species relative to monoclonal antibody (mAb) products was developed and applied for the successful enrichment of HCP from post-Protein A process pools for seven different mAb products. An advanced two-dimensional liquid chromatography/mass spectrometry platform (2D-LC/MS(E) ) was utilized to demonstrate that the HCP enriched material was representative, in terms of species content, to pre-enriched process pools. The HCP enrichment methodology was scaled up for two different mAb products, and this process relevant enriched HCP material was used to conduct advanced spike challenge studies to demonstrate the utility of the approach for the understanding of (1) quantitative HCP clearance, (2) individual species clearance, and (3) species clearance redundancy across polishing chromatography steps. The combined ability to enrich process relevant HCP, detect individual HCP species with 2D-LC/MS(E) technology, and conduct advanced challenge studies with process relevant material surmounts prior limitations to high integrity process challenge study implementation, and facilitates significant process understanding for development of risk-based control strategies and strategic process design. This also demonstrates implementation of a foundational strategy for conducting spike-challenge studies using process-relevant impurities isolated from processes of interest using orthogonal approaches.

At-line mid infrared spectroscopy for monitoring downstream processing unit operations

At-line mid infrared spectroscopy (FT-MIR) was used for monitoring monoclonal antibody (mAb) titer, host cell protein levels and antibody aggregate amount in samples, originating from different downstream processing unit operations, performed during technical laboratory scale development runs. Antibody aggregate quantification was possible down to 1% [w/w], while host cell protein levels could be monitored down to 700ngml−1. Antibody titer could be determined with high accuracy for samples >0.7gL−1, with the possibility to further increase sensitivity when using samples with highly similar matrix. Comparing these results to relevant levels during downstream processing, the most promising applicability of FT-MIR is within target protein (mAb) monitoring, while HCP monitoring following intermediate and polishing chromatography steps is currently not a well-suited application. FT-MIR for aggregate monitoring is more suitable for steps resulting in higher aggregate levels such as pH stress and shear stress- related purification steps. Additionally, FT-MIR might be considered for monitoring of samples with higher HCP and aggregate amount profiles, which can occur during initial purification steps and when using alternative purification methods such as precipitation.