A novel method for continuous chromatographic separation of monoclonal antibody charge variants by combining displacement mode chromatography and step elution

Charge heterogeneity of monoclonal antibodies is considered a critical quality attribute and hence needs to be monitored and controlled by the manufacturer. Typically, this is accomplished via separation of charge variants on cation exchange chromatography (CEX) using a pH or conductivity based linear gradient elution. Although an effective approach, this is challenging particularly during continuous processing as creation of linear gradient during continuous processing adds to process complexity and can lead to deviations in product quality upon slightest changes in gradient formation. Moreover, the long length of elution gradient along with the required peak fractionation makes process integration difficult. In this study, we propose a novel approach for separation of charge variants during continuous CEX chromatography by utilizing a combination of displacement mode chromatography and salt-based step elution. It has been demonstrated that while the displacement mode of chromatography enables control of acidic variants ≤26% in the CEX eluate, salt-based step gradient elution manages basic charge variant ≤25% in the CEX eluate. The proposed approach has been successfully demonstrated using feed materials with varying compositions. On comparing the designed strategy with 2-column concurrent (CC) chromatography, the resin specific productivity increased by 95% and resin utilization increased by 183% with recovery of main species >99%. Further, in order to showcase the amenability of the designed CEX method in continuous operation, the method was examined in our in-house continuous mAb platform.     

Displacement to separate host‐cell proteins and aggregates in cation‐exchange chromatography of monoclonal antibodies

An efficient and consistent method of monoclonal antibody (mAb) purification can improve process productivity and product consistency. Although protein A chromatography removes most host‐cell proteins (HCPs), mAb aggregates and the remaining HCPs are challenging to remove in a typical bind‐and‐elute cation‐exchange chromatography (CEX) polishing step. A variant of the bind‐and‐elute mode is the displacement mode, which allows strongly binding impurities to be preferentially retained and significantly improves resin utilization. Improved resin utilization renders displacement chromatography particularly suitable in continuous chromatography operations. In this study we demonstrate and exploit sample displacement between a mAb and impurities present at low prevalence (0.002%–1.4%) using different multicolumn designs and recycling. Aggregate displacement depends on the residence time, sample concentration, and solution environment, the latter by enhancing the differences between the binding affinities of the product and the impurities. Displacement among the mAb and low‐prevalence HCPs resulted in an effectively bimodal‐like distribution of HCPs along the length of a multi‐column system, with the mAb separating the relatively more basic group of HCPs from those that are more acidic. Our findings demonstrate that displacement of low‐prevalence impurities along multiple CEX columns allows for selective separation of mAb aggregates and HCPs that persist through protein A chromatography.     

A novel twin‐column continuous chromatography approach for separation and enrichment of monoclonal antibody charge variants

Downstream processing of mAb charge variants is difficult owing to their similar molecular structures and surface charge properties. This study aimed to apply a novel twin‐column continuous chromatography (called N‐rich mode) to separate and enrich acidic variants of an IgG1 mAb. Besides, a comparison study with traditional scaled‐up batch‐mode cation exchange (CEX) chromatography was conducted. For the N‐rich process, two 3.93 mL columns were used, and the buffer system, flow rate and elution gradient slope were optimized. The results showed that 1.33 mg acidic variants with nearly 100% purity could be attained after a 22‐cycle accumulation. The yield was 86.21% with the productivity of 7.82 mg/L/h. On the other hand, for the batch CEX process, 4.15 mL column was first used to optimize the separation conditions, and then a scaled‐up column of 88.20 mL was used to separate 1.19 mg acidic variants with the purity of nearly 100%. The yield was 59.18% with the productivity of 7.78 mg/L/h. By comparing between the N‐rich and scaled‐up CEX processes, the results indicated that the N‐rich method displays a remarkable advantage on the product yield, i.e. 1.46‐fold increment without the loss of productivity and purity. Generally, twin‐column N‐rich continuous chromatography displays a high potential to enrich minor compounds with a higher yield, more flexibility and lower resin cost.     

An Integrated Approach to Aggregate Control for Therapeutic Bispecific Antibodies Using an Improved Three Column Mab Platform-Like Purification Process

Single chain variable fragment-IgGs (scFv-IgG) are a class of bispecific antibodies consisting of two single chain variable fragments (scFv) that are fused to an intact IgG molecule. A common trend observed for expression of scFv-IgGs in mammalian cell culture is a higher level of aggregates (10%–30%) compared to mAbs, which results in lower purification yields in order to meet product quality targets. Furthermore, the high aggregate levels also pose robustness risks to a conventional mAb three column platform purification process which uses only the polishing steps (e.g., cation exchange chromatography [CEX]) for aggregate removal. Protein A chromatography with pH gradient elution, high performance tangential flow filtration (HP-TFF) and calcium phosphate precipitation were evaluated at the bench scale as means of introducing orthogonal aggregate removal capabilities into other aspects of the purification process. The two most promising process variants, namely Protein A pH gradient elution followed by calcium phosphate precipitation were evaluated at pilot scale, demonstrating comparable performance. Implementing Protein A chromatography with gradient elution and/or calcium phosphate precipitation removed a sufficient portion of the aggregate burden prior to the CEX polishing step, enabling CEX to be operated robustly under conditions favoring higher monomer yield. From starting aggregate levels ranging from 15% to 23% in the condition media, levels were reduced to between 2% and 3% at the end of the CEX step. The overall yield for the optimal process was 71%. Results of this work suggest an improved three-column mAb platform-like purification process for purification of high aggregate scFv-IgG bispecific antibodies is feasible. © 2018 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers. Biotechnol. Prog., 35: e2720, 2019     

Mechanistic modeling based process analytical technology implementation for pooling in hydrophobic interaction chromatography

A major challenge in chromatography purification of therapeutic proteins is batch-to-batch variability with respect to impurity levels and product concentration in the feed. Mechanistic model can enable process analytical technology (PAT) implementation by predicting impact of such variations and thereby improving the robustness of the resulting process and controls. This article presents one such application of mechanistic model of hydrophobic interaction chromatography (HIC) as a PAT tool for making robust pooling decisions to enable clearance of aggregates for a monoclonal antibody (mAb) therapeutic. Model predictions were performed before the actual chromatography experiments to facilitate feedforward control. The approach has been successfully demonstrated for four different feeds with varying aggregate levels (3.84%–5.54%) and feed concentration (0.6 mg/mL–1 mg/mL). The resulting pool consistently yielded a product with 1.32 ± 0.03% aggregate vs. a target of 1.5%. A comparison of the traditional approach involving column fractionation with the proposed approach indicates that the proposed approach results in achievement of satisfactory product purity (98.68 ± 0.03% for mechanistic model based PAT controlled pooling vs. 98.64 ± 0.16% for offline column fractionation based pooling). © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2758, 2019.     

Case study and application of process analytical technology (PAT) towards bioprocessing: Use of tryptophan fluorescence as at‐line tool for making pooling decisions for process chromatography

Process analytical technology (PAT) has been gaining momentum in the biopharmaceutical community due to the potential for continuous real time quality assurance resulting in improved operational control and compliance. Two imperatives for implementing any PAT tool are that “variability is managed by the process” and “product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions.” Recently, we have been examining the feasibility of applying different analytical tools to bioprocessing unit operations. We have previously demonstarted that commercially available online‐high performance liquid chromatography and ultra performance liquid chromatography systems can be used for analysis that can facilitate real‐time decisions for column pooling based on product quality attributes (Rathore et al., 2008 a,b). In this article, we review an at‐line tool that can be used for pooling of process chromatography columns. We have demonstrated that our tryptophan fluorescence method offers a feasible approach and meets the requirements of a PAT application. It is significantly faster than the alternative of fractionation, offline analysis followed by pooling. Although the method as presented here is not an online method, this technique may offer better resolution for certain applications and may be a more optimal approach as it is very conducive to implementation in a manufacturing environment. This technique is also amenable to be used as an online tool via front face fluorescence measurements done concurrently with product concentration determination by UV. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Increasing the activity of monoclonal antibody therapeutics by continuous chromatography (MCSGP)

The charged monoclonal antibody (mAb) variants of the commercially available therapeutics Avastin®, Herceptin® and Erbitux® were separated by ion‐exchange gradient chromatography in batch and continuous countercurrent mode (MCSGP process). Different stationary phases, buffer conditions and two MCSGP configurations were used in order to demonstrate the broad applicability of MCSGP in the field of charged protein variant separation. Batch chromatography and MCSGP were compared with respect to yield, purity, and productivity. In the case of Herceptin®, also the biological activity of the product stream was taken into account as performance indicator. The robustness of the MCSGP process against feed composition variations was confirmed experimentally and by model simulations. Biotechnol. Bioeng. 2010;107:652–662. © 2010 Wiley Periodicals, Inc.     

Large scale demonstration of a process analytical technology application in bioprocessing: Use of on‐line high performance liquid chromatography for making real time pooling decisions for process chromatography

Process Analytical Technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community because of the potential for continuous real time quality assurance resulting in improved operational control and compliance. In previous publications, we have demonstrated feasibility of applications involving use of high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) for real‐time pooling of process chromatography column. In this article we follow a similar approach to perform lab studies and create a model for a chromatography step of a different modality (hydrophobic interaction chromatography). It is seen that the predictions of the model compare well to actual experimental data, demonstrating the usefulness of the approach across the different modes of chromatography. Also, use of online HPLC when the step is scaled up to pilot scale (a 2294 fold scale‐up from a 3.4 mL column in the lab to a 7.8 L column in the pilot plant) and eventually to manufacturing scale (a 45930 fold scale‐up from a 3.4 mL column in the lab to a 158 L column in the manufacturing plant) is examined. Overall, the results confirm that for the application under consideration, online‐HPLC offers a feasible approach for analysis that can facilitate real‐time decisions for column pooling based on product quality attributes. The observations demonstrate that the proposed analytical scheme allows us to meet two of the key goals that have been outlined for PAT, i.e., “variability is managed by the process” and “product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions”. The application presented here can be extended to other modes of process chromatography and/or HPLC analysis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Characterization of a unique IgG1 mAb CEX profile by limited Lys-C proteolysis/CEX separation coupled with mass spectrometry and structural analysis

The unique cation exchange chromatography (CEX) charge variant profile of mAb1 is characterized by a combination of mass spectrometry, limited Lys-C digestion followed by CEX separation and structural analysis. During CEX method development, mAb1 showed several unexpected phenomena, including a unique profile containing two main species (acidic 2 and main) and significant instability during stability studies of the main species. Reduced Lys-C peptide mapping identified a small difference in one of the heavy chain peptides (H4) in acidic 2 and further mass analysis identified this difference as Asn55 deamidation. However, the amount of Asn55 deamidation in acidic 2 could account for only half of the species present in this peak. Lys-C limited digest followed by CEX separated several unique peaks in the acidic peak 2 including two pre Fab peaks (LCC1 and LCC2). Whole protein mass analysis suggested that both LCC1 and LCC2 were potentially deamidated species. Subsequent peptide mapping with MS/MS determined that LCC1 contained isoAsp55 and LCC2 contained Asp55. Combining LCC1 and LCC2 CEX peak areas could account for nearly all of the species present in acidic peak 2. Subsequent detailed sequence analysis combined with molecular modeling identified Asn55 and its surrounding residues are responsible for the different CEX behavior and instability of mAb1 following forced degradation at high pH. Overall, the combinatorial approach used in this study proved to be a powerful tool to understand the unique charge variant and stability profile of a monoclonal antibody.     

Characterization of a Monoclonal Antibody Cell Culture Production Process Using a Quality by Design Approach

The goal of quality by design (QbD) in cell culture manufacturing is to develop manufacturing processes which deliver products with consistent critical quality attributes (CQAs). QbD approaches can lead to better process understanding through the use of process parameter risk ranking and statistical design of experiments (DOE). The QbD process starts with an analysis of process parameter risk with respect to CQAs and key performance indicators (KPIs). Initial DOE study designs and their factor test ranges are based on the outcomes of the process parameter risk ranking exercises. Initial DOE studies screen factors for significant influences on CQAs as well as characterize responses for process KPIs. In the case study provided here, multifactor process characterization studies using a scale-down model resulted in significant variation in charge heterogeneity of a monoclonal antibody (MAb) as measured by ion-exchange chromatography (IEC). Iterative DOE studies, using both screening and response surface designs, were used to narrow the operating parameter ranges so that charge heterogeneity could be controlled to an acceptable level. The data from the DOE studies were used to predict worst-case conditions, which were then verified by testing at those conditions. Using the approach described here, multivariate process parameter ranges were identified that yield acceptable CQA levels and that still provide operational flexibility for manufacturing.     

A combined screening and in silico strategy for the rapid design of integrated downstream processes for process and product-related impurity removal

Integrated designs of chromatographic processes for purification of biopharmaceuticals provides potential gains in operational efficiency and reductions of costs and material requirements. We describe a combined method using screening and in silico algorithms for ranking chromatographic steps to rapidly design orthogonally selective integrated processes for purifying protein therapeutics from both process- and product-related impurities. IFN-α2b produced in Pichia pastoris containing a significant product variant challenge was used as a case study. The product and product-related variants were screened on a set of 14 multimodal, ion exchange, and hydrophobic charge induction chromatography resins under various pH and salt linear gradient conditions. Data generated from reversed-phase chromatography of the fractions collected were used to generate a retention database for IFN-α2b and its variants. These data, in combination with a previously constructed process-related impurity database for P. pastoris, were input into an in silico process development tool that generated and ranked all possible integrated chromatographic sequences for their ability to remove both process and product-related impurities. Top-ranking outputs guided the experimental refinement of two successful three step purification processes, one comprising all bind-elute steps and the other having two bind-elute steps and a flowthrough operation. This approach suggests a new platform-like approach for rapidly designing purification processes for a range of proteins where separations of both process- and product-related impurities are needed.     

Variant Identification in Multi-Sample Pools by Illumina Genome Analyzer Sequencing

Multi-sample pooling and Illumina Genome Analyzer (GA) sequencing allows high throughput sequencing of multiple samples to determine population sequence variation. A preliminary experiment, using the RET proto-oncogene as a model, predicted ≤30 samples could be pooled to reliably detect singleton variants without requiring additional confirmation testing. This report used 30 and 50 sample pools to test the hypothesized pooling limit and also to test recent protocol improvements, Illumina GAIIx upgrades, and longer read chemistry. The SequalPrep^TM method was used to normalize amplicons before pooling. For comparison, a single ‘control’ sample was run in a different flow cell lane. Data was evaluated by variant read percentages and the subtractive correction method which utilizes the control sample. In total, 59 variants were detected within the pooled samples, which included all 47 known true variants. The 15 known singleton variants due to Sanger sequencing had an average of 1.62±0.26% variant reads for the 30 pool (expected 1.67% for a singleton variant [unique variant within the pool]) and 1.01±0.19% for the 50 pool (expected 1%). The 76 base read lengths had higher error rates than shorter read lengths (33 and 50 base reads), which eliminated the distinction of true singleton variants from background error. This report demonstrated pooling limits from 30 up to 50 samples (depending on error rates and coverage), for reliable singleton variant detection. The presented pooling protocols and analysis methods can be used for variant discovery in other genes, facilitating molecular diagnostic test design and interpretation.     

Preferential precipitation of acidic variants from monoclonal antibody pools

Microheterogeneity of monoclonal antibodies (mAbs) can impact their activity and stability. Formation of charge variants is considered as the most important source of the microheterogeneity. In particular, controlling the content of the acidic species is often of major importance for the production process and regulatory approval of therapeutic proteins. In this study, the preferential precipitation process was developed for reducing the content of acidic variants in mAb downstream pools. The process design was preceded by the determination of phase behavior of mAb variants in the presence of different precipitants. It was shown that the presence of polyethylene glycol (PEG) in protein solutions favored precipitation of acidic variants of mAbs. Precipitation yield was influenced by the variant composition in the mAb feed solutions, the concentration of the precipitant and the protein, and the ionic strength of the solutions. To improve yield, multistage precipitation was employed, where the precipitate was recycled to the precipitation process. The final product was a mixture of supernatants pooled together from the recycling steps. Such an approach can be potentially used either instead or in a combination with chromatography for adjusting the acidic variant content of mAbs, which can benefit in improvement in throughput and reduction in manufacturing costs.     

Case study and application of process analytical technology (PAT) towards bioprocessing: II. Use of ultra-performance liquid chromatography (UPLC) for making real-time pooling decisions for process chromatography

Process analytical technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community due to the potential for continuous real-time quality assurance resulting in improved operational control and compliance. Two of the key goals that have been outlined for PAT are "variability is managed by the process" and "product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions". Recently, we have been examining the feasibility of applying different analytical tools for designing PAT applications for bioprocessing. We have previously shown that a commercially available online high performance liquid chromatography (HPLC) system can be used for analysis that can facilitate real-time decisions for column pooling based on product quality attributes (Rathore et al., 2008). In this article we test the feasibility of using a commercially available ultra- performance liquid chromatography (UPLC) system for real-time pooling of process chromatography columns. It is demonstrated that the UPLC system offers a feasible approach and meets the requirements of a PAT application. While the application presented here is of a reversed phase assay, the approach and the hardware can be easily applied to other modes of liquid chromatography.     

Charge-based analysis of antibodies with engineered cysteines

THIOMABs are antibodies with an engineered unpaired cysteine residue on each heavy chain that can be used as intermediates to generate antibody-drug conjugates. Multiple charge variant peaks were observed during cation-exchange chromatography (CEX) and imaged capillary isoelectric focusing (cIEF) analysis of several different THIOMABs. This charge heterogeneity was due to cysteinylation and/or glutathionylation at the engineered and unpaired cysteines through disulfide bonds formed during the cell culture process. Cysteine treatment followed by analysis using CEX, LC/MS and electrophoresis demonstrates that cysteine is a mild reductant that can remove glutathione and cysteine bound to the engineered cysteines without disrupting the inter- or intra-chain disulfide bonds of antibodies. We further demonstrated that using a cysteine/cystine redox pair (rather than cysteine alone) can not only effectively remove glutathione at the engineered cysteines, but also generate homogeneously cysteinylated species, which resulted in one main peak in both CEX-HPLC and imaged cIEF assays for antibodies with engineered and unpaired cysteines.     

Model-based design and control of a small-scale integrated continuous end-to-end mAb platform

A continuous integrated bioprocess available from the earliest stages of process development allows for an easier, more efficient and faster development and characterization of an integrated process as well as production of small-scale drug candidates. The process presented in this article is a proof-of-concept of a continuous end-to-end monoclonal antibody production platform at a very small scale based on a 200 ml alternating tangential flow filtration perfusion bioreactor, integrated with the purification process with a model-based design and control. The downstream process, consisting of a periodic twin-column protein A capture, a virus inactivation, a CEX column and an AEX column, was compactly implemented in a single chromatography system, with a purification time of less than 4 hr. Monoclonal antibodies were produced for 17 days in a high cell density perfusion culture of CHO cells with titers up to 1.0 mg/ml. A digital twin of the downstream process was created by modelling all the chromatography steps. These models were used for real-time decision making by the implementation of control strategies to automatize and optimize the operation of the process. A consistent glycosylation pattern of the purified product was ensured by the steady state operation of the process. Regarding the removal of impurities, at least a 4-log reduction in the HCP levels was achieved. The recovery yield was up to 60%, and a maximum productivity of 0.8 mg/ml/day of purified product was obtained.     

An NIR-based PAT approach for real-time control of loading in Protein A chromatography in continuous manufacturing of monoclonal antibodies

Control of column loading in Protein A chromatography is a crucial part of development of robust and flexible process platforms for continuous production of monoclonal antibody (mAb) products. In this paper, we propose a control system that uses near infrared spectroscopy (NIRS) flow cells to accomplish the above. Two applications have been demonstrated using a periodic counter-current continuous chromatography setup. The first application involves use of single NIR flow cell before the inlet of the loading column to measure the concentration of mAb in the harvested broth. Measurement was in real-time (every 3 s) and within ±0.05 mg/ml, significantly better than making UV-based concentration estimations. The second application involved use of an additional NIR flow cell at the outlet of the loading column to measure column breakthrough in real time. The concentration data was transferred to a Python-based monitoring and control algorithm layered over a Cadence BioSMB system. The program could successfully run a three-column periodic counter current method on the BioSMB whereas controlling loading to ensure optimal resin utilization in each loading cycle phase based on precharacterized dynamic binding capacity models, whereas maintaining periodic elutions. The system was tested with multiple perturbations in harvest concentration, modeled after deviations that could arise downstream of a perfusion cell culture system. The results show that the proposed control is a spectroscopy-based process analytical technology tool that facilitates real time monitoring and control of loading in process chromatography. It is adaptable to any continuous chromatography equipment and is very well suited for implementation in a continuous mAb production train.     

Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis

ABSTRACT

The preponderance and diversity of charge variants in therapeutic monoclonal antibodies has implications for antibody efficacy and degradation. Understanding the extent and impact of minor antibody variants is of great interest, and it is also a critical regulatory requirement. Traditionally, a combination of approaches is used to characterize antibody charge heterogeneity, including ion exchange chromatography and independent mass spectrometric variant site mapping after proteolytic digestion. Here, we describe charge variant native mass spectrometry (CVMS), an integrated native ion exchange mass spectrometry-based charge variant analytical approach that delivers detailed molecular information in a single, semi-automated analysis. We utilized pure volatile salt mobile phases over a pH gradient that effectively separated variants based on minimal differences in isoelectric point. Characterization of variants such as deamidation, which are traditionally unattainable by intact mass due to their minimal molecular weight differences, were measured unambiguously by mass and retention time to allow confident MS1 identification. We demonstrate that efficient chromatographic separation allows introduction of the purified forms of the charge variant isoforms into the Orbitrap mass spectrometer. Our CVMS method allows confident assignment of intact monoclonal antibody isoforms of similar mass and relative abundance measurements across three orders of magnitude dynamic range.     

Mid-manufacturing storage: Antibody stability after chromatography and precipitation based capture steps

Antibodies of the IgG2 subclass were captured from the clarified cell culture fluid either by protein A chromatography or by polyethylene glycol precipitation. The captured intermediates were stored as neutralized eluates (protein A chromatography) or in solid form as polyethylene glycol precipitates over a period of 13 months at three temperatures, −20°C, 5°C, and room temperature to compare the capture technologies in regard of the resulting product storability. Monomer content, high molecular mass impurities product loss and changes in the composition of the charge variants were determined at six time points during the storage. At the beginning and end of the study, samples were additionally tested by differential scanning calorimetry, differential scanning fluorimetry, and circular dichroism to determine structural alterations occurring during storage. Protein A purified material was highly stable at all tested temperatures in regard of monomer content and product losses. A transient, acidic isoform was formed during the chromatography step which re-converted to the main charged variant upon storage within a matter of days. Precipitated antibodies could be stored at −20 or 5°C for 3 months without product losses but afterwards recovery yields dropped to 65%. At room temperature, the precipitated antibody was not stable and degraded within 3 months.