Harvested Cell Culture Fluid Research Articles

Raman spectroscopy for monitoring free sulfhydryl formation during monoclonal antibody manufacturing

During production, harvested cell culture fluid (HCCF) can degrade due to reductases breaking interchain disulfide bonds, forming low molecular weight (LMW) impurities that contain free sulfhydryl and high molecular weight (HMW) impurities through disulfide shuffling. Thus, detecting and quantifying the free sulfhydryl increase in HCCF is critical. Herein, Raman spectroscopy is implemented as a process analytical technology, and multivariate data analysis is applied to characterize and quantify sulfhydryl formation in HCCF with disulfide-containing indicator molecules. Raman spectra qualitatively probe the presence or absence of disulfide bond breakage in antibodies, consistent with offline non-reduced capillary electrophoresis sodium dodecyl sulfate results. Between two antibodies studied, mAb A was identified for a higher risk of antibody reduction where sulfhydryl formation was observed within 16 h, while mAb B did not show similar concerns even after 1 week. The offline measurement of redox potential is below –100 mV in HCCF for mAb A, while the stable mAb B HCCF shows redox potentials above +20 mV. A multivariate partial least squares (PLS) model for quantification is developed using an offline free sulfhydryl assay, applying Raman spectra to predict free sulfhydryl concentration with high accuracy (R2 > 0.98) and expected mean error of 0.677 mM from the offline Ellman’s Assay. This work confirms the use of Raman PAT to monitor real-time disulfide reduction, enabling improvements to process understanding and product quality.

Purification of monoclonal antibodies using novel 3D printed ordered stationary phases

3D printing offers the unprecedented ability to fabricate chromatography stationary phases with bespoke 3D morphology as opposed to traditional packed beds of spherical beads. The restricted range of printable materials compatible with chromatography is considered a setback for its industrial implementation. Recently, we proposed a novel ink that exhibits favourable printing performance (printing time ∼100 mL/h, resolution ∼200 µm) and broadens the possibilities for a range of chromatography applications thanks to its customisable surface chemistry. In this work, this ink was used to fabricate 3D printed ordered columns with 300 µm channels for the capture and polishing of therapeutic monoclonal antibodies. The columns were initially assessed for leachables and extractables, revealing no material propensity for leaching. Columns were then functionalised with protein A and SO3 ligands to obtain affinity and strong cation exchangers, respectively. 3D printed protein A columns showed >85 % IgG recovery from harvested cell culture fluid with purities above 98 %. Column reusability was evaluated over 20 cycles showing unaffected performance. Eluate samples were analysed for co-eluted protein A fragments, host cell protein and aggregates. Results demonstrate excellent HCP clearance (logarithmic reduction value of > 2.5) and protein A leakage in the range of commercial affinity resins (<100 ng/mg). SO3 functionalised columns employed for polishing achieved removal of leaked Protein A (down to 10 ng/mg) to meet regulatory expectations of product purity. This work is the first implementation of 3D printed columns for mAb purification and provides strong evidence for their potential in industrial bioseparations.

Evaluating end-to-end continuous antibody manufacture with column-free capture alternatives from economic, environmental, and robustness perspectives.

Process intensification efforts have renewed interest in the potential of end-to-end continuous manufacture with column-free capture alternatives. This article describes a decisional tool that encompasses mass balance and design equations, process economics, stochastic simulation and multi-criteria decision-making and enables the evaluation of different batch, and continuous flowsheets for monoclonal antibody (mAb) manufacture. The traditional batch process was compared with end-to-end continuous bioprocesses with either protein A capture or column-free capture employing aqueous two-phase extraction or precipitation from economic, environmental, and robustness perspectives. The cost of goods analysis predicted that continuous flowsheets could offer substantial cost savings (~20%-40%) over the batch process at low and medium annual commercial demands (100-500 kg); however, at tonnage demands they resulted in either comparable or higher costs. Comparing the continuous options, the continuous flowsheets with protein A or precipitation yielded similar COG/g values, while aqueous two-phase extraction presented higher costs. The analysis of overall process mass intensities accounting for water and consumables suggested that the continuous flowsheet with protein A would result in the lowest environmental burden. When the economic, environmental, and operational criteria were reconciled using multi-criteria decision-making analysis, the continuous protein A-based flowsheet was found to be the most favorable. A target analysis highlighted the need for process improvements in the following parameters to reduce the manufacturing costs of the continuous column-free capture options below that of protein A: the perfusion volumetric productivity, the harvested cell culture fluid percentage in column-free operations, the column-free step yields along with the implementation of buffer concentrates.

Factors affecting product association as a mechanism of host-cell protein persistence in bioprocessing.

Product association of host-cell proteins (HCPs) to monoclonal antibodies (mAbs) is widely regarded as a mechanism that can enable HCP persistence through multiple purification steps and even into the final drug substance. Discussion of this mechanism often implies that the existence or extent of persistence is directly related to the strength of binding but actual measurements of the binding affinity of such interactions remain sparse. Two separate avenues of investigation of HCP-mAb binding are reported here. One is the measurement of the affinity of binding of individual, commonly persistent Chinese hamster ovary (CHO) HCPs to each of a set of mAbs, and the other uses quantitative proteomic measurements to assess binding of HCPs in a null CHO harvested cell culture fluid (HCCF) to mAbs produced in the same cell line. The individual HCP measurements show that the binding affinities of individual HCPs to different mAbs can vary appreciably but are rarely very high, with only weak pH dependence. The measurements on the null HCCF allow estimation of individual HCP-mAb affinities; these are typically weaker than those seen in affinity measurements on isolated HCPs. Instead, the extent of binding appears correlated with the initial abundance of individual HCPs in the HCCF and the forms of the HCPs in the solution, i.e., whether HCPs are present as free molecules or as parts of large aggregates. Separate protein A chromatography experiments performed by feeding different fractions of a mAb-containing HCCF obtained by size-exclusion chromatography (SEC) showed clear differences in the number and identity of HCPs found in the protein A eluate. These results indicate a significant role for HCP-mAb association in determining HCP persistence through protein A chromatography, presumably through binding of HCP-mAb complexes to the resin. Overall, the results illustrate the importance of considering more fully the biophysical context of HCP-product association in assessing the factors that may affect the phenomenon and determine its implications. Knowledge of the abundances and the forms of individual or aggregated HCPs in HCCF are particularly significant, emphasizing the integration of upstream and downstream bioprocessing and the importance of understanding the collective properties of HCPs in addition to just the biophysical properties of individual HCPs.

Fiber chromatographic enabled process intensification increases monoclonal antibody product yield.

The most straightforward method to increase monoclonal antibody (mAb) product yield is to complete the purification process in less steps. Here, three different fiber chromatographic devices were implemented using a holistic approach to intensify the mAb purification process and increase yield. Fiber protein A (proA) chromatography was first investigated, but traditional depth filtration was not sufficient in reducing the contaminant load as the fiber proA device prematurely fouled. Further experimentation revealed that chromatin aggregates were the most likely reason for the fiber fouling. To reduce levels of chromatin aggregates, a chromatographic clarification device (CCD) was incorporated into the process, resulting in single-stage clarification of harvested cell culture fluid and reduction of DNA levels. The CCD clarified pool was then successfully processed through the fiber proA device, fully realizing the productivity gains that the fiber technology offers. After the proA and viral inactivation neutralization (VIN) hold step, the purification process was further intensified using a novel single-use fiber-based polishing anion exchange (AEX) material that is capable of binding both soluble and insoluble contaminants. The three-stage fiber chromatographic purification process was compared to a legacy five-step process of dual-stage depth filtration, bead-based proA chromatography, post-VIN depth filtration, and bead-based AEX chromatography. The overall yield from the five-step process was 60%, while the fiber chromatographic-enabled intensified process had an overall yield of 70%. The impurity clearance of DNA and host cell protein (HCP) for both processes were within the regulatory specification (<100 ppm HCP, <1 ppb DNA). For the harvest of a 2000 L cell culture, the intensified process is expected to increase productivity by 2.5-fold at clarification, 50-fold at the proA step, and 1.6-fold in polishing. Relative to the legacy process, the intensified process would reduce buffer use by 1088 L and decrease overall process product mass intensity by 12.6%.

Development and Comparison of Alternative Methods for the Purification of Adalimumab Directly from Harvested Cell Culture Fluid.

Protein A affinity chromatography is a well-established method currently used in the pharmaceutical industry. However, the high costs usually associated with chromatographic separation of protein A and the difficulties in continuous operation make the investigation of alternative purification methods very important. In this study, extraction/back-extraction and precipitation/dissolution methods were developed and optimised. They were compared with protein A and cation exchange chromatography separations in terms of yield of monoclonal antibody (mAb) and amount of residual impurities, such as DNA and host cell proteins, and amount of mAb aggregates. For a comprehensive comparison of the different methods, experiments were carried out with the same cell-free fermentation broth containing adalimumab. Protein A and cation exchange chromatographic separations resulted in high yield and purity of adalimumab. The precipitation-based process resulted in high yield but with lower purity. The extraction-based purification resulted in low yield and purity. Thus, the precipitation-based method proved to be more promising than the extraction-based method for direct purification of adalimumab from harvested cell culture fluid. Although alternative purification methods may offer the advantages of simplicity and low-cost operation, further significant improvements are required to compete with the performance of chromatographic separations of adalimumab from true fermentation broth.

Analytical characterization of host-cell-protein-rich aggregates in monoclonal antibody solutions.

Host-cell proteins (HCPs) and high molecular weight (HMW) species have historically been treated as independent classes of impurities in the downstream processing of monoclonal antibodies (mAbs), but recent indications suggest that they may be partially linked. We have explored this connection with a shotgun proteomic analysis of HMW impurities that were isolated from harvest cell culture fluid (HCCF) and protein A eluate using size-exclusion chromatography (SEC). As part of the proteomic analysis, a cross-digest study was performed in which samples were analyzed using both the standard and native digest techniques to enable a fair comparison between bioprocess pools. This comparison reveals that the HCP profiles of HCCF and protein A eluate overlap substantially more than previous work has suggested, because hundreds of HCPs are conserved in aggregates that may be up to ~50 nm in hydrodynamic radius and that persist through the protein A capture step. Quantitative SWATH proteomics suggests that the majority of the protein A eluate's HCP mass is found in such aggregates, and this is corroborated by ELISA measurements on SEC fractions. The SWATH data also show that intra-aggregate concentrations of individual HCPs are positively correlated between aggregates that were isolated from HCCF and protein A eluate, and species that have generally been considered difficult to remove tend to be more concentrated than their counterparts. These observations support prior hypotheses regarding aggregate-mediated HCP persistence through protein A chromatography and highlight the importance of this persistence mechanism.

Systematic analytical workflow for characterisation and identification of partially reduced species in monoclonal antibody manufacturing

Fragmentation is a major degradation pathway ubiquitous to all therapeutic monoclonal antibody (mAb) and therefore, monitored throughout the manufacturing process. Here, we describe a three-step approach to 1) detect, 2) confirm and 3) characterize partially reduced fragment species in an immunoglobulin G1 (IgG1) mAb with prolonged hold time of harvested cell culture fluid (HCCF). Microchip capillary electrophoresis (MCE) and high-performance size exclusion chromatography (HPSEC) were used as fast and efficient screening methods to detect fragmentation. HPSEC was found to be underestimating fragmentation levels. To confirm and characterize the fragments, capillary electrophoresis-sodium dodecyl sulphate (CE-SDS) was employed. Interestingly, the absence of fragments in the reduced CE-SDS analysis suggested partial reduction of disulphide bonds contributing to fragmentation in this mAb lot. This was further confirmed using reverse phase high performance liquid chromatography (RP-HPLC) coupled with mass spectrometry, which established the presence of heavy-heavy-light (HHL), heavy-heavy (HH), light-light dimer (LL), light chain (LC) and half antibody (HL) fragments with good mass accuracy. In this study, we demonstrated a readily applicable systematic strategy to support process development and investigate anomalous events in manufacturing. An additional highlight of this work is the data-driven comprehensive comparison of modern and conventional analytical techniques for fragment analysis.

Single-stage chromatographic clarification of Chinese Hamster Ovary cell harvest reduces cost of protein production.

A single-stage clarification was developed using a single-use chromatographic clarification device (CCD) to recover a recombinant protein from Chinese Hamster Ovary (CHO) harvest cell culture fluid (HCCF). Clarification of a CHO HCCF is a complex and costly process, involving multiple stages of centrifugation and/or depth filtration to remove cells and debris and to reduce process-related impurities such as host cell protein (HCP), nucleic acids, and lipids. When using depth filtration, the filter train consists of multiple filters of varying ratios, layers, pore sizes, and adsorptive properties. The depth filters, in combination with a 0.2-micron membrane filter, clarify the HCCF based on size-exclusion, adsorptive, and charge-based mechanisms, and provide robust bioburden control. Each stage of the clarification process requires time, labor, and utilities, with product loss at each step. Here, use of the 3M™ Harvest RC Chromatographic Clarifier, a single-stage CCD, is identified as an alternative strategy to a three-stage filtration train. The CCD results in less overall filter area, less volume for flushing, and higher yield. Using bioprocess cost modeling, the single-stage clarification process was compared to a three-stage filtration process. By compressing the CHO HCCF clarification to a single chromatographic stage, the overall cost of the clarification process was reduced by 17%-30%, depending on bioreactor scale. The main drivers for the cost reduction were reduced total filtration area, labor, time, and utilities. The benefits of the single-stage harvest process extended throughout the downstream process, resulting in a 25% relative increase in cumulative yield with comparable impurity clearance.

Immunoreactivity profiling of Anti-Chinese hamster ovarian host cell protein antibodies by isobaric labeled affinity purification-mass spectrometry reveals low-recovery proteins

We evaluated the immunoreactivity profiles of eight commercial anti-host cell protein (anti-HCP) antibodies from different host animals and their antigens used for immunization by an isobaric labeled affinity purification-mass spectrometry (AP-MS) method. As a result, 34 proteins with high abundance but low recovery from harvest cell culture fluid were identified. Since they are likely to be underestimated in biopharmaceutical quality assessment, the features common to these proteins were investigated. Compared to other immunoprecipitated HCP proteins, proteins exhibiting lower molecular weight (ΔMW = -14600), lower isoelectric point (ΔpI = -0.86), and lower hydrophobicity (ΔGRAVY = -0.13) were enriched. This AP-MS method provides important information for HCP control strategies using immunological methods and is expected to contribute to the development of safe biopharmaceutics.

Tractable Method for Rapid Quality Assessment of Therapeutic Antibodies in Harvested Cell Culture Fluid based on FcγRIIIa-Immobilized Magnetic Microspheres.

FcγRIIIa-binding affinity is one of the key factors to ensure the efficacy of many antitumor therapeutic antibodies, which should be monitored along with the titer, protein aggregation, and other critical quality attributes. The conventional workflow for the quality assessment of therapeutic antibodies in harvested cell culture fluid (HCCF) is time-consuming and costly nevertheless. In this study, a tractable method was established for rapid quality assessment of a HCCF sample through differentially extracting IgG with different FcγRIIIa affinity levels using FcγRIIIa-immobilized magnetic microspheres, followed by size exclusion chromatography (SEC) to determine the amount and monomer percentage of IgGs in the preceding eluate. FcγRIIIa-immobilized magnetic microspheres with polydopamine (PDA) and hydrophilic dendrimer (PAMAM) coating (denoted as Fe3O4@PDA@PAMAM-FcγRIIIa) were synthesized for the first time as magnetic adsorbents. The PDA cladding endowed the composites with good chemical stability in acidic elution buffer, and the PAMAM dendrimer empowered the composites of high ligand immobilization capacity and hydrophilic surface. The labile FcγRIIIa was immobilized under mild conditions. By directly applying a simple magnetic solid phase extraction procedure to treat HCCF, favored IgG species with high FcγRIIIa affinity would be selectively captured by Fe3O4@PDA@PAMAM-FcγRIIIa composites for subsequent SEC analysis. The monomer peak area value in SEC, which was set as the read-out of the proposed method, correlated directly with the theoretical overall quality of standard-spiked HCCF samples.

Comprehensive assessment of host cell protein expression after extended culture and bioreactor production of CHO cell lines.

The biomanufacturing industry is advancing toward continuous processes that will involve longer culture durations and older cell ages. These upstream trends may bring unforeseen challenges for downstream purification due to fluctuations in host cell protein (HCP) levels. To understand the extent of HCP expression instability exhibited by Chinese hamster ovary (CHO) cells over these time scales, an industry-wide consortium collaborated to develop a study to characterize age-dependent changes in HCP levels across 30, 60, and 90 cell doublings, representing a period of approximately 60 days. A monoclonal antibody (mAb)-producing cell line with bulk productivity up to 3 g/L in a bioreactor was aged in parallel with its parental CHO-K1 host. Subsequently, both cell types at each age were cultivated in an automated bioreactor system to generate harvested cell culture fluid (HCCF) for HCP analysis. More than 1500 HCPs were quantified using complementary proteomic techniques, two-dimensional electrophoresis (2DE) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). While up to 13% of proteins showed variable expression with age, more changes were observed when comparing between the two cell lines with up to 47% of HCPs differentially expressed. A small subset (50 HCPs) with age-dependent expression were previously reported to be problematic as high-risk and/or difficult-to-remove impurities; however, the vast majority of these were downregulated with age. Our findings suggest that HCP expression changes over this time scale may not be as dramatic and pose as great of a challenge to downstream processing as originally expected but that monitoring of variably expressed problematic HCPs remains critical.

Development and application of an analytical approach to assess an antibody's potential for disulfide reduction.

Monoclonal antibody (mAb) interchain disulfide bond reduction has been observed in a recent large-scale clinical manufacturing operation. A massive reduction/precipitation at post-clarification steps has occurred. This note presents the development of a novel analytical approach to identify the "potential reduction"-a unique approach to predict the propensity of a monomeric-profiled mAb to be reduced in the post-harvest stage, such as harvest clarification and/or purification steps. The core of this new approach includes comparing the non-reducing capillary electrophoresis profiles of pre- and post-vacuum treated mAb in harvest cell culture fluid (HCCF). Using this approach, the potential reductions of two in-house mAbs in the unclarified and clarified cell culture harvest were assessed.

Profiling Active Enzymes for Polysorbate Degradation in Biotherapeutics by Activity-Based Protein Profiling.

Polysorbate is widely used to maintain stability of biotherapeutic proteins in pharmaceutical formulation development. Degradation of polysorbate can lead to particle formation in drug products, which is a major quality concern and potential patient risk factor. Enzymatic activity from residual host cell enzymes such as lipases and esterases plays a major role for polysorbate degradation. Their high activity, often at very low concentration, constitutes a major analytical challenge in the biopharmaceutical industry. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation. Using an optimized chemical probe, we established the first global profile of active serine hydrolases in harvested cell culture fluid (HCCF) for monoclonal antibodies (mAbs) production from two Chinese hamster ovary (CHO) cell lines. A total of eight known lipases were identified by ABPP with enzyme activity information, while only five lipases were identified by a traditional abundance-based proteomics (TABP) approach. Interestingly, phospholipase B-like 2 (PLBL2), a well-known problematic HCP was not found to be active in process-intermediates from two different mAbs. In a proof-of-concept study with downstream samples, phospholipase A2 group VII (PLA2G7) was only identified by ABPP and confirmed to contribute to polysorbate-80 degradation for the first time. The established ABBP approach is approved to be able to identify low-abundance host cell enzymes and fills the gap between lipase abundance and activity, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.

Metabolic understanding of disulfide reduction during monoclonal antibody production.

The disulfide reduction of intact monoclonal antibodies (mAbs) and subsequent formation of low molecular weight (LMW) species pose a direct risk to product stability, potency, and patient safety. Although enzymatic mechanisms of reduction are well established, an understanding of the cellular mechanisms during the bioreactor process leading to increased risk of disulfide reduction after harvest remains elusive. In this study, we examined bench, pilot, and manufacturing-scale batches of two mAbs expressed in Chinese hamster ovary (CHO) cells, where harvested cell culture fluid (HCCF) occasionally demonstrated disulfide reduction. Comparative proteomics highlighted a significant elevation in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels in a highly reducing batch of HCCF, compared to a non-reducing batch. Analysis during production cell culture showed that increased GAPDH gene and protein expression correlated to disulfide reduction risk in HCCF in every case examined. Additionally, glucose 6-phosphate dehydrogenase (G6PD) activity and an increased (≥ 300%) lactate/pyruvate molar ratio (lac/pyr) during production cell culture correlated to disulfide reduction risk, suggesting a metabolic shift to the pentose phosphate pathway (PPP). In all, these results suggest that metabolic alterations during cell culture lead to changes in protein expression and enzyme activity that in turn increase the risk of disulfide reduction in HCCF. KEY POINTS: • Bioreactor conditions resulted in reduction susceptible harvest material. • GAPDH expression, G6PD activity, and lac/pyr ratio correlated with mAb reduction. • Demonstrated role for cell metabolic changes in post-harvest mAb reduction. Graphical abstract.

Chromatographic clarification overcomes chromatin-mediated hitch-hiking interactions on Protein A capture column.

Protein A capture chromatography is a critical unit operation in the clearance of host cell protein (HCP) impurities in monoclonal antibody (mAb) purification processes. Though one of the most effective purification steps, variable levels of protein impurities are often observed in the eluate. Coelution of HCP impurities is suggested to be strongly affected by the presence of chromatin complexes (Gagnon et al., 2014; Koehler et al., 2019). We investigated the effect of removal of DNA complex and HCP reduction pre-Protein A on the HCP clearance performance of the Protein A capture step itself. We found that only reduction of DNA in the Protein A load consistently lowered HCP in the Protein A eluate. Reduction of HCP in the Protein A load stream did not produce a significant increase in the chromatography HCP clearance performance. These results are consistent across three different biosimilar therapeutic mAbs expressed by the same Chinese hamster ovary (CHO) cell line (i.e., CHOBC ® of Polpharma Biologics). This result demonstrates that optimization of the mAb purification process utilizing Protein A as the primary capture step depends primarily on being able to effectively clear DNA and associated complexes early in the process, rather than trying to incorporate HCP reduction at the harvest cell culture fluid.

High throughput solubility and redissolution screening for antibody purification via combined PEG and zinc chloride precipitation.

As upstream product titers increase, the downstream chromatographic capture step has become a significant "downstream bottleneck." Precipitation becomes more attractive under these conditions as the supersaturation driving force increases with the ever-increasing titer. In this study, two precipitating reagents with orthogonal mechanisms, polyethylene glycol (PEG) as a volume excluder and zinc chloride (ZnCl2 ) as a cross linker, were examined as precipitants for two monoclonal antibodies (mAbs), one stable and the other aggregation-prone, in purified drug substance and harvested cell culture fluid forms. Manual batch solubility and redissolution experiments were performed as scouting experiments. A high throughput (HTP) liquid handling system was used to investigate the design space as fully as possible while reducing time, labor, and material requirements. Precipitation and redissolution were studied by systematically varying the concentrations of PEG and ZnCl2 to identify combinations that resulted in high yield and good quality for the stable mAb; PEG concentrations in the range 7-7.5 wt/vol% together with 10 mM ZnCl2 gave a yield of 97% and monomer contents of about 93%. While yield for the unstable mAb was high, quality was not acceptable. Performance at selected conditions was further corroborated for the stable mAb using a continuous tubular precipitation reactor at the laboratory scale. The HTP automation system was a powerful tool for locating desired (customized) conditions for antibodies of different physicochemical properties.

Impacts on product quality attributes of monoclonal antibodies produced in CHO cell bioreactor cultures during intentional mycoplasma contamination events.

A mycoplasma contamination event in a biomanufacturing facility can result in costly cleanups and potential drug shortages. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and penetrate the standard 0.2‐µm filters used in the clarification of harvested cell culture fluid. Previously, we reported a study regarding the ability of Mycoplasma arginini to persist in a single‐use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G 1 (IgG1) antibody. Our previous work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Careful evaluation of certain identified process parameters over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before detection from a traditional method. In this report, we studied the changes in the IgG1 product quality produced by CHO cells considered to be induced by the M. arginini contamination events. We observed changes in critical quality attributes correlated with the duration of contamination, including increased acidic charge variants and high mannose species, which were further modeled using principal component analysis to explore the relationships among M. arginini contamination, CHO cell growth and metabolites, and IgG1 product quality attributes. Finally, partial least square models using NIR spectral data were used to establish predictions of high levels (≥104 colony‐forming unit [CFU/ml]) of M. arginini contamination, but prediction of levels below 104 CFU/ml were not reliable. Contamination of CHO cells with M. arginini resulted in significant reduction of antibody product quality, highlighting the importance of rapid microbiological testing and mycoplasma testing during particularly long upstream bioprocesses to ensure product safety and quality.

Fast and Automated Characterization of Monoclonal Antibody Minor Variants from Cell Cultures by Combined Protein-A and Multidimensional LC/MS Methodologies

Monitoring of post-translational modifications (PTMs) in therapeutic monoclonal antibodies (mAbs) is essential during their production in both upstream and downstream processes. However, characterization of PTMs using a conventional peptide mapping procedure requires time-consuming and labor-intensive offline sample preparation steps. This work describes for the first time, the implementation of a Protein-A affinity chromatography column as the first dimension (1D) in a multidimensional LC (3D and 4D) setup for the automated characterization of mAb variants from harvest cell culture fluid (HCCF) materials at different purification/production steps. A 4D-LC/MS method (Protein-A-Reduction-RPLC-Digestion-RPLC/MS) was developed to determine PTM levels including oxidation, deamidation, and succinimide formation by online peptide mapping analysis. To obtain an accurate and comprehensive profiling of mAb glycosylation patterns at the reduced level, a 3D-LC/MS method (Protein-A-Reduction-RPLC-HILIC/MS) was also developed on the same chromatographic system. Overall, the full workflow (data acquisition and analysis) for both 3D and 4D-LC/MS setups can be completed within less than 1-2 days, compared to weeks with the conventional manual approach. This proof of concept study demonstrates that mD-LC/MS has the potential to be used as a powerful tool to perform a fast and reliable monitoring of PTMs during the manufacturing process for both bioreactor control or as a monitoring assay.

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator.

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.