Viral clearance and inactivation are critical steps in ensuring the safety of biological products derived from mammalian cell culture and are a component of an adventitious agent control strategy ...which spans both upstream and downstream processes. Although these approaches have been sufficient to support the development of biologics to date, the empirical and semi-quantitative nature of the approach leaves some potential gaps. For example, the concept of performing a quantitative risk assessment for the downstream components of virus safety was introduced in ICH Q5A for XMuLV. An ideal future state would be to perform a similar quantitative risk assessment for a range of viruses based on an assessment of potential virus risk in both upstream and downstream processes. This assessment combined with an integrated control strategy (including monitoring) would be extremely beneficial in minimizing potential adventitious agent risks. Significant progress has been achieved towards this goal in the last several years including recent advances in quantification of virus sequences in cell banks (ADVTIG), development of truly modular or generic viral clearance claims for specific unit operations, enhanced controls of upstream media (HTST/nanofiltration) and the use of RVLP for in-process monitoring. The recent shift towards continuous processing has the potential to enhance the criticality of in-line monitoring and the complexity of viral clearance and inactivation (owing to a wide range of potential ‘worst case’ viral clearance scenarios). However, gaps exist in, firstly, the ability to quantify potential virus risk levels in process streams in real-time, secondly, mechanistic understanding of virus/chromatography media interactions, and thirdly, mechanistic understanding of virus/filter interactions. Some new technologies may also need to be developed to allow for real-time confirmation of virus inactivation and clearance to support process development (both batch and continuous) and assessment of the impact of process deviations during manufacturing.
This review paper provides an overview of the current state of an overall integrated control strategy for upstream and downstream processing and highlights the investments that could be pursued to achieve the future state of a quantitative virus risk assessment for a range of viruses. One potential approach to address these gaps is the use of data mining from large, comprehensive and diverse data sets to establish heuristic rules for virus detection, clearance and inactivation followed by specific hypothesis-driven experiments for cases that fall outside of the normal paradigm. Once this approach reaches a mature state suitable for implementation, there is an opportunity to update regulatory guidance (e.g. ICH Q5A) accordingly.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPUK, ZRSKP
The increased cell density and product titer in biomanufacturing have led to greater use of depth filtration as part of the initial clarification of cell culture fluid, either as a stand‐alone unit ...operation or after centrifugation. Several recent studies have shown that depth filters can also reduce the concentration of smaller impurities like host cell proteins (HCP) and DNA, decreasing the burden on subsequent chromatographic operations. The objective of this study was to evaluate the HCP removal properties of the Pall PDH4 depth filter media, a model depth filter containing diatomaceous earth, cellulose fibers, and a binder. Experiments were performed with both cell culture fluid (CCF) and a series of model proteins with defined pI, molecular weight, and hydrophobicity chosen to match the range of typical HCP. The location of adsorbed (fluorescently labeled) proteins within the depth filters was determined using confocal scanning laser microscopy. Protein binding was greater for proteins that were positively charged and more hydrophobic, consistent with adsorption to the negatively charged diatomaceous earth. The lowest degree of binding was seen with proteins near their pI, which were poorly removed by this filter. These results provide new mechanistic insights into the factors governing the filter capacity and performance characteristics of depth filters containing diatomaceous earth that are widely used in the clarification of CCF.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
•Evaluated mAb retention on MM resins at various salt and temperature conditions.•Data accurately represented by non-linear van't Hoff plots at all the conditions.•Enthalpic contributions were ...dominant at low salt concentrations on all resins.•The binding of mAbs was entropically driven at higher salts on some of the resins.•The heat capacity change upon adsorption was dependent on the salt concentration.
In this study, the thermodynamics of binding of two industrial mAbs to multimodal cation exchange systems was investigated over a range of buffer and salt conditions via a van't Hoff analysis of retention data. Isocratic chromatography was first employed over a range of temperature and salt conditions on three multimodal resins and the retention data were analyzed in both the low and high salt regimes. While mAb retention decreased with salt for all resins at low salts, retention increased at high salts for two of the resins, suggesting a shift from electrostatic to more hydrophobic driven interactions. The retention data at various temperatures were then employed to generate non-linear van't Hoff plots which were fit to the quadratic form of the van't Hoff equation. At low salts, retention of both mAbs decreased with increasing temperature and the van't Hoff plots were concave downward on Capto MMC and Nuvia cPrime, while being concave upward on Capto MMC ImpRes. Different trends were observed on some of the resins with respect to both the concavity of the van't Hoff plots as well as the impact of temperature on the favorable enthalpies in the low salt regime. Interestingly, while increasingly favorable enthalpy with temperature was observed with Capto MMC and Nuvia cPrime at low salt, favorable enthalpy decreased with temperature for Capto MMC ImpRes. At high salts, binding of both mAbs on the two Capto resins were consistently entropically driven, consistent with desolvation. While the negative heat capacity data at low salts indicated that desolvation of polar/charged groups were important in Capto MMC and Nuvia cPrime, the positive data suggested that desolvation of non-polar groups were more important with Capto MMC ImpRes. Finally, the data at high salts indicated that desolvation of non-polar groups was the major driver for binding of both mAbs to the Capto resins under these conditions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
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•New Direct RT qPCR method for CHO Retrovirus-like particles.•No RNA extraction prior to RT qPCR.•Thermolabile RNase and DNase digestion of CHO culture samples.•Denaturation of ...enzymes prior to RT qPCR.•All operations done in one PCR plate.
Chinese hamster ovary (CHO) cells are the host cell of choice for manufacturing biologic drugs, like monoclonal antibody, in the biopharmaceutical industry. Retrovirus-like particles (RVLPs) are made during the manufacturing process with CHO cells and it is incumbent upon the manufacturer to perform risk assessment based on levels of RVLP in unprocessed bulk. Quantification of RVLP using electron microscopy (EM) is the standard method. However, reverse transcription based real-time PCR (RT qPCR) is an alternative method available. This method involves RNase digestion of cell culture fluid to remove free RNA, followed by extraction of total nucleic acid and digestion with DNase to remove extracted DNA molecules, and then finally reverse transcription and PCR. Here we report a method where the nucleic acids extraction step is eliminated prior to qPCR. In this method the cell-free culture supernatant sample is digested with thermolabile DNase and RNase at the same time in a 96-well PCR plate; subsequently the enzymes are heat-denatured; then RT qPCR reagents are added to the wells in the PCR plate along with standards and controls in other wells of the same plate; finally the plate is subjected to RT qPCR for analysis of RVLP RNA in the samples. This direct RT qPCR method for RVLP is sensitive to 10 particles of RVLP with good precision and accuracy and has a wide linear range of quantification. The method has been successfully tested with different production batches, shown to be consistent, and correlates well with the extraction-based method. However, the results are about 1-log higher compared to EM method. This method simplifies the RVLP quantification protocol, reduces time of analysis and leads to increased assay sensitivity and development of automated high-throughput methods. Additionally, the method can be an added tool for viral clearance studies, by testing process-intermediate samples like Protein A column and ion-exchange column eluates, for increased confidence in purification of biologics manufactured in CHO cell culture.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
The growth of advanced analytics in manufacturing monoclonal antibodies (mAbs) has highlighted the challenges associated with the clearance of host cell proteins (HCPs). Of special concern is the ...removal of “persistent” HCPs, including immunogenic and mAb‐degrading proteins, that co‐elute from the Protein A resin and can escape the polishing steps. Responding to this challenge, we introduced an ensemble of peptide ligands that target the HCPs in Chinese hamster ovary (CHO) cell culture fluids and enable mAb purification via flow‐through affinity chromatography. This study describes their integration into LigaGuard™, an affinity adsorbent featuring an equilibrium binding capacity of ~30 mg of HCPs per mL of resin as well as dynamic capacities up to 16 and 22 mg/ml at 1‐ and 2‐min residence times, respectively. When evaluated against cell culture harvests with different mAb and HCP titers and properties, LigaGuard™ afforded high HCP clearance, with logarithmic removal values (LRVs) up to 1.5, and mAb yield above 90%. Proteomic analysis of the effluents confirmed the removal of high‐risk HCPs, including cathepsins, histones, glutathione‐S transferase, and lipoprotein lipases. Finally, combining LigaGuard™ for HCP removal with affinity adsorbents for product capture afforded a global mAb yield of 85%, and HCP and DNA LRVs > 4.
A novel purification paradigm based on the selective capture of host cell proteins (HCPs) from CHO cell culture fluids containing monoclonal antibodies (mAbs) was demonstrated using a novel adsorbent (LigaGuard™) developed by Prof. Menegatti and coworkers. Using an ensemble of analytical techniques—including proteomics, enzyme‐linked immunosorbent assay, and analytical chromatography—the authors demonstrated the ability of LigaGuard™ to remove persistent and high‐risk HCPs via “flow‐through affinity chromatography,” thus enabling a next‐generation mAb purification process with superior robustness and performance.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
In the production of biopharmaceuticals such as monoclonal antibodies (mAbs) and vaccines, the residual amounts of host‐cell proteins (HCPs) are among the critical quality attributes. In addition to ...overall HCP levels, individual HCPs may elude purification, potentially causing issues in product stability or patient safety. Such HCP persistence has been attributed mainly to biophysical interactions between individual HCPs and the product, resin media, or residual chromatin particles. Based on measurements on process streams from seven mAb processes, we have found that HCPs in aggregates, not necessarily chromatin‐derived, may play a significant role in the persistence of many HCPs. Such aggregates may also hinder accurate detection of HCPs using existing proteomics methods. The findings also highlight that certain HCPs may be difficult to remove because of their functional complementarity to the product; specifically, chaperones and other proteins involved in the unfolded protein response (UPR) are disproportionately present in the aggregates. The methods and findings described here expand our understanding of the origins and potential behavior of HCPs in cell‐based biopharmaceutical processes and may be instrumental in improving existing techniques for HCP detection and clearance.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Depth filters are used throughout downstream bioprocessing, with specialized filter chemistries developed for removal of host cell proteins, DNA, and protein aggregates. These filters often show very ...broad breakthrough curves, but there have not been any previous analyses of this phenomenon. Protein breakthrough curves were evaluated for Ribonuclease A and α-chymotrypsin in two small-scale depth filter modules using PDH4 media containing diatomaceous earth. Computational fluid dynamics (CFD) simulations were developed, with flow in the porous media described using the modified Navier-Stokes equations incorporating a Brinkman flow term and a Langmuir binding model. The breakthrough curve in the stainless-steel module was much sharper than that in the commercial Supracap™ 50 capsule due to the more uniform axisymmetric flow field. The behavior in the Supracap™ 50 capsule was more complex, with multiple inflection points observed due to the different flow paths and the time-dependent saturation of the two layers of the depth filter media. These results provide important insights into the factors controlling protein binding/breakthrough in commercial depth filter modules.
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•Protein binding behavior evaluated in small-scale Supracap™ 50 depth filters.•CFD analysis shows presence of four distinct flow paths through capsule.•Multiple flow paths lead to broad and complex breakthrough curve.•Computational/experimental results provide insights into performance of depth filters.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPUK, ZAGLJ, ZRSKP
Nonionic surfactant polysorbates, including PS-80 and PS-20, are commonly used in the formulation of biotherapeutic products for both preventing surface adsorption and acting as stabilizer against ...protein aggregation. Trace levels of residual host cell proteins (HCPs) with lipase or esterase enzymatic activity have been shown to degrade polysorbates in biologics formulation. The measurement and control of these low abundance, high-risk HCPs for polysorbate degradation are an industry-wide challenge to achieve desired shelf life of biopharmaceuticals in liquid formulation, especially for high-concentration formulation product development. Here, we reviewed the challenges, recent advances, and future opportunities of analytical method development, risk assessment, and control strategies for polysorbate degradation during formulation development with a focus on enzymatic degradation. Continued efforts to advance our understanding of polysorbate degradation in biologics formulation will help develop high-quality medicines for patients.
Significant increases in cell density and product titer have led to renewed interest in the application of depth filtration for initial clarification of cell culture fluid in antibody production. The ...performance of these depth filters will depend on the local pressure and velocity distribution within the filter capsule, but these are very difficult to probe experimentally, leading to challenges in both process design and scale‐up. We have used a combination of carefully designed experimental studies and computational fluid dynamics (CFD) to examine these issues in both lab‐scale (SupracapTM 50) and pilot‐scale (StaxTM) depth filter modules, both employing dual‐layer lenticular PDH4 media containing diatomaceous earth. The SupracapTM 50 showed a more rapid increase in transmembrane pressure and a more rapid DNA breakthrough during filtration of a Chinese Hamster Ovary cell culture fluid. These results were explained using CFD calculations which showed very different flow distributions within the modules. CFD predictions were further validated using measurements of the residence time distribution and dye binding in both the lab‐scale and pilot‐plant modules. These results provide important insights into the factors controlling the performance and scale‐up of these commercially important depth filters as well as a framework that can be broadly applied to develop more effective depth filters and depth filtration processes.
The authors evaluated the performance characteristics for both lab‐and pilot‐scale depth filtration modules for Chinese Hamster Ovary (CHO) cell clarification. The pilot‐scale module showed greater capacity and DNA removal due to more uniform flow distribution, which was verified by computational fluid dynamics, residence time distribution, and dye‐binding studies.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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