Strategies for developing design spaces for viral clearance by anion exchange chromatography during monoclonal antibody production.

The quality-by-design (QbD) regulatory initiative promotes the development of process design spaces describing the multidimensional effects and interactions of process variables on critical quality attributes of therapeutic products. However, because of the complex nature of production processes, strategies must be devised to provide for design space development with reasonable allocation of resources while maintaining highly dependable results. Here, we discuss strategies for the determination of design spaces for viral clearance by anion exchange chromatography (AEX) during purification of monoclonal antibodies. We developed a risk assessment for AEX using a formalized method and applying previous knowledge of the effects of certain variables and the mechanism of action for virus removal by this process. We then use design-of-experiments (DOE) concepts to perform a highly fractionated factorial experiment and show that varying many process parameters simultaneously over wide ranges does not affect the ability of the AEX process to remove endogenous retrovirus-like particles from CHO-cell derived feedstocks. Finally, we performed a full factorial design and observed that a high degree of viral clearance was obtained for three different model viruses when the most significant process parameters were varied over ranges relevant to typical manufacturing processes. These experiments indicate the robust nature of viral clearance by the AEX process as well as the design space where removal of viral impurities and contaminants can be assured. In addition, the concepts and methodology presented here provides a general approach for the development of design spaces to assure that quality of biotherapeutic products is maintained.

Understanding the mechanism of virus removal by Q sepharose fast flow chromatography during the purification of CHO-cell derived biotherapeutics.

During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non-electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science-based process validation strategies to ensure viral safety of biotechnology products.

Removal of endogenous retrovirus-like particles from CHO-cell derived products using Q sepharose fast flow chromatography.

Retrovirus-like particles (RVLPs) that are expressed during the production of monoclonal antibodies in Chinese hamster ovary (CHO) cell cultures must be removed during product recovery. Anion exchange chromatography (AEX) performed in product flow-through mode, a common component in the purification of monoclonal antibodies, has been shown to provide robust removal of a related retrovirus model, but it's ability to remove the actual RVLP impurities has not been directly investigated. We have determined the ability of a typical Q sepharose process to remove actual CHO RVLP impurities. Using small scale experiments with three model antibodies, we observe that this AEX process is capable of effectively removing both in-process and spiked RVLPs from different feedstocks containing different mAb products. In addition, we show that this AEX process also achieves a similarly high degree of RVLP removal during large scale manufacturing operations.

Anion exchange chromatography provides a robust, predictable process to ensure viral safety of biotechnology products.

The mammalian cell-lines used to produce biopharmaceutical products are known to produce endogenous retrovirus-like particles and have the potential to foster adventitious viruses as well. To ensure product safety and regulatory compliance, recovery processes must be capable of removing or inactivating any viral impurities or contaminants which may be present. Anion exchange chromatography (AEX) is a common process in the recovery of monoclonal antibody products and has been shown to be effective for viral removal. To further characterize the robustness of viral clearance by AEX with respect to process variations, we have investigated the ability of an AEX process to remove three model viruses using various combinations of mAb products, feedstock conductivities and compositions, equilibration buffers, and pooling criteria. Our data indicate that AEX provides complete or near-complete removal of all three model viruses over a wide range of process conditions, including those typically used in manufacturing processes. Furthermore, this process provides effective viral clearance for different mAb products, using a variety of feedstocks, equilibration buffers, and different pooling criteria. Viral clearance is observed to decrease when feedstocks with sufficiently high conductivities are used, and the limit at which the decrease occurs is dependent on the salt composition of the feedstock. These data illustrate the robust nature of the AEX recovery process for removal of viruses, and they indicate that proper design of AEX processes can ensure viral safety of mAb products.

Examination of Salmonella and Escherichia coli translocation from hog manure to forage, soil, and cattle grazed on the hog manure-treated pasture.

Use of hog (Sus scrofa) manure as a fertilizer is a practical solution for waste re-utilization, however, it may serve as a vehicle for environmental and domestic animal contamination. Work was conducted to determine whether pathogens, naturally present in hog manure could be detected in cattle (Bos taurus) grazed on the manure-treated pasture, and whether forage contamination occurred. During two 3 mo summer trials manure was applied to yield < or = 124 kg available N per hectare in a single spring or split spring and fall application. Samples of hog manure, forage, soil, and cattle feces were analyzed for naturally occurring Salmonella, Yersinia enterocolitica, and Escherichia coli. To follow movement of Salmonella in the environment isolates were identified to serovar and serotyped. Transfer of E. coli from hog manure to soil and cattle was examined by randomly amplified polymorphic DNA (RAPD) analysis of >600 E. coli isolates. While Y. enterocolitica was absent from all samples, in both years S. enterica Derby and S. enterica Krefeld were found in most hog manure samples, but were only on forage samples in the second year. Salmonella enterica Typhimurium, absent from hog manure was present on some forage in the first year. Cattle feces and soil samples were consistently Salmonella negative. These contaminations could not be traced to manure application. During this study, Salmonella and E. coli found in hog manure had different RAPD genomic profiles from those found in the feces of cattle grazing on manure-treated pasture.

Salmonella survival in manure-treated soils during simulated seasonal temperature exposure.

Addition of animal manure to soil can provide opportunity for Salmonella contamination of soil, water, and food. This study examined how exposure of hog manure-treated loamy sand and clay soils to different simulated seasonal temperature sequences influenced the length of Salmonella survival. A six-strain cocktail of Salmonella serovars (Agona, Hadar, Heidelberg, Montevideo, Oranienburg, and Typhimurium) was added to yield 5 log cfu/g directly to about 5 kg of the two soils and moisture adjusted to 60 or 80% of field capacity (FC). Similarly, the Salmonella cocktail was mixed with fresh manure slurry from a hog nursery barn and the latter added to the two soils at 25 g/kg to achieve 5 log cfu/g Salmonella. Manure was mixed either throughout the soil or with the top kilogram of soil and the entire soil volume was adjusted to 60 or 80% FC. Soil treatments were stored 180 d at temperature sequences representing winter to summer (-18, 4, 10, 25 degrees C), spring to summer (4, 10, 25, 30 degrees C), or summer to winter (25, 10, 4, -18 degrees C) seasonal periods with each temperature step lasting 45 d. Samples for Salmonella recovery by direct plating or enrichment were taken at 0, 7, and 15 d post-inoculation and thereafter at 15-d intervals to 180 d. Salmonella numbers decreased during application to soil and the largest decreases occurred within the first week. Higher soil moisture, manure addition, and storage in the clay soil increased Salmonella survival. Salmonella survived longest (> or = 180 d) in both soils during summer-winter exposure but was not isolated after 160 d from loamy sand soil exposed to other seasonal treatments. For all but one treatment decimal reduction time (DRT45d) values calculated from the first 45 d after application were < or = 30 d and suggested that a 30-d delay between field application of manure in the spring or fall and use of the land would provide reasonable assurance that crop and animal contamination by Salmonella would be minimized.

Survival of pathogenic bacteria in pesticide solutions and on treated tomato plants.

The ability of Salmonella, Escherichia coli O157:H7, Listeria monocytogenes, and Shigella to survive or grow in pesticide solutions (Ambush 240EC, Benlate T-N-G, Bravo 500, Botran 75WP, Captan 80WDG, Parasol, and Vendex 50W) used by the horticultural industry was examined. In the laboratory, individual cultures were inoculated at 4 log CFU/ml in pesticides diluted with sterile saline to the lowest recommended spray concentrations. During 21 degrees C incubation for < or =96 h, bacterial survivors in the samples and a control consisting of saline were enumerated either by agar surface plating or hydrophobic grid membrane filtration. Most formulations tested were somewhat inhibitory to the pathogenic bacteria. All inoculated bacteria survived or grew in Bravo 500. Among bacteria tested, Salmonella spp. were best able to survive and Listeria spp. were least able to survive in pesticide solutions. When the incubation temperature or pesticide concentration was increased, survival of Salmonella varied depending on the type of formulation. In the field, when a bacterial cocktail containing E. coli O157:H7 and Salmonella Enteritidis was added to Bravo 500 at 6 log CFU/ml, both organisms were recovered from leaves and fruit skins of sprayed tomato plants after the recommended 1 day-to-harvest interval. E. coli and Salmonella survived longer on tomato leaves when sprayed in saline (at least 26 and 56 days, respectively) than when sprayed in Bravo 500 (>45 h and <15 days, respectively). While Salmonella serovars Typhimurium and Heidelberg grew in the fungicide Bravo, and Enteritidis grew in the insecticide Vendex within 96 h at 21 degrees C in the laboratory, pathogen growth in other pesticide formulations did not occur. Higher temperature (< or =30 degrees C) or doubling pesticide concentrations had either no or a negative effect on Salmonella Heidelberg survival. Use of unexpired pesticide formulations may have contributed to the reduced bacterial survival and growth found in the laboratory and during the field trials with Bravo.

Generic/matrix evaluation of SV40 clearance by anion exchange chromatography in flow-through mode.

The potential of viral contamination is a regulatory concern for continuous cell line-derived pharmaceutical proteins. Complementary and redundant safety steps, including an evaluation of the viral clearance capacity of unit operations in the purification process, are performed prior to registration and marketing of biotechnology pharmaceuticals. Because process refinement is frequently beneficial, CBER/FDA has published guidance facilitating process improvement by delineating specific instances where the bracketing and generic approaches are appropriate for virus removal validation. In this study, a generic/matrix study was performed using Q-Sepharose Fast Flow (QSFF) chromatography to determine if bracketing and generic validation can be applied to anion exchange chromatography. Key operational parameters were varied to upper and lower extreme values and the impact on viral clearance was assessed using simian virus 40 (SV40) as the model virus. Operational ranges for key chromatography parameters were identified where an SV40 log(10) reduction value (LRV) of >or=4.7 log(10) is consistently achieved. On the basis of the apparent robustness of SV40 removal by Q-anion exchange chromatography, we propose that the concept of "bracketed generic" validation can be applied to this and potentially other chromatography unit operations.