Adenoviral vectors have been widely used in gene therapy clinical trials and subjected to rigorous testing to ensure safety and efficacy. Like therapeutic proteins, aggregation of adenoviral vectors needs to be quantified for process consistency and stability monitoring. The sucrose gradient sedimentation method of adenovirus particles using disc centrifugation, which is a modification of a method described by Bondoc and Fitzpatrick, was used. It proved to be quantitative and reproducible in evaluating a variety of samples including deliberately cross-linked adenovirus particles and process development lots of various ages. This aggregation assay revealed that most aggregates detected in the production lots were dimers, trimers, and tetramers; and the number of these small oligomers was easily reduced with the addition of 300 mM salt, thus demonstrating the reversible nature of a portion of the aggregate population. This method was validated to demonstrate that it was appropriate for final product lot release and stability monitoring…
BioProcessing Journal Posts
Nematode worms release pheromones to communicate information about food, stress, and sex to other individuals. The nematode species C. elegans is a genetic system for studying the biology underlying these pheromone-mediated behaviors. Identifying the chemical structure of C. elegans pheromones requires purifying them directly from large quantities of conditioned growth media. Release of pheromones may depend on growth conditions, population density, and developmental stage. However, published studies only report nematode pheromones from developmentally asynchronous cultures at variable population densities. Here, we have developed reliable methods for liquid culture of developmentally-synchronous populations of C. elegans at controlled population densities from 5,000 to 10,000 worms/mL at the scale of 10 to 100 L. Generating synchronous populations requires harvesting eggs from adults using alkaline bleach. In this study, we developed modified tangential flow filtration (TFF) systems to rapidly bleach adults and recover large quantities of healthy, viable eggs. This method achieved synchronous cultures at scales 2.5 x greater than cultures based on agar plates, and 5 x greater than previously reported for synchronous liquid cultures. We routinely harvested 2.5 x 10E7 animals (total) from a single 5 L large-scale culture. Furthermore, our TFF system effectively recovered pheromone-containing media at all scales tested. This work makes it feasible to isolate other biologically useful secreted molecules from transgenic C. elegans…
Contract testing organizations (CTOs) offer considerable value by providing testing services and expertise that may not be available within organizations that are developing and producing biopharmaceuticals, from virtual companies to fully integrated international organizations. CTOs are constantly challenged by the breadth of services that are requested as well as the levels of regulatory compliance that are expected and the degree of oversight that various companies intend to impose. And knowing this variability of needs and expectations often results in a CTO trying to become “all things to all people” regarding assay tech transfer, validation, and qualification. For example, with the same type of sample from the same stage of product development, one company may expect its contracted testing to comply with 21 CFR Part 210 and 211 (good manufacturing practice [GMP]) while another may expect its testing to comply with 21 CFR Part 58 (good laboratory practice [GLP]) with different assay validation expectations for each approach…
Economic conditions are driving a greater demand for improvements in productivity, cost management, and outsourcing. Technology also continues to advance and change the biomanufacturing environment. This was documented in the recently released 2010 7th Annual Report and Survey of Biopharmaceutical Manufacturing with input provided by 327 biomanufacturers globally. “Productivity improvements” is the #1 area where companies have been focusing on resources this year. The survey also documents an evolutionary change in the basic approaches and technologies used for upstream bioprocessing. While in recent years, the industry has largely been dominated by fed batch (or batch fed) bioprocessing, with fermentation performed in a bioreactor containing the same medium until ready for harvest, the BioPlan survey shows a definite shift towards greater acceptance and use of perfusion bioprocessing…
Since its beginnings in the 1970s, recombinant DNA technology has become integral in the production of pharmaceutically important proteins such as blood factors, hormones, growth factors, interferons, interleukins, and vaccines. Recombinant technology was developed in Escherichia coli, and because of its long history of use and ease of manipulation, the bacteria remains a common expression system. However, E. coli is limited in the types of proteins it can express, and production can be hampered by the formation of intracellular inclusion body aggregates which complicate the processes of protein recovery and purification. The non-pathogenic Corynebacterium glutamicum can accumulate amino acids extracellularly and has historically been used for the commercial production of amino acids glutamate and lysine. More recently, C. glutamicum has been developed as an expression system with the ability to secrete properly folded, functionally active proteins into the fermentation broth. Production in C. glutamicum has the same advantages of scalability and ease of culturing as in E. coli, but with more simplified recovery and purification processes…
J&J’s recent recall of Motrin and other pharmaceutical products is more than a cautionary tale in crisis management. It raises a very important question: Why do companies hesitate to address known risks (in this case the QC problems at a specific manufacturing facility)? As Douglas McIntyre, editor of 24/7 Wall Street, stated in a recent posting, “Acting early would probably be harmless at worst and might be critical to success.” However, he admits that few companies have gotten the message. But why?…
Process optimization is a key development step that precedes scale-up and tech-transfer in a manufacturing environment. New England Biolabs (NEB) and GE Healthcare (GEHC) set the objective of improving filtration clarification and concentration process steps with the target of improving overall efficiency, and reducing the labor time and expense associated with these unit operations…
This investigation into the light isomerization of tropolone was initiated after a method for determining residual tropolone in the bulk drug substance (BDS) of a therapeutic monoclonal antibody was developed and validated at our laboratory. This method, developed at a client’s request, required a detection limit of 50 parts per billion (ppb) in the BDS to meet their regulatory requirements. The method was developed using liquid chromatography and tandem mass spectrometry (LC/MS/MS) because of the selectivity and sensitivity of the technique when using selective reaction monitoring (SRM). During development, however, it was discovered that only after creating a photoisomer of tropolone could successful separation, detection, and quantitation of tropolone be achieved. During validation, the method showed good performance…
Classical filtration fouling models do not accurately predict fouling behaviors for sterilizing filtration applied to some bioprocess fluids, particularly when filters are operated in series. To overcome the limitations of these models, we extended a previously developed single-stage fouling model that combined blocking and adsorption mechanisms to dual filters operated in series. Our model demonstrates improvements in predicting bioprocess fluid filtration performance accuracy when applied to microfiltration membranes operated in series, from measured performance of each membrane filter operated individually. In addition, the model and developed method allows for rapid and efficient optimization of prefiltration to final filter area ratios. In redundant sterile filtration, two filters with the same pore size rating are operated in series to protect against an integrity failure of the primary sterilizing filter. Prefilters can also be used to protect final sterilizing-grade membrane filters from excessive fouling which in turn offer the benefits of reduced costs and filtration time…