A perfusion approach at N-1, where cells stay in the exponential growth phase throughout the entire culture duration, is becoming more common as a strategy for process intensification. This is because the higher cell densities it generates allows manufacturers to skip seed stages and reduce process transfer time through multiple bioreactor sizes, thus providing more cost-effective biologics production in smaller facilities. However, this N-1 perfusion approach requires optimization. In this article, we describe the development and proof-of-concept studies with single-use rocking motion perfusion bioreactors in which we have achieved a ten-fold increase in viable cell count in N-1 seed stage, compared to the fed-batch control process, in just 6–8 days. We also mention in detail how we inoculated a 50 L bioreactor production run using this intensified seed train and show comparable growth kinetics and yield with a control process, also at 50 L scale. Using this intensification approach in the future will help our manufacturing facility, the Biopharma Division of Intas Pharmaceuticals Ltd., reach 4000 L production-scale volumes with fewer process transfer steps, and without changing the feeding strategy or production bioreactors of our biologics’ portfolio.
T issue-derived products are a class of biological materials harvested directly from animal or human tissue, in contrast to recombinant DNA materials grown in cell culture bioreactors. Tissue-derived products are often used for structural purposes and are typically regulated as medical devices. However, when used to treat human patients, tissuederived products are subject to many of the same concerns as recombinant DNA biotherapeutics, with viral safety being one of them. To address this, the tissue source material must undergo a risk analysis and testing regimen for the presence of viral contaminants. In addition, viral clearance studies must be performed to evaluate whether the purification process is robust enough to remove and/or inactivate viruses that may be present in the starting material. The goals of viral clearance studies are the same for tissuederived products and biotherapeutics, but the design and performance of these studies can be quite different because of the diverse nature of the materials. In this article, we will present an overview of viral clearance studies for tissue-derived products based on our experience in performing a large number of such studies. Rather than discussing the issues related to viral clearance in general, our focus will be on the unique challenges that tissue-derived products pose.
Due to its antioxidant properties and favorable safety profile, glutathione (GSH) finds use in protein formulations by improving overall protein stability. Once degraded, primarily by oxidation into glutathione disulfide (GSSG), the protecting effect of GSH is lost. A simple, direct method using reversed-phase separation and charged-aerosol detection (RP-CAD) to quantitate GSH is described in this paper. The analytical methodology is also capable of monitoring several by-product degradants of GSH, both oxidative and non-oxidative. For high-concentration protein formulations, the method provides direct analysis of GSH and its degradants in the presence of protein at up to 225 mg/mL simply through a dilution of the sample. Quantitation of many amino acids typically included in pharmaceutical protein formulations is also possible. Use of an online diverting valve in the method prevents interference in the detector from the high protein concentration in formulation. Accuracy and effectiveness of this method is demonstrated through monitoring the stability of GSH in high-concentration protein formulations through confirmation of GSH concentration and mass-balance of its loss over time. Monitoring GSH stability in protein formulations is necessary, as GSH concentration is indicative of protein stability.
OPEN ACCESS ARTICLE www.bioprocessingjournal.com 1 FOOTNOTES 1. Footnotes 2–5 provide examples of the complexity of importation requirements. Additional requirements exist for other importing countries with respect to certain exporting regions, and this table should not be regarded as complete or necessarily current. Exporters should contact the relevant border inspection post to confirm current importation requirements. 2. Must come from a registered facility and meet importation eligibility requirements for sterility. Must be negative for mycoplasma, bluetongue virus, bovine viral diarrhea virus, bovine leukemia virus, cytopathic effect, and hemadsorption. 3. Requires three-month residency. 4. USDA Safety Testing – bluetongue and Akabane virus (must be negative after testing). 5. USDA Safety Testing – bluetongue virus (must be negative after testing). 6. Excluding Guatemala, El Salvador, and Belize. Introduction It is a common belief that fetal bovine serum (FBS) collected from certain geographical regions, such as New Zealand, is of superior quality to material collected from South America. Whilst it is true that origin does have an impact on the price of serum, it does not affect the quality or biological performance of the product. FBS collected under similar conditions from any geographical region will demonstrate comparable ability to support cell growth. For FBS, the term “quality” is frequently confused with “health status.” It is the health status of the geographical region from which the serum is collected that will dictate its potential use, the availability of material for import, and eventually, the price. It should be noted that health status should be considered a result of more than just the geographical source of the material, but also the regulatory infrastructure and how well regulations are enforced by the countries within that region. The health status of a country is determined by the World Organization for Animal Health (OIE). The mission of the OIE is to ensure transparency in the global animal disease situation. The OIE issues information concerning the health status of various countries with regard to animal diseases of concern, including foot-and-mouth disease, bovine spongiform encephalopathy (BSE), and other diseases affecting cattle populations globally.[1] The status of a country, with regard to the presence of an animal disease of concern, together with interagency government agreements, will determine where serum collected within that country may be exported. Individual countries have varying requirements for importation of serum from the same geographies based on the animal health status of the region from which the serum was collected. Table 1 provides an overview of the requirements for moving FBS from one part of the world to another. As can be seen, these requirements are extremely complex and are continually changing as regulations evolve. Exporters are strongly encouraged to contact border Fetal Bovine Serum – Geographical Origin and International Trade By Jennifer A. Murray and Rosemary J. Versteegen
M edicago manufactures influenza vaccine virus-like particles (VLPs) in an unusual production platform consisting of Nicotiana benthamiana plants. During the in vitro adventitious agent test (AAT) of certain Medicago B strain influenza vaccine VLP test samples, positive hemagglutination of guinea pig red blood cells was observed on day 14, but not on day 28. The positive result in the assay was surprising because the production process uses no animal-derived raw materials and contains a viral inactivation step. Plant-associated viruses would not be expected to infect the mammalian cell-based assay. No cytopathic effects or hemadsorption of red blood cells was observed in these AATs. The positive hemagglutination was observed at 2–8°C, but not at 36–38 °C, and only in a few of the six detector cell lines used in the assay. Because this is quite an unusual pattern of responses for an AAT, Medicago and the contract testing lab, Eurofins Lancaster Laboratories (ELLI) investigated the positive responses thoroughly for the presence of an adventitious agent or an alternative explanation not involving a viral contaminant. Investigation results indicated that the hemagglutinating activity associated with the vaccine test sample itself was responsible for the positive hemagglutination response. The positive hemagglutination on day 14 of these AATs was deemed an assay artifact, and preventive actions were taken to prevent recurrence of this type of false positive response.
