In-line conditioning (IC) is a form of dilution where a process buffer is formulated in-line from concentrated stock solutions of acids, bases, and salts that are mixed with the correct amount of water-for injection (WFI). This new buffer preparation strategy must prove its equivalency to buffers made the traditional way (i.e., weighing salts, stirring in water, titrating with acid or base). In this paper, such a demonstration is presented using two control modes: (1) ratio control with flow feedback; and (2) pH/conductivity feedback. To obtain the necessary parameters for an error propagation analysis, a robustness study has been performed. Our analysis showed that with low incoming variability, or when the uncertainty of the stock solutions is below 2%, the two modes of control give comparable performance. When the uncertainty increases, so does the uncertainty of ratio control with flow feedback, more with respect to conductivity than pH, while the precision of pH/conductivity feedback remains at the same level. The choice of control should therefore take into consideration the critical process parameters, their tolerances, and the input variability in the stock solution concentration. In situations where there are higher variabilities in stock solution concentrations or process temperatures, this study suggests that pH/conductivity feedback might be a better option.
Category: <span>Biologics Production</span>
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.
Almost 75 years after implementing the industrial ethanol fractionation process, based on the pioneering work of Edwin J. Cohn’s research group, this niche biotechnology process has not lost its importance in helping to supply patients with life-saving biotherapies. Clearly, the focus has shifted from albumin, which was first used, to the indispensable immunoglobulin preparations produced for the effective, long-term treatment of patients suffering from immunodeficiencies. In addition, the widespread and safe therapeutic use of immunoglobulins has paved the way for the development of monoclonal antibodies, now used not only for the treatment of various autoimmune diseases, but also for cancer treatment. The Cohn fractionation process, based on the different solubilities of plasma proteins, depends on the five parameters of ethanol concentration, pH, temperature, protein, and salt concentration, which are the basis of this development. Ethanol concentration can clearly be considered an essential critical parameter for this process. Therefore, it is surprising that even after the advent of process analytical technology, there is still no fast, precise, and accurate procedure at hand to determine the alcohol content of Cohn fractionation intermediates. In this paper, we will describe the implementation of an old methodology for a new purpose, which is designed to close this gap.
The rapid and seemingly uncontrolled spread of African swine fever (ASF) throughout China and many of its neighboring countries within the last 19 months (August 2018–March 2020) has put the rest of the world on high alert. The geographic distribution of viruses of importation concern, like ASF virus (ASFV), can change very quickly, putting at risk conventional sources of porcine serum and other porcine-derived products used as ingredients in research, the manufacture of biologics, and other biomedical applications. This article reviews the 2019 information from the World Organization for Animal Health (OIE) regarding the presence or absence of eight viruses of importation concern in the swine populations of 30 countries from animal serum-producing regions of the world. Companies importing porcine raw materials for formulation into porcine products – and their customers – should be aware of the geographic location of swine diseases of importation concern. The article also identifies ten adventitious viruses of concern cited in United States Department of Agriculture (USDA) and European Union (EU) regulations that need to be tested for or eliminated through one or more barrier treatments when porcine ingredients are used in the manufacture of biologics.
When working on biotherapeutic process development, the analysis of spent cell culture media is often a daily practice during the optimization of bioreactor conditions and media composition. The introduction of parallel microbioreactor systems has decreased the complexity and costs of process development by allowing for concurrent studies of multiple bioreactor and media variables. However, the bioreactors’ small volumes (typically less than 250 mL) limit the volume of media one can extract for daily sampling. We describe a means to analyze spent media with an integrated microchip capillary electrophoresis mass spectrometer (CE-MS) analyzer with minimal sample volume requirements and rapid analysis time. The platform was evaluated with a parallel microbioreactor system (ambr® 250) culturing a Chinese hamster ovary (CHO) cell line stressed by varying levels of ammonia (NH3).
The spent media analysis identified net increases in the levels of the amino acids (AA) Ala, Arg, Asp, Glu, Gly, His, Ile, Leu, Lys, Phe, Thr, Trp, Tyr, and Val in all bioreactors, with Gly levels showing increases in excess of 8-fold initial levels in all bioreactors. Other media components either steadily decreased in concentration or were completely depleted by the end of culture. For example, Asn was depleted in all of the unstressed and 10 mM NH3-stressed bioreactors, but was approximately twice as high as the initial levels in the 30 mM NH3-stressed bioreactors at the end of the culture periods. Also, the 30 mM NH3-stressed condition may have caused either complete degradation or rapid consumption of choline, since it was no longer present starting at the t = 36 h sampling. Overall, the monitored media components were observed to have independent trajectories based on feeding and consumption by the cells, and depending on the stressed condition. The capability to have more frequent spent media analyses would allow for real-time observation of these process changes and associated control strategies.
Process characterization using qualified scale-down models (SDM) offers time and resource-saving advantages to companies developing biotherapeutics. Current approaches with glass benchtop bioreactors as SDMs have demonstrated the ability to predict process performance and product quality, but are throughput- limited by infrastructure that requires significant operational input, as well as large volumes of media and reagents. In this article, the Sartorius Stedim Biotech ambr®250 high-throughput, single-use mini bioreactor system will be discussed for its suitability as an SDM for process characterization.
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…
In the past 20 years, mammalian cell lines have been utilized to produce many viral veterinary vaccines. Cell lines such as baby hamster kidney (BHK)-21, Vero, and Madin Darby canine kidney (MDCK) are widely used because they help facilitate shorter manufacturing lead times and tighter process controls. As compared to other biotech products, viral vaccine manufacturing processes present some specific constraints linked to the cell substrates used. With the global veterinary vaccine market value predicted to be almost $7 billion per year by 2021[2], to remain competitively priced as well as profitable, bioprocess scientists are under pressure to develop methods for faster and more cost-efficient cell culture production. This has led to a shift from the use of expensive, two-dimensional T-flask and roller bottles to single-use, stirred tank bioreactors with microcarriers, or the adaptation of attachment-dependent cell lines such as BHK-21 for suspension culture. This requires time-consuming optimization and scale-up development experiments, which are real drawbacks. However, utilizing automated, single-use mini bioreactors as a scale-down model can enable more efficient use of time and optimization of media, feed, and culture conditions to de-risk upstream process development. In this article, single-use, mini bioreactors are evaluated to determine if they are geometrically comparable to benchtop bioreactors (both glass and single-use vessels) and pilot-scale, single-use bioreactors for effectively modelling mammalian cell culture at 2 L and 50 L scale…