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Design and Performance of Viral Clearance Studies with Tissue-Derived Products

by Stephen Stoltzfus and Katherine F. Bergmann
Volume 20, Open Access (Feb 2021)

Tissue-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, tissue-derived 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 tissue-derived 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.

Citation:
Stoltzfus S, Bergmann KF. Design and performance of viral clearance studies with tissue-derived products. BioProcess J, 2021; 20.
https://doi.org/10.12665/J20OA.Stoltzfus

Posted online February 5, 2021.

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A Direct Method to Monitor Glutathione Stability in High Concentration Protein Formulations

by Seth Keever, Bassam Nakhle, and Bernice Yeung
Volume 20, Open Access (Jan 2021)

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.

Citation:
Keever S, Nakhle B, Yeung B. A direct method to monitor glutathione stability in high concentration protein formulations. BioProcess J, 2021; 20.
https://doi.org/10.12665/J20OA.Keever

Posted online January 12, 2021.

 
Defining Therapeutic Window for Viral Vectors: A Statistical Framework to Improve Consistency in Assigning Product Dose Values

by Nancy Sajjadi and Janice D. Callahan
Volume 19, Open Access (Oct 2020)

Pre-clinical and clinical trials conducted to establish the minimum effective dose and the maximum tolerated dose of a viral vector assume that the assigned dose values are comparable across studies. Toxicity has been associated with high dose administration of both adenovirus and adeno-associated virus-based vectors, and increased attention must be paid to assays used to measure dose. High assay variability can be mitigated by replication and the reporting of a mean value for product lot release. The establishment of a dose specification and a testing strategy must take into account the risk of errant quality control decisions. This can be accomplished by linking assay qualification information to measurement uncertainty through a statistical framework. By adopting an equivalence approach, the risk of releasing lots with unacceptably high or low dose values is minimized by reducing measurement uncertainty. This article provides a worked-through example to introduce applicable statistical concepts and the equations necessary to facilitate their implementation in the field.

Citation:
Sajjadi N, Callahan J. Defining therapeutic window for viral vectors: A statistical framework to improve consistency in assigning product dose values. BioProcess J, 2020; 19.
https://doi.org/10.12665/J19OA.Sajjadi

Posted online October 25, 2020.

 
Alcohol Determination in Protein Fractionation Intermediates by Steam Distillation and Digital Refractometry

by Heinz Anderle, Matthias Spork, David Horn, Theresa Bauer, Lucia Gnauer, and Alfred Weber
Volume 19, Open Access (May 2020)

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.

Citation:
Anderle H, Spork M, Horn D, Bauer T, Gnauer L, Weber A. Alcohol determination in protein fractionation intermediates by steam distillation and digital refractometry. BioProcess J, 2020; 19. https://doi.org/10.12665/J19OA.Anderle

Posted online May 23, 2020.

 
Porcine Serum, Trypsin, and Other Porcine-Derived Products: Swine Viruses of Importation and Adventitious Concern

by Percy W. Hawkes
Volume 19, Open Access (Apr 2020)

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.

Citation:
Hawkes PW. Porcine serum, trypsin, and other porcine-derived products: swine viruses of importation and adventitious concern. BioProcess J, 2020; 19. https://doi.org/10.12665/J19OA.Hawkes

Posted online Apr 16, 2020.

 
Spent Media Analysis with an Integrated CE-MS Analyzer of Chinese Hamster Ovary Cells Grown in an Ammonia-Stressed Parallel Microbioreactor Platform

by Kathryn Elliott, Ji Young L. Anderson, Colin M. Gavin, Kenion H. Blakeman, Sarah W. Harcum, and Glenn A. Harris
Volume 19, Open Access (Jan 2020)

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.

Citation:
Elliott K, Anderson JYL, Gavin CM, Blakeman KH, Harcum SW, Harris GA. Spent media analysis with an integrated CE-MS analyzer of Chinese hamster ovary cells grown in an ammonia-stressed parallel microbioreactor platform. BioProcess J, 2020; 19. https://doi.org/10.12665/J19OA.Elliott

Posted online Feb 29, 2020.

 
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