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A Comparison of Yield, Quality, and Functional Integrity for Human Genomic DNA Extracted from Fresh and Frozen Whole Blood Using Frozen Aliquotting Technology

by Michael G. Barnes, PhD, Kenneth Kaufman, PhD, Richard Ballweg, Stephen Esslinger, Justin Hogan, and Joseph Fraone
Volume 13, Issue 1 (Spring 2014)

As the institutionalized collection, storage, and distribution of bio-samples becomes more commonplace, the attention and adherence to strict and systematic sample handling practices is paramount. An important component of such efforts is the maintenance of bio-samples in a safe and controllable state (e.g., –80°C storage) apart from any downstream processes the samples could be subjected to in the future. CryoXtract Instruments’ CXT 750 Frozen Aliquotter provides repeated and reproducible access to a variety of frozen bio-fluid samples by maintaining samples at –80°C, thus eliminating undesirable freeze-thaw cycles during sample handling processes. The following study was performed by the Cincinnati Biobank Core Facility and the Center for Autoimmune Genomics and Etiology (CAGE) at Cincinnati Children’s Hospital Medical Center, in order to assess the feasibility of frozen aliquotting in conjunction with DNA extraction services provided by the Cincinnati Biobank. The Cincinnati Biobank planned and independently executed the experiment while CryoXtract’s role was to provide technical guidance running the CXT 750. Both groups participated in preparation of the manuscript...

Citation:
Barnes MG, Kaufman K, Ballweg R, Esslinger S, Hogan J, Fraone J. A Comparison of Yield, Quality, and Functional Integrity for Human Genomic DNA Extracted from Fresh and Frozen Whole Blood Using Frozen Aliquotting Technology. BioProcess J, 2014; 13(1): 12-17. http://dx.doi.org/10.12665/J131.Barnes.

Posted online April 15, 2014.

 
A Working Definition of Quality by Design (QbD): Understanding its Application to the Development and Manufacturing of Biopharmaceuticals

by Mark F. Witcher, PhD
Volume 13, Issue 1 (Spring 2014)

This paper places the “Quality by Design” (QbD) in an overall context and provides the following straightforward definition so that QbD can be effectively used to solve a wide variety of important problems:
Quality by Design–During the design stage, to achieve a well-defined goal, iteratively apply science and engineering methods to anticipate, identify, understand, resolve, and control problems that will be encountered during testing, operating, and verifying the goal over its lifecycle.
The viewpoint of the paper is to view QbD as “Success by Design.” The definition is based on answering the following question: What will be required to provide assurance that the enabler developed during the design stage will successfully reach the goal over the entire lifecycle before leaving the design stage? The paper argues that QbD should not be implemented as a program, but used as a tool. To provide understanding, the paper explores underlying concepts, the history of QbD, develops a working definition, and then applies it to biopharmaceutical development and manufacturing...

Citation:
Witcher MF. A Working Definition of Quality by Design (QbD): Understanding its Application to the Development and Manufacturing of Biopharmaceuticals. BioProcess J, 2014; 13(1): 6-11.
http://dx.doi.org/10.12665/J131.Witcher.

Posted online April 2, 2014.

 
Efficient Recombinant Antibody Production Platform Using Lentiviral Vector-Transduced CHO-K1 Cells

by Lajos Baranyi, PhD, Darrel E. Menking, James Carney, PhD, Patricia Buckley, PhD, Andre Roy, and Boro Dropulic, PhD
Volume 12, Issue 4 (Winter 2013/2014)

Hybridoma cell lines are highly unpredictable and often, an unreliable source of important antibodies of national security interest. There is an urgent need to convert antibody production from these cell lines into robust, recombinant platforms that can reliably produce large quantities of antibody on demand, and abandon methods based on murine ascites production. This work describes the use of lentiviral vectors, Chinese hamster ovary (CHO)-K1 cells, and high-density perfusion cultures for antibody production. Cell line development was rapid, high insertion copy numbers were achievable, and the heavy/light chain ratios could be rapidly optimized. The mammalian cells provided an appropriate environment for IgG folding and obviated the difficult purification steps such as removal of endotoxins, refolding, or dealing with abnormal post-synthetic modifications common to other production systems. We found that even in the absence of an optimized cell line, in high-density cultures, routine productivities in the 1–5 g/L range were achieved. As expected, productivity was independent of the performance of the original hybridomas. We conclude that the lentiviral vector system can achieve high copy numbers of immunoglobulin genes with optimized heavy and light chain ratios to appropriately assemble and secrete the immunoglobulins, achieving high productivity. This observation suggests that substantial advances can be made by selecting and optimizing the cell line used for immunoglobulin production. The lentiviral vector-based method of antibody production offers substantial improvements over traditional murine ascites-based antibody production in terms of reliability, productivity, and cumulative cost—if the antibody need exceeds 1–3 grams within the shelf life of the product...

Citation:
Baranyi L, Menking DE, Carney J, Buckley P, Roy A, Dropulic B. Efficient Recombinant Antibody Production Platform Using Lentiviral Vector-Transduced CHO-K1 Cells. BioProcess J, 2014; 12(4): 6-23. http://dx.doi.org/10.12665/J124.Dropulic.

Posted online January 13, 2014.

 
Virus Clearance in Your Process from Start to Finish

by Patricia Greenhalgh, PhD and Ushma Mehta
Volume 12, Issue 4 (Winter 2013/2014)

The safety of biological products (biologics) derived from in vitro cultured cell lines of animal origin is dependent both on clinical studies to evaluate efficacy, and a matrix of controls throughout the manufacturing process to assure consistency, quality, and safety of the marketed product for human use. One major area of concern is virus safety. Through the combination of: (A) careful selection of raw materials; (B) testing of process intermediates; and (C) virus clearance evaluations of the individual steps in the manufacturing process; biologics manufacturers can demonstrate that their products are free from detectable adventitious and endogenous viruses. Comprehensive regulatory guidance suggests approaches for virus testing of biologics at early and later stages of clinical development and, although some countries have specific requirements, many of these approaches are harmonized worldwide. In general, regulatory authorities expect purification processes to include multiple steps with complementary, or orthogonal, methods for virus reduction including inactivation and separation or removal. Each step in the process may be more or less effective for reducing levels of different test viruses, and therefore selection of the appropriate panel of test viruses for the specific product is critical for viral clearance studies...

Citation:
Greenhalgh P, Mehta U. Virus Clearance in Your Process from Start to Finish. BioProcess J, 2014; 12(4): 48-52. http://dx.doi.org/10.12665/J124.Greenhalgh.

Posted online January 13, 2014.

 
Microcarrier-Based Expansion of Adipose-Derived Mesenchymal Stem Cells in Shake Flasks

by Khandaker Siddiquee, PhD and Ma Sha, PhD
Volume 12, Issue 4 (Winter 2013/2014)

The expansion of stem cells, including mesenchymal stem cells (MSCs), has been successfully demonstrated using microcarrier-based small bioreactors such as spinner flasks. In this study, we explored a simple alternative for microcarrier-based MSC expansion using conventional shake flasks. This method relies on a new type of shaker with built-in CO2 gas control capability, the New Brunswick™ S41i incubator shaker. The expansion of adipose-derived mesenchymal stem cells (AdMSCs) was compared between shake and spinner flasks containing microcarriers. The AdMSCs were seeded at a density of 3 × 103 cells/cm2 in both setups, each containing 0.5 g of plastic microcarriers and 50 mL of stem cell growth medium. The cell culture experiments were conducted over 12 days with samples collected daily for cell growth, biochemistry, and metabolite analysis. The study revealed that AdMSCs cultured under shake flask conditions achieved excellent growth under 12-day batch-culture conditions. Finally, the AdMSCs expanded using the shake flask method retained high quality stem cell characteristics, as indicated by CD44 and CD90 stem cell marker assays, and the ability of these cells to differentiate into either adipocytes or osteocytes...

Citation:
Siddiquee K, Sha M. Microcarrier-Based Expansion of Adipose-Derived Mesenchymal Stem Cells in Shake Flasks. BioProcess J, 2014; 12(4): 32-38. http://dx.doi.org/10.12665/J124.Sha.

Posted online January 13, 2014.

 
Biosimilars in the Rest of the World: Developments in Lesser-Regulated Countries

by Ronald A. Rader
Volume 12, Issue 4 (Winter 2013/2014)

Biosimilars, and related biopharmaceutical biobetters and biogenerics, are still relatively new, but are already starting to impact worldwide biopharmaceutical markets. Most discussions of biosimilars center on developed regions where markets are mature and manufacturing capabilities allow for the cost-efficient manufacture of these complex molecules. This article covers the development of these products outside the United States (US), European Union (EU), and other developed, generally rather affluent and high-technology economy-based countries. To start, we first offer some definitions...

Citation:
Rader RA. Biosimilars in the Rest of the World: Developments in Lesser-Regulated Countries. BioProcess J, 2014; 12(4): 41-47. http://dx.doi.org/10.12665/J124.Rader.

Posted online January 13, 2014.

 
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