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Biopharmaceutical Manufacturing: Historical and Future Trends in Titers, Yields, and Efficiency in Commercial-Scale Bioprocessing

by Ronald A. Rader and Eric S. Langer
Volume 13, Issue 4 (Winter 2014/2015)

This article documents the progress, current state, and projected future trends in titer and yield as industrial and technological benchmarks for commercial-scale biopharmaceutical manufacture. Biopharmaceutical product commercial-scale manufacturing (bioprocessing) was benchmarked by tracking titers and yields over time, from the 1980s to the present, and further out ten years. This study compiled commercial-scale titer and yield data for a set of 39 major biopharmaceuticals, nearly all mammalian-expressed proteins, particularly, monoclonal antibody products. This included extensive searches of many potential data sources, including contacting knowledgeable bioprocessing professionals. In the 1980s and early 1990s, average titers at commercial scale started out at < 0.5 g/L. The current average reported commercial-scale titer is 2.56 g/L. We also confirmed that the manufacture of commercial products has, over the years, undergone repeated cycles of technical production upgrades, with titers and yields increasing incrementally, even for the oldest products. BioPlan estimates that ≥3 g/L is now the industry standard titer for new bioprocesses being developed, with ≤7 g/L now presumed to be the general industry top-end titer level that, while not unusual, is not often achieved. In terms of yields, we found a 70% yield to be the current industry average yield, not the often-cited 75%. Improvements in downstream purification technologies (e.g., as demonstrated by higher yields) have been fewer and adopted more slowly than upstream production...

Citation:
Rader RA, Langer ES. Biopharmaceutical manufacturing: historical and future trends in titers, yields, and efficiency in commercial-scale bioprocessing. BioProcess J, 2015; 13(4): 47–54. http://dx.doi.org/10.12665/J134.Langer.

Posted online January 20, 2015.

 
In Situ Hybridization: The Importance of Ultrapure Water for RNA Technologies

by Frauke Nitzki and Elmar Herbig
Volume 13, Issue 4 (Winter 2014/2015)

In situ hybridization (ISH) for localization of DNA/RNA hybrids in cytological preparations was first described in 1969 by Gall and Pardue. This method enables mRNA transcripts to be detected in tissue sections. Unlike expression analyses based on polymerase chain reactions, the exact localization of the target transcripts can be identified within the tissue...

Citation:
Nitzki F, Herbig E. In situ hybridization: the importance of ultrapure water for RNA technologies. BioProcess J, 2015; 13(4): 56–9. http://dx.doi.org/10.12665/J134.NitzkiHerbig.

Posted online January 20, 2015.

 
Use of Interactive Laser-Scanning Imaging Cytometry (ILIC) for Real-Time Cytotoxicity Assessment of Bacteriocins Against Cultured Mammalian Cells

by Shelton E. Murinda, PhD, Robert F. Roberts, PhD, Elaine M. Kunze, and Kamal A. Rashid, PhD
Volume 13, Issue 4 (Winter 2014/2015)

The evaluation of bacteriocin cytotoxicity is a critical first step in guaranteeing its safe use in food and therapeutic applications. In this study, the bacteriocins nisin, pediocin, and colicin ([Col] E1, E3, E6, E7, and K) were evaluated for cytotoxicity against cultured mammalian cells. Cellular membrane potential (ΔΨ) changes of bacteriocin-treated cells were used as an index of cytotoxicity. Simian virus 40-transfected human colon (SV40-HC) cells and African green monkey kidney epithelial cells (Vero) cells were cultured (35°C, 10% CO2, humidified air) as monolayers on tissue culture plates. Log phase cells (~104 cells/mL) were treated with partially purified, individual bacteriocin preparations at 170, 350, and 700 activity units (AU)/mL. Perturbations in cellular ΔΨ were measured using interactive laser-scanning imaging cytometry (ILIC) with 3,3’-dipentyloxacarbocyanine iodide (DiOC5[3]), a potential-sensitive, fluorescent dye. Cellular ΔΨ studies indicated significant levels of dose-dependent toxicity with nisin, pediocin, and colicins E6, E7, and K. In contrast, Col E1 and E3 had no definitive toxic impact on SV40-HC cells. The only colicin to significantly impact Vero cells negatively was the E6. Overall, SV40-HC cells demonstrated greater sensitivity to bacteriocins than Vero cells. Among all the fluorescence parameters and peak statistics measured, the integrated area under the curve appeared to correlate best with the prediction of cytotoxic events. Consequences were quickly apparent with maximum changes occurring within 3–5 minutes following the addition of test substances. Refinements in the ILIC protocol could potentially afford a unique tool for rapid, real-time cytotoxicity testing of bacteriocins in food preservation, and in human and animal health applications...

Citation:
Murinda SE, Roberts RF, Kunze EM, Rashid KA. Use of interactive laser-scanning imaging cytometry (ILIC) for real-time cytotoxicity assessment of bacteriocins against cultured mammalian cells. BioProcess J, 2015; 13(4): 21–32. http://dx.doi.org/10.12665/J134.Murinda.

Posted online January 20, 2015.

 
Statistical Verification that One Round of Fluorescence-Activated Cell Sorting (FACS) Can Effectively Generate a Clonally-Derived Cell Line

by Lara E. Krebs, Jinxin Gao, PhD, and Christopher C. Frye, PhD
Volume 13, Issue 4 (Winter 2014/2015)

Mammalian cell line generation is foundational to protein-based therapeutic product development. Ensuring production cell line “clonality” (i.e., clonal derivation of the production cell line) is a requirement for product registration. Many single-cell deposition approaches have been developed since the inception of traditional limiting-dilution. However, regulatory expectations, in line with the capability of this original cloning technique, still lead many institutions to either execute multiple rounds of single-cell plating or conduct extensive screening immediately post-cloning to demonstrate the single-cell origin of a production cell line. Using fluorescence-activated cell sorting (FACS), we have developed a strategy to verify single-cell deposition that, when combined with statistical analysis, creates a highly-effective and efficient process for generating clonally-derived production cell lines in just one round of plating...

Citation:
Krebs LE, Gao J, Frye CC. Statistical verification that one round of fluorescence-activated cell sorting (FACS) can effectively generate a clonally-derived cell line. BioProcess J, 2015; 13(4): 6–19. http://dx.doi.org/10.12665/J134.Krebs.

Posted online January 20, 2015.

 
Characterization of a Lyophilized Immunoaffinity Chromatography Matrix Employed to Purify Hepatitis B Surface Antigen for Pharmaceutical Use

by Miguel Castillo et al.
Volume 13, Issue 4 (Winter 2014/2015)

Immunoaffinity chromatography is an indispensable purification tool. However, its use has been limited by cost, purification cycle numbers, and storage requirements. Therefore, authors speculated that a possible solution to these problems could be CB.Hep-1 monoclonal antibody (mAb)-immunosorbent lyophilization. This study sought to assess the impact of the CB.Hep-1 mAb quantification by enzyme-linked immunoadsorbent assay and the CB.Hep-1 mAb-immunosorbent lyophilization process for its impact on hepatitis B virus surface antigen purification for pharmaceutical use. Study results found that CB.Hep-1 mAb lyophilization did not affect mAb purity and antigen recognition capacity. CB.Hep-1 mAb-immunosorbent lyophilization did not modify volume-weight factor, infrared spectrum, particle-size distribution, particle density and viscosity, antigen adsorption capacity, antigen elution capacity, antigen recovery, antigen purity, gamma immunoglobulin (IgG) leakage, and purification cycle number. Therefore, the lyophilized CB.Hep-1 mAb and CB.Hep-1 mAb-immunosorbents can be successfully used for hepatitis B vaccine production...

Citation:
Castillo M et al. Characterization of a lyophilized immunoaffinity chromatography matrix employed to purify hepatitis B surface antigen for pharmaceutical use. BioProcess J, 2015; 13(4): 35–45. http://dx.doi.org/10.12665/J134.Valdes.

Posted online January 20, 2015.

 
Which Factors to Consider When Selecting an Analytical Method for Cell Culture Fermentation: A Comparison of Four Different Metabolic Analyzer Instruments

by Caitlin Kurtz and John Paul Smelko
Volume 13, Issue 3 (Fall 2014)

Mammalian cell culture processes require an in depth understanding of inputs and outputs in order to maximize productivity, efficiency, and product quality. Daily monitoring of essential metabolites, nutrients, and protein titer using at-line analyzers are the building blocks of drug manufacturing process development and characterization. In order to better understand how these instruments perform, a comparative analysis was conducted using two different classes of metabolicsensing technologies, membrane-based technology (MBT) and absorption photometric-based technology (APBT). Four commercially available instruments were examined using various samples and maintenance conditions in order to emphasize the following testing criteria: specificity, linearity, range, accuracy, and precision. Samples included standard solutions with known metabolite concentrations, cell culture supernatant, and supernatant liquid spiked with additional metabolite solutions. The results from our testing indicated that the photometric analyzers yielded more accurate and consistent results than the membrane-based analyzers and were easier and less time-consuming to maintain. Ultimately, these studies summarized the capabilities and limitations for both types of analyzers and provide a critical summary for instruments used in everyday bioprocess monitoring.

Citation:
Kurtz C, Smelko JP. Which factors to consider when selecting an analytical method for cell culture fermentation: a comparison of four different metabolic analyzer instruments. BioProcess J, 2014; 13(3): 12–31. http://dx.doi.org/10.12665/J133.Kurtz.

Posted online October 13, 2014.

 
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