BioProcessing Journal

A Comparative Bioreactor Vessel Study: Conventional Reusable Glass and Single-Use Disposables for the Production of Alkaline Phosphatase

Single-use, stirred-tank bioreactor systems have been used in large-scale production for a number of years. Bench-scale, stirred-tank bioreactors have not been commercially-available for single-use until recently. The New Brunswick™ CelliGen® BLU pitched-blade bioreactor was introduced in 2009, and the CelliGen BLU packed-bed bioreactor, in 2012. Little information is currently available on the utility of these bioreactors for bench-scale production of recombinant products. Thus, we designed this study to perform multiple comparisons with these single-use bioreactors and their traditional glass vessel counterparts. The data comparisons included: (1) CelliGen BLU pitched-blade vs. glass pitched-blade; and (2) CelliGen BLU pitched blade in batch mode vs. CelliGen BLU packed-bed in perfusion mode. Chinese hamster ovary (CHO) cells were used to measure alkaline phosphatase (ALKP) production in each bioreactor. The final measured concentration of ALKP, after eight days of batch-mode culture in the single-use, pitched-blade bioreactor, was 1.6 U/mL compared to 2.1 U/mL in the reusable bioreactor. After six perfusion harvests in the single-use, packed-bed bioreactor, the combined ALKP production was 16.2 U/mL compared to 17.4 U/mL in the reusable bioreactor in batch mode. Multiple batch culture runs in the pitched-blade bioreactor would be required to match the output of a single run in the packed-bed bioreactor in perfusion mode. Results demonstrate that there are no significant differences between the reusable and single-use systems for bench-scale production of recombinant proteins. Our results also suggest that the CelliGen BLU packed-bed bioreactor, when operated in perfusion mode, is superior to the CelliGen BLU pitched-blade bioreactor when operated in batch mode, confirming our studies from 2012…

Biologics Production

alkaline phosphatase alkp production batch mode batch production bench-scale bioproduction biologics celligen blu cho cells glass bioreactors glass vessels perfusion mode single-use single-use bioreactor stirred-tank bioreactor

Bioburden Control in Bioprocessing Using Hydrogen Peroxide Vapor

The emerging use of hydrogen peroxide vapor (HPV) biodecontamination technology in the late 1990s was initially implemented as part of an alternate strategy for disinfecting rooms, areas, and whole buildings. The technology was developed with rapid cycles and was adopted by barrier isolator users allowing for rapid, effective, and validated bio-decontamination cycles inside critical zones. In the years to follow, the use of HPV technology spread throughout the development and production pipelines of small- and large-molecule biopharmaceutical products. This article will consider the current and future uses of HPV technology from the perspective of the development through manufacture of a biologically-derived product…

Biologics Production

alternative disinfection strategy barrier isolator bio-decontamination biodecontamination biopharmaceutical production hpv hydrogen peroxide vapor

Spinning Out: From Concept to Actualization

ExpreS2ion, established in January 2010, is located in the Hørsholm Science Park, north of Copenhagen. It was formed as a spin-out from Affitech A/S (Affitech) which holds an equity position in the company. ExpreS2ion operates as a contract research organization (CRO) offering services related to vector and cell line development, cloning, upstream development, optimization, and production of GLP material using its S2 (Drosophila Schneider 2) cell-based ExpreS2 platform. Two of the company’s founders, Drs. de Jongh and Dyring, and early employees of ExpreS2ion were colleagues for many years at Affitech and Pharmexa A/S (Pharmexa) before the two companies combined. It was during this period that they developed and optimized a S2 expression system for use in the production of therapeutic vaccines. ExpreS2ion’s proprietary protein expression platform, ExpreS2, consists of high-yielding expression vectors, a S2 cell line that grows to higher cell densities than standard S2 cells, an optimized culture media, and a highly efficient transfection reagent specifically optimized for S2 cells…

Biologics Production

cell line development drosophila s2 drosophila schneider 2 glp material life sciences protein expression therapeutic vaccines therapeutics vaccine production vectors

Patent Exhaustion: A Tireless Limit on Patent Rights

Technology-dependent industries such as biotechnology, nanotechnology, and digital signal processing rely heavily on patent law to secure protection for innovation and ensure returns adequate to sustain costly research and development programs. It can be a daunting task to stay current with patent law in the United States, and then throughout the world, the challenges increase exponentially. A case in point is the US, where significant patent law changes brought by the America Invents Act in 2011 are now being implemented. Beyond the revised US patent statute, seemingly lurking in the weeds, is the common law, also known as case or precedent law. One common law doctrine that can exert powerful influence over patent rights and economic behavior is the doctrine of patent exhaustion. In February 2013, the Supreme Court heard arguments in an especially relevant patent exhaustion case involving genetically modified plant seed via viral vector. The article will touch on this case and then move backward in time to review several historic court judgments that have played an integral part in shaping current interpretation of the law. Lastly, Bowman will be revisited…

Biologics Production

america invests act biotech digital signal processing gmo innovation nanotechnology patent exhaustion patent limits patent protection patent statutes r&d viral vector

Enhanced Recovery of L-Asparaginase by the Optimization of a Three-Phase Partitioning System Using the Taguchi DOE Methodology

L-asparaginase, produced by Pseudomonas fluorescens, was purified by a three-phase partitioning method of t-butanol in the presence of ammonium sulphate. Enzyme recovery was enhanced by the optimization of process parameters (i.e., ammonium sulphate concentration, t-butanol ratio, temperature, and pH) using the Taguchi design of experiment (DOE) methodology. The enhanced recovery of 31.1% with 96.0% yield and purification of 10-fold was obtained…

Biologics Production

doe enzyme recovery l-asparaginase life sciences pseudomonas fluorescens taguchi design three-phase partitioning

Synthetic Antibodies: The Emerging Field of Aptamers

The current global market for aptamers is approximately $99 million annually and is anticipated to increase at an astonishing compound annual growth rate (CAGR) of 106.3% for the next five years resulting in an estimated value of $3.7 billion by the year 2017. Sometimes referred to as a “synthetic antibody,” an aptamer is a nucleic acid or peptide molecule that binds to a target or antigen with high affinity and specificity. Aptamers have a wide range of applications including diagnostics, therapeutics, forensics, and biodefense. To date, hundreds of aptamer sequences have been identified and can now be chemically synthesized in the lab on demand, faster and less expensively, without the traditional issues associated with producing recombinant antibodies. This article will review aptamer technology, its advantages and limitations, as well as highlight a few of its many applications in the life sciences…

Biologics Production

aptamers biodefense forensics life sciences nucleic acid peptide molecule synthetic antibody therapeutics

Quantitative Endotoxin Assay of Ultrapure Water

The cell wall components of gram-negative bacteria, such as E. coli and Pseudomonads, are designated as endotoxins. They have a hydrophilic polysaccharide and a lipophilic lipid component and, unlike the bacteria from which they originate, are highly heat and pH-stable. Endotoxins are pyrogens (i.e., they can cause fever if they come in contact with mucous membranes or enter the bloodstream). According to the prevalent
pharmacopeias, the defined limit values for endotoxin content may not be exceeded during the manufacturing process of pharmaceuticals. In mammalian cell cultures that are used to produce biopharmaceuticals, such as immunoglobulins, the presence of endotoxins can lead to cell death. For this reason, ultrapure media (i.e., ultrapure water, with levels proven to be below the limits) must be used to manufacture biopharmaceuticals or to propagate cell lines or cell cultures. The objective of this study is to demonstrate that the ultrapure water produced by the arium pro VF system exhibits an endotoxin content that is far below the prescribed limits and that can be used for the applications mentioned above.

Biologics Production

biopharmaceuticals bioproduction endotoxin assay endotoxins gram-negative bacterial h20 pharmaceuticals pyrogen ultrapure water

Induced Patent Infringement Breaks Free from Direct Infringement: The Implications for Bioprocessing

Infringement is the unauthorized copying of patented subject matter. Infringement can be either direct or indirect (e.g., induced or contributory), and the issue becomes complicated when the patented subject matter relates to methods. To illustrate, consider the fictional J.P. Jones, PhD, who has developed a burgeoning business in the bioinformatics field by dividing the practice of patented methods amongst several actors. Dr. Jones encouraged medical practitioners to augment their diagnostic resources by utilizing his for-profit laboratory to perform specific DNA analyses. The diagnostic methods collectively being followed by the clinicians and the independent laboratory were patented by another company, but in dividing performance of the method steps among the clinicians and the lab, Dr. Jones was able to ignore the patent rights by exploiting what has often been perceived as a loophole in US patent law. Under these circumstances, the patented protocols were not directly infringed. Consequently, Dr. Jones could not be held liable for inducing patent infringement. However, a September 2012 Appellate Court decision has changed things for Dr. Jones and his contract research organization (CRO.

Biologics Production

biomedicine contract research cro intellectual property patent infringement patent law patents

The Pervasiveness of Bovine Viral Diarrhea Virus in Commercial Bovine Serum

The procurement of lots of bovine serum that are free of infectious bovine viral diarrhea virus (BVDV) and of neutralizing antibodies to BVDV can still be problematic for manufacturers of biologics. For cell-culture based applications requiring the use of bovine serum as a cell culture medium supplement, the BVDV issue has plagued the industry for decades. Has there been any improvement over the years? The literature from the past four decades has been reviewed to answer this question. There is some evidence that the frequency of detection of infectious BVDV in commercial bovine serum lots has decreased in recent years. There are, however, insufficient data for making conclusions in the case for neutralizing antibodies to BVDV. There are several complementary approaches for mitigating the risk of introducing infectious BVDV into a manufacturing process. These include eliminating the use of serum, pre-treatment of the serum by the vendor or end-user, or treatment of media formulated with the serum to inactivate any BVDV that might be present.

Biologics Production

animal-derived materials biologics bovine serum bvdv cell culture media medium supplement viral inactivation

Process Intensification of Protein A Affinity Step for Monoclonal Antibody Purification Using Membrane and Multi-Column Chromatography

With growing interest in process intensification in the biopharmaceutical industry, implementing cost-effective purification strategies has become increasingly important, particularly for the expensive protein A affinity capture step in monoclonal antibody production. This study compares traditional resin-based batch chromatography (rbBC), multi-column chromatography (MCC), and membrane chromatography (MC) using small-scale experiments with commercially available formats. Performance metrics including yield, elution volume, and impurity reduction were evaluated. The best-performing conditions for MC and MCC were used to project cost and productivity for mAb purification at the 1,000 L bioreactor scale. Both MC and MCC demonstrated significant advantages over rbBC. MC achieved the highest productivity (234–236 g/L/h, 19–20-fold higher than rbBC) and the lowest media costs, resulting in up to 91% reduction in cost-of-goods (CoG) per batch. MCC also showed notable improvements, with 4.6–5.1-fold higher productivity than rbBC, and up to 72% CoG savings. Due to its operational similarity to rbBC and compatibility with existing infrastructure, MC was selected for further evaluation. Optimization was performed using a 3.5 mL membrane over 50 cycles, followed by scale-up to a 58 mL membrane tested on a pilot-scale skid representative of clinical or commercial manufacturing. The process was successfully run for 55 cycles, demonstrating comparable yield, impurity reduction, and elution profiles to rbBC. These results support the feasibility of membrane chromatography as a scalable and economical alternative for protein A capture in intensified downstream processing.

Biologics Production

biologics downstream purification membrane chromatography multi-column chromatography process intensification protein a removal

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