Biologics Production – BioProcessing Journal

Standardized and GMP-Ready, Closed-System Processing of Expanded Adipose-Derived Stem Cells Using the Gibco CTS Rotea System Across 2D and 3D Bioreactor Expansions

The manufacturing of cell-based therapies requires harmonized processing protocols to ensure consistent quality. Expanded adipose-derived mesenchymal stromal cells (ASC) are among the most promising candidates for such therapies due to their regenerative and immunomodulatory properties. However, transitioning these therapies to large-scale production presents challenges related to cell recovery, formulation, and in-process cell counting (IPC). The Gibco CTS™ Rotea™ multipurpose Counterflow Centrifugation System enables multiple workflow operations—including cell separation, concentration, washing, and buffer exchange—and is designed to operate within a GMP environment. From a cell concentration perspective, the Rotea system can also formulate concentrated cells in different types of media, including fetal bovine serum…

Biologics Production

2D 3D Adipose Derived Mesenchymal Stromal adipose stem cell adipose-derived mesenchymal ASC automation biologics bioreactor cell expansion cell therapy cellular therapies centrifugation closed system downstream purification GMP manufacturing Mesenchymal Stromal Cells msc rotea Wash Volume Reduction xeno-free media

Myriad Further Limits Patent Eligibility

The recent decision of the US Supreme Court in Assoc. Mol. Pathol. v. Myriad Genetics, Inc., No. 12-398 (June 13, 2013) continued the Court’s efforts to clarify those innovations eligible for patent protection. Myriad unanimously held that “isolated” BRCA1/2 DNAs were patent ineligible under 35 USC § 1 01 while BRCA1/2 complementary DNAs (cDNAs) were patent eligible under that statutory provision. In considering patent eligibility, the Court proposed a test for isolated DNAs that balances incentives and impediments to innovation arising from patent protection, without placing any weight on whether the innovation relates to nature or to an abstract idea. The Court concluded that the mere act of isolating DNA segments claimed in terms of their genetic information was insufficient to render the claims patent eligible. Although the full effect of Myriad won’t be fully known for some time, it is already apparent that reliance expectations developed over 30 years have been upset. Taking a closer look, Myriad discovered the BRCA1 and BRCA2 genes and their influence on the risks of breast and ovarian cancers. Myriad also located these genes in the human genome, isolated the genes, and determined the sequences of several alleles, or versions, of these genes, with some alleles associated with higher cancer risks than others. This led Myriad to develop and market a diagnostic test for assessing breast and ovarian cancer risk, and to seek patent protection for the technology. Myriad refused to license the technology to competitors, which led to a declaratory judgment suit against Myriad. The district court granted summary judgment to the challengers, holding that Myriad’s isolated BRCA1/2 DNA claims and BRCA1/2 cDNA claims were invalid for patent ineligibility under 35 USC § 101. For reasons not relevant to the patent eligibility of DNA composition claims, the Court of Appeals for the Federal Circuit (CAFC) had two cracks at this case on appeal, and both times it held that isolated BRCA1/2 DNAs and BRCA1/2 cDNAs were patent eligible. On its own second review of the case, the US Supreme Court unanimously and finally decided the appeal of summary judgment, holding isolated BRCA1/2 DNAs patent ineligible, but BRCA1/2 cDNAs patent eligible…

Biologics Production

brca1 gene brca1/2 brca2 gene cDNA court of appeals diagnostic test human genome myriad genetics patent protection patent violation supreme court

Adventitious Virus Contamination Testing: Massively Parallel Sequencing in a Multimodal Solution for Biopharmaceutical Safety Testing

Virus contamination of commercially valued cell cultures can be a health risk to the general population and imposes financial burdens on manufacturing and biopharmaceutical companies. We investigated the use of massively parallel sequencing/next-generation sequencing (MPS/NGS) and bioinformatics for the detection and subsequent identification of adventitious contamination. Specifically, the Illumina® HiSeq 2000 instrument, in the 2 × 100 base pair (bp) paired end (PE) run configuration, was used to determine the identity and limit of detection of a DNA virus spiked into a virus vaccine, and an RNA virus spiked into a mammalian master cell bank (MCB). This configuration provided sufficient sequence read lengths and depth of coverage to detect and identify spiked-in SV40 and measles viruses in a background of vaccine, MCB, and host nucleic acid that make up the bulk of these samples. Furthermore, the detection of 30 SV40 reads within one tested sample suggested a < 1 plaque forming unit (pfu) sensitivity in a background of 2.8 × 107 infectious adenovirus serotype 5 (Ad5) particles. Results from subsequent virus vaccine testing suggested the presence of non-viable virus DNA contamination in the sample. Therefore, we propose that a multimodal approach, in which broad-range screening for known or unknown adventitious agents by MPS/NGS is complemented by targeted virus detection assays (e.g., PCR-based and infectivity assays), should provide the most useful safety monitoring information. We suggest that MPS/NGS and the accompanying bioinformatics is a sensitive, broad-range, and long-lasting tool with the ability to improve upon existing biosafety testing within a larger testing program…

Biologics Production

bioinformatics cell culture dna contamination host cell proteins mammalian cell bank massively parallel sequencing master cell bank mcb measles virus next generation sequencing PCR plaque forming unit SV40 vaccine production vaccines viral contaminants virus contamination virus detection assays virus spikes

A Proposed Modeling Approach for Comparing the Heat Inactivation Susceptibility of Viruses

Heat inactivation is dependent both on temperature and time at temperature, making inter-assay and intervirus comparisons of heat sensitivity of viruses problematic. Historically, heat inactivation data for pathogens, including viruses, have been evaluated by determining decimal reduction value ([D] the time required to inactivate 1 log10 of the organism at a given temperature) and the incremental temperature required to decrease the D by 1 log10 (z). We recommend the use of a straightforward approach for extrapolating heat inactivation (i.e., inactivation vs. time at fixed temperature) data from measured to non-measured temperatures that is based not on the z value, but on a power function fit of the D vs. temperature plots. There needs to have been at least three temperatures evaluated in the inactivation vs. time kinetics studies in order to conduct these modeling analyses. For inter-assay and inter-virus comparisons of heat inactivation sensitivity, we propose the use of two modeled parameters: (1) temperature required to inactivate 1 log10 of virus in 0.5 minutes; and (2) time required for 1 log10 reduction in infectivity at 80°C. By using both modeled parameters, we have calculated consensus heat inactivation values for two caliciviruses (feline calicivirus and murine norovirus)…

Biologics Production

calicivirus feline calicivirus heat inactivation kinetics studies log10 reduction modeling analyses murine norovirus virus inactivation virus sensitivity

An Overview of Metal–Peptide Complexes Used in Biomedical Research

Metal peptide complexes play an important role in understanding the mechanisms of complex diseases at a molecular level. Peptides are good chelators for metal ions and can form important tools in the drug and medical device industries. Transition metal ions are characterized by their ability to assume different oxidation states and serve as centers for metalloenzymes that catalyze electron transfers. Some transition metals act as Lewis acids and
become an active site on enzymes and also catalyze reactions (e.g., peptidases, phosphatases). Metal peptide complexes are involved in Alzheimer’s disease (AD) and are used as cancer diagnostics. Many biomolecules (molecules produced by living cells) like metabolites, hormones, and vitamins are capable of forming strong, multidentate metal ion complexes. Species in the plant and animal kingdoms synthesize specific metal ion chelators that bind to essential metals with extremely high affinities. Plants and animals including humans possess a number of unique biomolecules that can coordinate metal ions. Peptide conjugation is a widely used and effective method for improving both cellular and nuclear entry of a variety of molecules…

Biologics Production

biomolecules catalyze electron transfers chelation metal ions metal peptide complexes metalloenzymes peptide conjugation peptides

Rapid Manufacture and Release of a GMP Batch of Avian Influenza A(H7N9) Virus-Like Particle Vaccine Made Using Recombinant Baculovirus-Sf9 Insect Cell Culture Technology

Novavax reported that, in 28 days following public release of the avian influenza A/Anhui/1/2013 (H7N9) virus gene sequences, its recombinant DNA and baculovirus-Spodoptera frugiperda cell culture-based technology was used to produce a virus-like particle vaccine to avian influenza A(H7N9) virus, and murine animal challenge studies were initiated. This report describes Novavax’s manufacturing process and the coordinated timing of critical activities necessary to produce and release a clinical batch of avian influenza A(H7N9) virus virus-like particle vaccine, under current good manufacturing practices, within three months from the time that the virus genomic sequences for this potential pandemic influenza virus were reported…

Biologics Production

avian flu avian influenza a baculovirus expression biopharmaceuticals h7n9 pandemic influenza protein expression recombinant sf9 sf9 sf9 baculovirus Spodoptera frugiperda vaccine development virus-like particles vlp vlp vaccine

Five Insurance Tips for Biotechnology Companies Facing Product Recalls or Liability Claims

Biotechnology companies — those that research and manufacture products through the use of biological techniques such as genetic engineering and the development of specialized strains of biological substances — constitute an increasing segment of the US economy. These companies might: (a) create a new type of insect- or drought-resistant corn by the modification of genes; (b) alter naturally occurring enzymes to aid in manufacturing or to help produce foods; (c) use recombinant DNA to create medicines that are remarkably effective in curing or treating disease; or (d) use any of a number of other techniques to create beneficial and potentially lucrative products. Just like other industries, biotechnology groups can be faced with recalls of their products, either voluntary or government-mandated, or claims that their products have caused bodily injury or property damage to their customers. Because biopharmaceuticals are produced by — or extracted from — a biological source, the chances of a product recall are higher than that of a synthesized drug. In April 2012, a report by GBI Research, an independent research firm, concluded that biologics were involved in more recalls, voluntary or mandatory, than drugs from other sources for the four-year period from 2007 through 2010…

Biologics Production

biological product recall biological substances biologics biopharmaceutical recall biotechnology biotechnology insurance DNA gene engineering liability issues recombinant dna therapeutics

Effective Site-Specific Isotopic Labeling: Expression Optimization Using Specialized Media

For decades, recombinant genes have been expressed in a variety of cellular systems to generate protein reagents that are the potential targets for new small-molecule drugs. As these targets become more complex, researchers have developed innovative methods to study the structure of these proteins and their interactions with potential drugs. BioExpress® 2000 media (Cambridge Isotope Laboratories, CIL) incorporates isotope-labeled amino acids
into recombinant proteins expressed in baculovirus-infected insect cells to assist nuclear magnetic resonance (NMR) structural studies. In order to use this labeled media most efficiently, expression conditions must be optimized at a small-scale prior to production…

Biologics Production

baculovirus-infected cells bioexpress media cellular systems isotope-labeled amino acids NMR nuclear magnetic resonance protein reagents recombinant genes recombinant proteins small-molecule drugs

Bovine Serum Albumin Partitioning in Aqueous Two-Phase Systems: Effects of Variables and Optimization

The objective of this study was to optimize process conditions for the effective partitioning of bovine serum albumin (BSA) using response surface methodology (RSM). Initially, four different salts (tripotassium citrate, tripotassium phosphate, sodium carbonate, and sodium sulphate) were tested for the ability to partition BSA. Among the salts chosen, tripotassium citrate was observed to yield a high partition coefficient. The effect of phase forming components: concentration, PEG molecular weight, and pH were studied for a PEG/tripotassium citrate system and the information obtained was utilized to fix the ranges in RSM studies. Four different independent variables (PEG 2000, tripotassium citrate, NaCl concentrations, and pH) were considered for RSM studies and the responses generated were partition coefficient (k) and percentage yield. A statistical model was developed and the values obtained were 99% within the confidence level. Optimal conditions of the system were found as: 0.25 M sodium chloride, 32% PEG 2000 (w/w), 16% tripotassium citrate (w/w), pH 6.0, a partition coefficient of 6.03, a recovery of 91.76%, and a controlled operating temperature of 303.15 K…

Biologics Production

biologics bioprocess bovine serum albumin bsa partitioning phase forming components response surface methodology rsm salts for partitioning

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

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