Upstream Processing

Viral Vector Production in the Integrity® iCELLis® Disposable Fixed-bed Bioreactor from Bench-scale to Industrial Scale

Recombinant viruses (e.g. lentivirus and adeno-associated-virus) can be used as human gene therapy vectors. They are mainly produced in adherent cell cultures (e.g. HEK293T, A549, VERO) in Roller Bottles or multiple-tray-stacks using either transient transfection or infection strategies. Therefore, the Integrity® iCELLis® (ATMI LifeSciences) line of bioreactors offers a new production alternative with stronger process controls and ease of scale-up.   The iCELLis bioreactor is designed for adherent cell culture applications. Cells grow on microfibers carriers packed in a fixed-bed…

Is the Gold Standard Tarnished? Weaknesses in the Standard Method Compromise Your RMM Validation

Introduction of a new Rapid Microbial Method (RMM) requires a full validation. The purpose of the validation is to prove that equivalent or better results are obtained using the new method when compared to the compendia. One assumes that the result obtained with the compendial method is absolute and is the “Gold Standard” by which to compare the new test. In reality, the compendial method exhibits a number of weaknesses that compromise the integrity of the validation. The standard requires…

Standards for Ancillary Materials Used in Cell- and Tissue-Based Therapies

Cell- and tissue-based therapies are being used increasingly to treat many diseases for which currently no other adequate treatment options are available. These products contain human or animal cells that can replace, regenerate, or augment a recipient’s diseased, dysfunctional, or injured cells, tissues, or organs. Cells or tissues might be unmanipulated, or their biological characteristics can be altered ex vivo before administration of the final product to patients. Examples of cell therapies range from traditional blood transfusions to recent approaches…

Protein A

The number of blockbuster monoclonal antibody (MAb) drugs continues to grow. In 2008, MAbs generated revenues in excess of US$15 billion (1), making them the highest-earning category of all biotherapeutics. The world MAb market will reach $62.3 billion in 2015, with next-generation therapeutic antibody revenues reaching $2.3 billion in 2015 according to Visiongain reports published in September and November 2011 (2, 3). Biosimilar antibodies will also begin to enter established markets as regulatory authorities clear approval pathways for them. Most…

Enhanced 2-D Electrophoresis and Western Blotting Workflow for Reliable Evaluations of Anti-HCP Antibodies

Biologic drugs are subject to unique regulatory and technical requirements because of their origin and expression in genetically engineered host cells, as well as their underlying physicochemical properties and elaborate purification processes. One such requirement is the accurate monitoring and effective removal of process-derived impurities such as host-cell proteins (HCPs) and DNA/RNA, viruses, cell culture media, chromatographic leachates, and so on (1). Of those impurities, HCPs are perhaps the most challenging to accurately monitor. Each expression system’s proteome consists of…

Upstream Chemistry Analysis in Cell-Based Process Development

Cell line selection is important to any pharmaceutical company’s development pathway for biological compounds (1). In cell-line selection laboratories, many different, slightly variable cell lines are tested in parallel for desired characteristics. Candidate cell lines are chosen for further development on the basis of their performance in basic tests of critical quality attributes (CQAs). Historically, such cell lines were selected in large-volume containers because it was necessary to have sufficient volume in culture to allow repeated sampling without damaging the…

Automation of Microbioreactors

Current methodologies in genetics and microbiology enable researchers to influence metabolic pathways of microbial cells in many directions. Beside the academic interest in investigating fundamental functions in metabolic pathways, commercial production of valuable compounds by microbial hosts is state of the art. For example, such products include enzymes (lipases, proteases, phytases), therapeutic agents (insulin, antibodies), bulk chemicals (lysine, glutamate, citric acid), or the microbial cells themselves (used in brewing or milk processing), with therapeutic agents probably the fastest growing market.…

Mirus TransIT-PRO® Transfection Kit for Biotherapeutic Protein Production

Decrease time to produce usable protein by maximizing target protein yields through transient transfection. The TransIT-PRO® Transfection Kit uses animal origin free components designed for high and reproducible nucleic acid delivery into suspension CHO and 293 derived cells. Since it is compatible with varied media formulations, the same media can be used for both transient and stable expression. The TransIT-PRO outperforms linear PEI in protein yield, while providing a cost-effective alternative to FreeStyle™ MAX and 293Fectin™ Transfection Reagents.

Scale-Up of Adherent Vero Cells Grown on Cytodex™ Microcarriers Using ReadyToProcess Equipment

In cell culture-based vaccine production, scale-up of adherent cells is challenging. This study shows a process for scaling up adherent Vero cells from static cell factories to influenza production at 50 L scale using WAVE Bioreactor™ systems and ReadyToProcess singleuse equipment. Vero cells were grown to high cell density on Cytodex microcarriers in 10 L working volume. The cells were detached with trypsin and used to seed a 50 L production culture with the same microcarrier concentration. The cells were allowed to reattach and grow on the new microcarriers in a larger Cellbag™ bioreactor chamber. Cells were subsequently infected with influenza virus. The results show a repeatable scaleup procedure.