BioProcessing Journal

Engineering Innovations Improve Process Column Chromatography

Over the past decade there has been a steady increase in the number of biotherapeutics requiring high doses and long term administration. Most notable among these are monoclonal antibodies (MAbs) and fusion proteins comprised partially of antibody molecules. Column chromatography is a commonly applied purification method for downstream processing of biotherapeutics, and there is considerable pressure to process much greater volumes at a faster rate. For recombinant proteins and MAbs, a variety of chromatographic methods are employed, including affinity, ion exchange, hydrophobic interaction, and to a lesser extent, immobilized metal affinity and gel filtration. Improving process control for chromatography operations is essential for biopharmaceutical manufacturers to process larger volumes and overcome capacity shortfalls. As the past ten years have seen increasing volumes of MAb-based drugs, there have been significant innovations to address growing productivity requirements. Dominant among these has been high throughput media capable of isolating product at faster rates than previously achievable…

Biologics Production

chromatography clean-in-place high throughput media pack-in-place process control

FTA Filter Applications: A PCR Format to Alleviate Technical Barriers in the Detection of Foodborne Pathogens in Complex Matrices

The safety of our food supply is a major public health concern for consumers, government regulatory agencies, and the food industry. Earlier generations may recall when fresh produce was largely domestic and seasonal. Today we live in a global marketplace, where fresh fruits and vegetables may be on the vine overseas one day and on our grocer’s shelf the next. Although this has provided more yearround variety of foods for the consumer, a lack of uniformity in established agricultural standards and practices among international trading partners (e.g., sanitary issues and inspections) may ultimately lead to deleterious health effects. This is evident in the number of food-borne illness outbreaks and associated deaths. Negative economic consequences also result through lost wages and productivity, and health care costs. The impact can be far greater in developing nations. Therefore, refining domestic and international food safety policies is at the forefront of many government agencies’ efforts toward protecting the public health. Reducing the number of such incidences has become a priority for government regulatory agencies and the food industry…

Regulatory

food safety food-borne illness food-borne pathogen FTA filter PCR PCR- based assay standards

Gene Therapy with Viral Vectors

The modern era of interest in gene transfer as a methodology for treating disease began around 1985 with the first use and publication of mouse-based retroviruses that could transduce human cells. In fact, the use of gene transfer as a clinically useful method is probably older than any other therapy commonly used today — it forms the basis for the vaccinia vaccination against smallpox, popularized in Western medicine by Jenner. Another antecedent is phage therapy for bacterial infection, which was largely but not completely superseded by antibiotics (although it may make a comeback in this era of drug-resistant pathogenic bacterial strains.) Other examples include the other live viral vaccines: measles/mumps/rubella, polio, varicella, tuberculosis, influenza, the use of bacillus Calmette-Guérin (BCG) as a therapeutic for bladder cancer bone marrow transplants, and even the use of maggots to clean wounds…

Cell & Gene Therapy Viral Vectors

gene therapy vectors gene transfer targeted delivery

The Complete Nucleic Acid Sequence of the Adenovirus Type 5 Reference Material (ARM) Genome

The Adenovirus Reference Material Working Group (ARMWG) oversaw development of an adenovirus reference material (ARM) with the intent to provide a way to standardize assay measurements from different laboratories. The ARM, which was manufactured in stages by various organizations including Canji (San Diego, CA) and Introgen Therapeutics (Houston, TX), is available from American Type Culture Collection (Manassas, VA). Upon completion of its manufacture, the characterization phase primarily defined viral particle concentration as well as infectious titer for this product. However, many other concurrent characterization studies were conducted including an assessment of vector purity (e.g., host cell DNA, host cell protein, reversed-phase HPLC), a short-term field use and shipping stability study, and a long-term stability study. Also included in these studies was a coordinated effort to determine the complete DNA sequence of the ARM vector genome…

Nucleic Acids Viral Reference Materials Viral Vectors

adenovirus serotype 5 analysis characterization genome nucleic acids purification sequencing

Role of the Container/Closure System and Formulation on Agitation-Induced Aggregation Phenomena in Recombinant Adenoviral Products

The biologic activity of protein therapeutics is often compromised by the formation of soluble aggregates and protein precipitates. While the detailed molecular mechanism of protein aggregation remains unclear, substantial evidence suggests that factors that promote protein unfolding and exposure of hydrophobic residues play a significant role in aggregate formation. These can vary according to the particular protein of interest, the solvent system, and storage conditions. Slight changes in temperature, pH, and ionic strength have been documented to have a significant effect on the aggregation phenomena of numerous proteins. Freezing and subsequent thawing of a preparation (whether by design or accident) during processing, shipping, and storage can induce subtle changes in the ionic strength, solute concentration, and pH of a given preparation and induce aggregate formation…

Biologics Production Viral Vectors

adenovirus aggregation protein aggregation recombinant proteins therapeutic proteins

A Rational Approach to Monoclonal Antibody Development According to Predefined Assay Criteria

Monoclonal antibodies (MAbs) destined for use in drug-specific assays must meet specific binding criteria, and therefore require much more in the way of development than many protein-specific antibodies. This article describes one way to facilitate the development of well-characterized, high specificity MAbs. Essentially the same techniques have also been used for producing MAbs that are cancer markers and MAbs that are specific for infectious agents. Monoclonal antibody development, as in other endeavors, requires clearly defined goals and an examination of proposed methods to attain them. This has been succinctly stated by the phrase “Start with the end in mind.” Unfortunately, the vast majority of hybridoma development could be more appropriately described by Yogi Berra, “If you don’t know where you are going, you will wind up somewhere else.” In most laboratories, fusions are carried out with the goal of reducing the amount of cell culture and handling required and using the minimum amount of screening. Little or no consideration is given to ensuring that single clones are subjected to the screening tests. Under these conditions, it is little wonder that dozens of fusions can be carried out, each with the same result — no specific antibody found…

Biologics Production

cell culture media characterization fusion hybridoma development monoclonal antibody Poisson distribution screening

The Study of GPCR Activity Using Insect Cell Membranes and AlphaKey Peptide Probes

G protein-coupled receptors (GPCRs) comprise a “superfamily” of cell surface receptors that play a prominent role in cell signalling and are classified into more than 100 subfamilies according to sequence, ligand structure, and receptor function. They are cell surface receptor proteins with seven transmembrane domains which transduce extracellular signals to the interior of cells through heterotrimeric G proteins. GPCRs’ exposure at the exterior cell surface and strong role in cell regulation has provided a rich target family for small compound therapeutics. Of the estimated 35,000 genes in the human genome, approximately 750 encode for GPCRs; half likely encoding sensory receptors, the remaining half representing potential drug targets. Only about 30 of these potential targets are currently modulated by existing pharmaceuticals with approximately 400 remaining potential pharmaceutical targets for validation…

Baculovirus Expression Technology Research

BEVS cell surface receptors expression G protein-coupled receptors insect cell technology recombinant baculovirus sf9 transfection

The Use of Experimental Design Methods in the Development of a High Concentration Lyophilized Formulation for a Humanized Monoclonal Antibody

lation components that stabilize the molecule in order to provide the desired product storage stability. Generally, an aqueous formulation is preferred; however, the instability of proteins, both physical (e.g. aggregation) and chemical (e.g. deamidation and oxidation), often necessitates the development of lyophilized formulations. In these formulations, selection of the appropriate stabilizing cryoprotectants, lyoprotectants, and bulking agents is critical. Accelerated stability studies are typically used to evaluate the effect of a single factor at a time in order to identify the optimum pH, buffer, and stabilizing excipients. This approach is limited in that many independent time-consuming experiments must be run, the results are obtained only at the evaluated set points, and additional experiments are required to assess potential interactions between the evaluated factors…

Biologics Production

chromatography differential scanning calorimetry lyophilization monoclonal antibody stability therapeutic proteins

Production of a Recombinant Influenza Vaccine Using the Baculovirus Expression Vector System

Influenza is a highly contagious, acute viral respiratory disease that occurs seasonally in most parts of the world. The infection resides primarily in the respiratory tract (nose, throat and bronchi), but causes both local and systemic symptoms including fever, chills, cough, headache, myalgia, sore throat, and malaise. Influenza-related pneumonia is the main complication of infection. Annual epidemics cause significant morbidity and mortality worldwide. Each year, influenza infections result in an average of 110,000 hospitalizations, approximately 20,000 of which result in death. These deaths are heavily concentrated (>90%) among persons who are at highest risk for influenza-related complications — elderly adults (over 65), children under age five, patients with pre-existing respiratory or cardiovascular disease, and women in the third trimester of pregnancy. Thus, the prevention of influenza virus infection is a major public health priority…

Baculovirus Expression Technology Viral Vectors

BEVS influenza purification recombinant proteins transfection trivalent vaccine vaccines

Advanced Methods of Adenovirus Vector Production for Human Gene Therapy: Roller Bottles, Microcarriers, and Hollow Fibers

Various types of viral vectors are being employed extensively as gene therapeutics to treat cancer and genetic diseases. Among the viruses that have been produced for human clinical trials (i.e. retrovirus, adenovirus, poxvirus, adeno-associated virus, and herpesvirus vectors) adenoviruses exhibit the lowest pathogenicity yet still infect an extensive range of cell types with high efficiency. These key characteristics make recombinant adenoviruses efficient gene-delivery vehicles and excellent research tools. However, the time-consuming and complex processes of generation, amplification, purification, and quality testing associated with production of recombinant adenoviruses make it difficult for many researchers to utilize these vectors. This is particularly true with respect to cell culture optimization and the virus propagation protocols employed in vector production. In this regard, the development of innovative cell culture techniques has become vital for optimizing vector production for gene therapy…

Biologics Production Cell & Gene Therapy Viral Vectors

adenoviral vectors analysis cell culture enumeration propagation purification recombinant proteins

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