BioProcessing Journal Posts

At the onset of modern-day biotechnology, products typically fell into two distinct categories, the traditional high volume, low value products (e.g. beer and industrial enzymes) that had come to characterize the biotechnology industry, and low volume, high cost products. Recombinant proteins, the result of technological advances in molecular biology, have come to typify these latter products. Recombinant protein therapeutics have been hugely successful, potentially outstripping production capacity and continue to drive much of the biotechnology. Meanwhile, many recombinant proteins, those characterized as research tools and reagents, are governed by a price-volume relationship typical of industrial enzymes. In a competitive environment, they are fast becoming commodities ā€” price sensitive, packaged as kits, coupled to instrumentation, and relying on heavy marketing and brand recognition. Ominously, the advantage protein therapeutics have enjoyed with patent protection and regulatory constraints on production is being threatened as patents expire and competition from generics increases…

Biologics Production

Validating the safety of biological preparations requires thorough testing for contamination by adventitious agents. Utilizing mammalian cell cultures to produce recombinant proteins as biopharmaceuticals requires testing for viral contamination. The polymerase chain reaction (PCR) may be employed to specifically detect the presence of viral DNA or RNA with great sensitivity. PCR assays are particularly useful for the qualification of recombinant cell banks. Regulatory agencies recommend that mammalian cell banks be tested for a variety of possible human viral contaminants. In most cases the cells used to produce the cell bank have been previously analyzed for viral contamination. The use of PCR for the detection of viruses in the final banked cells can alleviate the need for difficult, costly, and time-consuming infectivity assays. In some cases the relevant viruses cannot be cultured, eliminating the ability to perform infectivity assays. The PCR assay can provide a sensitive and specific method for detection of viral contamination when standard infectivity assays are unsatisfactory…

Cell & Tissue Banking

In general, the industry has gone through another of its realignment periods, where much was learned, but a lot of restructuring and refocusing took place. Driven by the need to keep the doors open, small to medium sized firms had to do some severe belt tightening, or completely redefine themselves as to technologies, products, and personnel. Many of the larger firms reevaluated their product pipelines, and then made the changes they felt were necessary to assure future revenues, or to make themselves attractive merger partners. Numerous large mergers took place with some that were the largest the biopharmaceutical industry has ever seen. In addition, several medium-sized companies merged, or otherwise found strategic alliances that energized their product pipelines, or simply provided the cash they needed to keep going. Antibody products did very well with a number of blockbusters receiving license approval in 2003…

Biologics Production

Xcyte Therapies has recently introduced a bioreactor-based process for the GMP manufacture of autologous activated T cells, Xcellerated T Cellsā„¢, for clinical trials. Using a single customized disposable 20-L Cellbagā„¢ with a working volume of 10 L on a customized Wave Bioreactor platform (Wave Biotech, Bridgewater, NJ), the Xcellerateā„¢ III Process has supplanted the 60-L static Xcellerate II Process that used 60 bags cultured in a standard incubator. Compared to the Xcellerate IIā„¢ Process, the Xcellerate III Process significantly reduces the overall labor, the number of culture containers, bag spikes, and sterile connections required, as well as reducing the process volume and the cost of goods, while more than quadrupling the final cell density and doubling the facility capacity. These process improvements are achieved without compromising final product composition or quality…

Biologics Production Manufacturing

Cation exchange chromatography (CEX) is a versatile method for separation of proteins based on exploiting differences in positive electrostatic charges. In CEX, proteins are bound to the negatively charged stationary phase (cation exchangers) and then eluted using a salt gradient. Typically, the liquid-phase pH in CEX is lower than the isoelectric points (pI) of the proteins. CEX has been used to monitor various post-translational modifications such as glycosylation, deamidation, phosphorylation, truncation, oxidation, C-terminal and N-terminal clipping, and N-terminal cyclization. Some of these variants may exhibit different bioactivity. Therefore, it is important to characterize protein variants and monitor the stability of these variants throughout the process of drug discovery, development, and manufacture. Characterization of complex proteins such as antibodies, has traditionally been performed using slab gel-based techniques such as isoelectric focusing (IEF). This technique is qualitative and time consuming. It also generates large quantities of chemical waste from the staining process…

Biologics Production

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

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 grocerā€™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 agenciesā€™ 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

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 ā€” 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-GuĆ©rin (BCG) as a therapeutic for bladder cancer bone marrow transplants, and even the use of maggots to clean wounds…

Cell & Gene Therapy Viral Vectors

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

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