Raw materials as a source of contamination in large-scale cell culture.

Large-scale cell culture operations for biotechnology products use millions of litres of complex media and gases as well as huge quantities of organic and inorganic raw materials. These raw materials must always be assumed to contain contamination by adventitious agents such as Minute Virus of Mice (MVM). Genentech has had experience in dealing with two such contaminations.

Specifications from a biotechnology industry perspective.

The emergence of new analytical technology and the production of pharmaceuticals for a global market in a cost-effective manner necessitate the establishment of worldwide specifications that are appropriate for the product and the manufacturing process. This requires a thorough knowledge of the protein and control of the systems that produce it as well as an understanding of the accuracy and precision of the assays used for testing. Harmonization of specifications among the worldwide regulatory authorities is critical for the future development of new pharmaceuticals.

Novel assays based on human growth hormone receptor as alternatives to the rat weight gain bioassay for recombinant human growth hormone.

Two methods, High-Performance Receptor Binding Chromatography (HPRBC) and Cell Proliferation (CP), have been developed as alternatives to the classical hypophysectomized rat weight gain bioassay for the determination of potency for recombinant human growth hormone (rhGH). In the HPRBC assay, rhGH is combined with an excess of the soluble extracellular domain of the recombinant human growth hormone receptor (referred to as 'receptor' in the discussion of the HPRBC assay). Nondenaturing size-exclusion chromatography is used to analyzed the resulting complex, which forms in a 2:1 receptor to rhGH ratio.

Experience with viral contamination in cell culture.

Genentech has had direct experience with two contaminations of large-scale cell cultures by Minute Virus of Mice (MVM). No definitive source of either contamination has been identified, although the conclusions of the investigation were consistent with a raw material used in cell culture as the source. Effective analytical barriers were developed following the first contamination using polymerase chain reaction (PCR) and cell culture-based methodology for MVM, and these were employed on a routine basis.

Genetic stability and recombinant product consistency.

A committee of U.S. industry scientists from the Pharmaceutical Manufacturers Association has reviewed the recent suggestions of Galibert and Center for Biologics Evaluation and Research (CBER) regarding assurances of product consistency by cloning and sequencing efforts.

Quality control issues in the analysis of lyophilized proteins.

The assessment of protein stability requires the use of many sophisticated analytical techniques. Lyophilization procedures, commonly used to improve the stability profile of protein products, may potentiate undesirable protein degradation. The potential effects of lyophilization on proteins may include denaturation, decreased potency, aggregation, oxidation, and deamidation.

Peptide mapping for detecting variants in protein products.

The genetic stability of biotechnology production systems is of importance in evaluating the safety and efficacy of protein products. The analysis methods currently available for assessing the genetic stability of such systems are limited by the complexity of the eukaryotic genome and by their inability to predict the effects of post-transcriptional and post-translational events, such as glycosylation or host cell protein level changes, on the protein produced. Therefore, it is important to focus attention on the protein product itself in evaluating product consistency rather than on nucleotide sequence analysis for genetic stability of the production systems involved.

A total organic carbon analysis method for validating cleaning between products in biopharmaceutical manufacturing.

The validation of cleaning procedures for biopharmaceutical products produced by recombinant DNA (rDNA) technology presents a diverse analytical challenge. This is because of the need for quantitation of a broad range of potential residual cellular components, including proteins, carbohydrates, and nucleic acids, as well as trace levels of detergents at various manufacturing stages. The validation of a Total Organic Carbon (TOC) analysis method for use in cleaning validation studies is presented.

Safety aspects in the quality control of recombinant products from mammalian cell culture.

The recent approval of the recombinant DNA-derived biological, human tissue-type plasminogen activator (rt-PA), obtained from large scale mammalian cell culture, addressed a number of issues concerning the safety of recombinant products. The assurance of the safety of such highly complex proteins requires that the following topics be investigated: the characterisation of the recombinant production organism; control of large scale cell culture production conditions; design of the purification process and consistency of manufacture; the purity, safety, and stability testing of the final product; and clinical studies. Each of the topics described above is discussed with respect to those key quality control issues that ensure the safety of the product.

The determination of recombinant human tissue-type plasminogen activator activity by turbidimetry using a microcentrifugal analyzer.

A method is described for determining the activity of recombinant human tissue-type plasminogen activator (rt-PA) by turbidimetry using a microcentrifugal analyzer (MCA). A mixture of thrombin and rt-PA is centrifuged into a mixture of fibrinogen and plasminogen to initiate clot formation and subsequent clot dissolution. The resultant profile of absorbance versus time is analyzed to determine the assay endpoint.

High-performance liquid chromatographic assay for sodium levothyroxine in tablet formulations: content uniformity applications.

Sodium levothyroxine was quantitated in 25-300 micrograms/tablet formulations. The procedure consisted of pulverization of a suitable sample, extraction into acetonitrile-water (40:60, v/v) containing 0.05% o-phosphoric acid, and injection onto a bonded-phase cyanopropyl column; the effluent was monitored by UV detection at 225 nm.