Gary Walsh

Biopharmaceutical benchmarks 2010

Over the past four years, several new types of experimental biologic treatment have received commercial registration, but the emergence of biosimilars represents the biggest shift in the biologic approval landscape.

Post-translational modifications in the context of therapeutic proteins

The majority of protein-based biopharmaceuticals approved or in clinical trials bear some form of post-translational modification (PTM), which can profoundly affect protein properties relevant to their therapeutic application. Whereas glycosylation represents the most common modification, additional PTMs, including carboxylation, hydroxylation, sulfation and amidation, are characteristic of some products. The relationship between structure and function is understood for many PTMs but remains incomplete for others, particularly in the case of complex PTMs, such as glycosylation. A better understanding of such structural-functional relationships will facilitate the development of second-generation products displaying a PTM profile engineered to optimize therapeutic usefulness.

Biopharmaceutical benchmarks 2006

The rate of biopharmaceutical approvals has leveled off, but some milestones bode well for the future.

Post-translational modifications of protein biopharmaceuticals.

The majority of therapeutic proteins display one or more post-translational modifications (PTMs). These modifications normally influence the biochemical and therapeutic properties of such proteins. Choosing an expression system capable of generating an appropriate product PTM profile remains one of the most crucial decisions a drug developer must make. This review considers the PTMs most often associated with therapeutic proteins and focuses upon more recent advances in engineering PTMs with the aim of improving the application-relevant functional characteristics of these drugs.

Therapeutic insulins and their large-scale manufacture

Biotechnological innovations over the past 25 years have underpinned the rapid development of a thriving biopharmaceutical sector. Therapeutic insulin remains one of the most commonly used products of pharmaceutical biotechnology and insulin-based products command annual global sales in excess of

Purification and characterization of extracellular phytase from Aspergillus niger ATCC 9142

4.5 billion. Innovations in its method of production and in particular the advent of engineered insulin analogues provide a fascinating insight into how scientific and technological advances have impacted upon the pharmaceutical biotechnology sector as a whole. Current insulin-based diabetes research is increasingly focused not on the insulin molecule per se, but upon areas such as the development of non-parenteral insulin delivery systems, as well as organ-/cell-based and gene therapy-based approaches to controlling the disease.

Pharmaceutical biotechnology products approved within the European Union

Extracellular phytase produced by Aspergillus niger ATCC 9142 was purified to homogeneity by employing an initial ultrafiltration step, followed by chromatography using ion exchange, gel filtration and chromatofocusing steps. The purified enzyme was an 84 kDa, monomeric protein. It possessed a temperature optimum of 65 degrees C, and a pH optimum of 5.0. Km and Vmax values of 100 microM and 7 nmol/s, respectively, were recorded and these values fall well within the range of those previously reported for microbial phytases. Substrate specificity studies indicated that, while the enzyme could hydrolyse a range of non-phytate-based phosphorylated substrates, its preferred substrate was phytate. Phytase activity was moderately stimulated in the presence of Mg2+, Mn2+, Cu2+, Cd2+, Hg2+, Zn2+ and F- ions. Activity was not significantly affected by Fe2- or Fe3- and was moderately inhibited by Ca2+. The enzyme displayed higher thermostability at 80 degrees C than did two commercial phytase products. Initial characterisation of the purified enzyme suggested that it could be a potential candidate for use as an animal feed supplement.

The industrial production of enzymes

The manufacture of therapeutic proteins represented the first true industrial application of recombinant DNA technology. Thus far some 88 recombinant proteins/monoclonal antibody-based products have gained marketing approval within the European Union (EU). This represents 36% of all new drug approvals since the introduction of the new centralized European drug approval system in 1995. More recently, an increasing proportion of approved proteins are engineered, tailored to display altered pharmacokinetic profiles or reduced immunogenicity in man. Currently no nucleic acid-based products are approved in the EU. Technical innovations/milestones likely characterizing the biopharmaceutical industry within the next decade include approval of some products produced in transgenic systems, approval of some products administered by non-parenteral means, approval of at least some nucleic acid-based products and the identification of novel biopharmaceuticals/biopharmaceutical targets through discoveries in functional genomics and proteomics.

Comparison of Selected Physicochemical Characteristics of Commercial Phytases Relevant to their Application in Phosphate Pollution Abatement

The production of enzymes is a pursuit central to the modern biotechnology industry. Markets for traditional industrial enzymes continue to grow while the continued emphasis on biotechnological endeavours has generated demand for an ever increasing number of additional biocatalysts. The advent of genetic engineering has now facilitated the large-scale production of enzymes and other proteins which are produced naturally only in minute quantities. This development is particularly significant with regard to the production of enzymes and other proteins of therapeutic significance, which are now available in clinically useful quantities.The level of downstream processing to which any enzyme is subjected is dependent upon its intended application. Industrial enzymes produced in bulk generally require little downstream processing, and hence are relatively crude preparations. Enzymes destined for therapeutic applications are subject to a far higher degree of downstream processing, often incorporating 3-4 chromatographic steps. While enzymology is one of the longest established branches of the biochemical sciences, it continues to be an area of ongoing, active research. The continual discovery of new enzymes and a greater understanding of previously discovered enzymes and their functional significance suggests many novel applications for these catalytic activities. The intestinal production and utilization of enzymes will continue to be of central importance in the biotechnology industry.

Physicochemical characteristics of commercial lactases relevant to their application in the alleviation of lactose intolerance

The enzyme phytase catalyses the dephosphorylation of phytic acid and its salts, phytates. Supplementation of monogastric animal feed with microbial-derived phytase increases the bioavailability of phytic acid bound phosphate. This facilitates a reduction in the addition of inorganic phosphate to the feed and reduces phosphorus excretion. To achieve maximum efficacy in terms of phosphate pollution abatement, supplemental phytases added to animal feed must survive thermal processing of the feed, resist inactivation by the proteolytic enzymes encountered in the animals digestive tract and display high activity at physiological temperature and pH. A series of in vitro experiments were carried out to determine the relative suitability of four major commercial phytase products for use in animal feed. The enzymes assessed lost between 14% and 72% of their original activities after heating to 80°C for 5 minutes. After exposure to simulated upper digestive tract conditions, the phytases assessed retained between 0 and 28% of their original activities. The commercial phytases displayed between 98% and 67% of their maximum activities at 39°C and all phytases assessed had an optimum pH between pH 4 and pH 5. None of the phytases assessed satisfied all of the criteria of an ideal phytase for use in animal feed.