Expression systems for therapeutic glycoprotein production

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There are slightly over 165 recombinant pharmaceuticals currently approved for human use. Another 500 protein candidates are in preclinical and clinical development, about 70% of these being glycosylated proteins. The need for expression systems allowing the efficient manufacturing of high quality glycoproteins is thus becoming imperative. Recent developments with CHO cells, the predominant mammalian expression system, have focused on either increasing cell specific productivity or prolonging the life span of cells in culture that translates to high integrated viable cell densities. These two factors have allowed volumetric productivities in excess of 5 g/L under conditions of controlled nutrient feeding. In addition to glycoengineering strategies, which are offering considerable advantage in producing proteins with enhanced therapeutic properties, several alternative expression systems are being developed for their manufacture, each with their advantages and limitations.

Introduction

Protein therapeutics are the largest class of new products being developed by the biopharmaceutical industry. In 2006, this market was US $57 billion and is expected to grow at a compound annual growth rate of 12% till 2010 [1]. Animal cell expression systems are preferred for the production of large, complex proteins requiring post-translational modification (PTM) for their biological activity. Monoclonal antibodies and Fc-fusion proteins, a major class of licensed therapeutics, are mainly produced in Chinese Hamster Ovary cells (CHO), mouse myeloma cells (NS0, SP2/0) and hybridomas [2]. Most of the recombinant protein (r-protein) therapeutics currently under development are also expressed in CHO cells because of their extensive characterization and ability for human-like glycosylation. Other animal cell lines used for production include Baby Hamster Kidney (BHK21), human fibrosarcoma (HT1080), human lymphoma (Namalwa) and Human Embryo Kidney 293 (HEK293). Many yeast-derived non-glycosylated proteins are already on the market and recent developments in glycoengineering pave the way for glycoprotein manufacturing. Expression systems such as insect cells, filamentous fungi and plants are used predominantly for vaccine production, but many glycoproteins are also in clinical phases. Finally, transgenic animals recently gained approval for the manufacture of a human therapeutic, offering an additional platform for production. To meet the increasing demand for production of protein therapeutics, efforts are being applied to these expression systems to increase productivity and protein quality.

Section snippets

Chinese hamster ovary cells

CHO cells are the most widely used mammalian host for the production of biotherapeutics. They have not been implicated in any obvious adverse effects and there is now a history of regulatory approval for r-protein drugs produced with these cells. Other cell lines offering alternative platform technologies for production are being pursued and may offer scientific advantages but the major hurdle can be to convince the regulatory authorities for their approval. Here we present some of the issues

BHK cells

While many vaccines are manufactured in BHK21 cells, only a few r-protein therapeutics are produced in this cell line including the coagulation Factor VIIa (NovoSeven®) and FVIII (Kogenate®). Factor VIII is probably the largest (∼300 kDa), most complex (it possesses 25 potential N-linked glycosylation and many O-glycosylation sites, 6 tyrosine sulfation sites and 7 disulfide bonds) and most challenging r-protein therapeutic to manufacture [38]. Sulfation of FVIII on tyrosine residues was shown

Human cells

Even though HEK293 is probably the most widely used cell line to express research grade r-proteins, only one licensed therapeutic (Xigris®, activated protein C) is manufactured in this host. Other approved protein therapeutics currently produced in human cells include Dynepo (epoetin delta), Elaprase® (iduronate-2-sulfatase) and Replagal® (α-galactosidase A), all expressed in the human fibrosarcoma cell line HT-1080, and Alferon N® and Sumiferon (both a mixture of 10–14 alpha interferon

Insect cells

The insect cell-baculovirus expression system is a powerful platform to rapidly produce high level of r-proteins. The main insect cell lines used are Spodoptera frugiperda SF9 or SF21 and Trichoplusia ni BTI 5B1-4 (High Five™). Owing to the high-mannose and paucimannose type of glycosylation that is obtained in insect cells, no therapeutic protein is currently produced in this system as this would compromise in vivo bioactivity and potentially induce allergenic reactions. Engineering those

Yeasts

Yeasts are more robust than mammalian cells, provide high titers (often in the g/L ranges) and are easily adapted to fermentation processes in simple and cost-effective culture media. Many approved non-glycosylated therapeutic proteins are currently produced in Saccharomyces cerevisiae and the methylotrophics Hansenula polymorpha and Pichia pastoris, including insulin (and analogues), growth hormone, hirudin (a leech-derived anticoagulant) and albumin. However, as they provide N- and O-linked

Plant-based systems

Genetically modified plants are especially well suited for the production of edible oral vaccines [71, 72]. Since they offer a significant potential in reducing the cost of manufacturing glycosylated and pathogen-free therapeutics, many plant-based expression systems are under development [73]. Currently, owing to low productivities, growing sufficient quantities of transgenic plants to meet clinical needs would require outdoor field growth. However, issues such as field containment (especially

Transgenic animals

The first therapeutic proteins were initially extracted from animal tissues or blood (e.g. insulin from pig pancreas, clogging factors from human plasma). It is almost nine decades later that ATryn® (human anti-thrombin α), the first licensed therapeutic produced in transgenic goat milk, was approved in Europe and the US (in 2006 and 2009, respectively) [89]. Other systems like eggs from transgenic hens are also under development [90]. It is noteworthy that the glycosylation profile may again

Conclusions

Advances in the production of biopharmaceuticals have evolved from cell engineering and bioprocess development of efficient expression systems that produce glycoproteins with appropriate structures for efficacious clinical application. Future developments in the expression systems will focus on the enhancement of productivity to meet the growing demand for these products and the refinement of their function so that the dosage requirements are minimized.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

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