Engineered antibodies remain the most common engineered product category to gain approval, with one chimeric and five humanized products entering the market over the last five years (Table 1). The most novel engineering approach witnessed in latter years is the acylation of insulin (Levemir, Novo Nordisk, Bagsvaerd, Denmark). The principle engineering feature of Levemir (insulin detemir) is the covalent attachment (acylation) of the C14 fatty acid (myristic acid) to the side chain of the lysine residue found at position 29 of insulin's B chain. Human serum albumin (HSA) harbors three high affinity fatty acid binding sites and the engineering rationale was to facilitate tight but reversible binding of the insulin to HSA, both at the site of injection and in the blood. This occurs in practice, ensuring a constant and prolonged release of free insulin, which gives the product an activity duration of 24 hours. Levemir was the first and, so far, only approved biopharmaceutical to be engineered in this way.

The development of another engineered product, Somavert (pegvisomant, Pfizer, New York, NY), is notable because it involves PEGylation combined with amino acid substitutions (nine in all). Somavert is indicated for the treatment of acromegaly, a rare endocrine disorder characterized by elevated blood hGH concentrations. The product is a hGH analogue capable of binding to the hGH cell surface receptor without triggering an intracellular response. Somavert acts as a hGH antagonist, reducing the effects of endogenous hGH while PEGylation increases the product's serum half-life.

Several disease conditions are triggered or acerbated by the inappropriate over expression of a gene product. Several biopharmaceuticals are now approved, which treat such conditions by inhibiting the activity of the overexpressed protein. In addition to Somavert, antibody-based products such as Kineret (Amgen, Thousand Oaks, CA) and Erbitux (ImClone Systems, New York, NY) function by direct binding and inactivation of the target protein. On the other hand, products such as Humira (Abbott, Chicago, IL) and Enbrel (discussed before) bring about their effect when the biopharmaceutical binds to the protein's target receptor, acting as an antagonist.

Products engineered by amino acid sequence modification or by attachment of polyethylene glycol or some other chemical moiety will continue to gain approval. Because of increasing appreciation of the importance of post-translational modifications (PTMs) on the therapeutic characteristics of many proteins, knowledge-based alteration of native PTM profiles is now receiving more attention.²

The majority of protein-based biopharmaceuticals bear some form of PTM, with glycosylation being the most complex and widespread modification. It is estimated that up to 50% of all native human proteins are glycosylated and that 1–2% of the human genome encodes proteins that contribute to glycosylation. Approximately one third of all approved biopharmaceuticals are glycosylated, with antibodies as well as blood factors and some hormones (e.g., gonadotropins and erythropoietin) representing the most prominent categories. A protein's glycocomponent can have many significant influences on its therapeutic characteristics, including influencing stability, ligand recognition or binding, serum half-life, and immunogenicity.³

Cerezyne (as discussed earlier) and Aranesp (Amgen, Thousand Oaks, CA) are two products for which glycocomponent has been engineered and are already on the market. Aranesp is a recombinant human erythropoietin (EPO) indicated for the treatment of anaemia. Produced in a Chinese hamster ovary (CHO) cell line, the product displays an increased carbohydrate content when compared to native EPO. The native molecule contains three carbohydrate side chains whereas Aranesp contains five. This increased carbohydrate content extends the product's half-life, facilitating once weekly or once fortnightly administration schedules.

Protein glycosylation occurs naturally in the endoplasmic reticulum (ER) and the Golgi, and is undertaken by a multienzyme pathway comprising 2–3 dozen glycosyltransferase and glucosidase enzymes.⁴ The characteristic heterogeneity associated with a glycocomprotein's glycocomponent is mainly a reflection of incomplete glycosyl processing. Furthermore, different cell types vary in terms of the exact glycosylation pathways they harbor and this can have profound implications on the suitability of any given cell type as a production platform for therapeutic proteins. For example, yeast-derived glycosylation patterns tend to be of a high mannose type, which generally confers a short circulating half-life on the protein in humans. Plant-based glycosylation tends to be more complex and extensive when compared with mammalian-derived patterns and contains sugar motifs that are immunogenic in humans.