Engineering cells and proteins – creating pharmaceuticals
Posted: 5 September 2014 | | No comments yet
Pharmaceutical biotechnology is big business; it currently consists of 1/6 of the total volume of the pharmaceutical market and continues to grow steadily. Expression of therapeutic proteins is mainly done in living cells, although ‘cell free protein synthesis’ (CFPS) or ‘in-vitro transcription translation’ (IVTT) is beginning to emerge as an alternative for commercial production. In this article we will highlight some of the more recent advances in protein expression systems for the production of pharmaceutical proteins. We will also discuss current trends in the engineering of pharmaceutical proteins with improved properties.
Pharmaceutical protein production
While proteins of interest are still isolated from natural extracts, the advent of modern molecular biology techniques has made recombinant protein expression the mainstream methodology for pharmaceutical production. The main drivers for the use of recombinant proteins are: low availability of native protein (e.g. the annual use of amylase and xylose isomerase is over 95,000 tons each); livestock infections for the production of vaccines and subsequent economic loss; immune responses to animal proteins after injection (e.g. insulin); and reproducibility of protein production in relation to its quality.
Since the implementation of recombinant DNA in the early-1970s, proteins have been expressed in many different organisms and (derived) cell types, such as bacteria, yeasts, moulds, insects, protozoa, mammals, plants, transgenic plants and animals and with the use of cell lysate in CFPS. During recombinant protein expression a gene is introduced in an organism (or its derivative) followed by constitutive- or induced-translation and transcription. Choosing the correct expression system is protein dependent and factors such as protein quality, functionality, production speed and yield are most important.
As of 2014, overall trends in recombinant protein production show an increase in the use of Chinese hamster ovary (CHO) and yeast cells, while there has been a decrease in the use of other mammalian and Escherichia coli (E. coli) systems. Production of non-glycosylated proteins is conducted mainly in bacteria or yeast (45%, May 2014), while the production of glycosylated proteins is carried out predominantly in CHO cells (29% compared to mammalian cells, insect cells and transgenic-animals and -plants at 26%. More than a decade ago about 20 pharmaceutical proteins were produced by transgenic technology for clinical trials14, however the high developmental costs for these production systems clearly hindered the advancement of this method. Other emerging systems in the market include the use of P. pastoris and H. polymorpha – both of which are discussed hereafter.
Escherichia coli (E.coli)
During the 1980s, E. coli was the dominating organism of choice for recombinant protein production15, and E. coli derived pharmaceutical proteins still account for 29% of marketed biologics. Due to the long history of its use and deep understanding of the E. coli genetics, much progress has been made in the engineering of E. coli strains for the production of proteins an plasmids10,12,16-19.
E. coli is a good choice for the first effort to produce a recombinant protein15, and a consensus protocol has been developed recently as a guide to start E. coli protein expression20. It can be cultivated with a relatively cheap defined media (e.g. glucose, ammonia phosphate and some minerals) and strategies for low cost production have been developed15,21. E. coli has clear advantages; however drawbacks include the incorrect formation of multiple disulfide bonds, non-extracellular production, and limited PTMs (e.g. glycosylation is not possible). Moreover, E. coli produces pyrogenic endotoxins, although various methods can be employed for their removal22. Typical protein yields are in the range of 20-400 mg/L medium (see Figure 2).
Saccharomyces cerevesiae (S. cerevesiae), Pichia pastoris (P. pastoris) and Hansenula polymorpha (H. polymorpha) are currently the only three expression strains used in the production of marketed pharmaceutical proteins (see Figure 1). Yeasts are single-celled eukaryotic fungal organisms, and the major advantages of its use are: high yield (see Figure 2); stable production strains; durability; cost effective; high density growth; high productivity; suitability for production of isotopically-labelled protein; rapid growth in chemically defined media; product processing similar to mammalian cells; can handle S–S rich proteins; can assist protein folding; and its ability to glycosylate proteins12. Proteins that do not fold correctly in E. coli or that require glycosylation for its function are often produced in yeast.
Like E. coli, S. cerevesiae has a comparatively well-characterised genome and since the organism has no pathogenic properties it is classified as GRAS (generally regarded as safe). In general, S. cerevesiae is good alternative to E. coli, however the complex glycosylation patterns are often unacceptable for mammalian proteins because the O-linked oligosaccharides contain only mannose whereas higher eukaryotic proteins have sialylated O-linked chains. Additionally, N-linked sites are over-glycosylated, high mannose type structures which can lead to immunological responses and rapid clearance rates12,23.
The methylotrophic yeast P. pastoris is an effective and versatile system for the expression of heterologous proteins24. Its growing popularity can be attributed to several factors: (i) easy accessibility to well-established molecular biology techniques developed for S. cerevesiae; (ii) its ability to express proteins at high levels (either intracellular or extracellular); (iii) performs PTMs (i.e. glycosylation, disulfide bond formation, and proteolytic processing); (iv) the expression system is available as a commercial kit; (v) tightly regulated promoter systems were developed (e.g. AOX1, up to 1000 fold up-regulation); and (vi) capable of enduring high cell density cultivations in a bioreactor and the preference of a respiratory- over a fermentative-growth model25,26. Glycosylation is less extensive in P. pastoris than in S. cerevisiae27. N-linked high-mannose oligosaccharides are usually up to 20 residues. Human-like hybrid and complex N-glycans were generated in P. pastoris28,29, and these systems are currently optimised30-32.
An in-depth review of H. polymorpha by Gotthard and co-workers highlights its strengths for pharmaceutical protein production33. In short, the generation of recombinant H. polymorpha strains typically employs vectors, traditionally circular plasmids, which are mitotically stable being integrated into the genome of the host. Integration over a number of generations may result in strains with as many as 100 integrated plasmids present in tandem repeats. A major advantage of H. polymorpha is that proteins can be secreted into the media, or into a pre-selected cell compartment, such as the peroxisome, the vacuole, or targeted to the cell surface. For secreted phytase, product levels of up to 13.5 g/L have been obtained.
Compared to S. cerevisiae, the high mannose glycan chains of N-linked oligosaccharides generally appear to be much shorter in H. polymorpha; typical oligosaccharide species attached to the recombinant protein have Man8-12 GlcNAc2-structures without terminal α-1,3-linked mannose residues. Therefore, the outer chain processing in the N-linked glycosylation pathway in H. polymorpha appears to be similar to that in P. pastoris, with the addition of shorter mannose structures and lack of any terminal α-1,3-linked mannose residues.
Today over 50% of all recombinant protein pharmaceuticals are produced in mammalian cells (see Figure 1). Driven by the need of PTMs of pharmaceutical proteins, these relatively complex expression systems have increased their yields tremendously due to developments in bioprocess engineering, media optimisation, and strain engineering since the 1980s34 . While adherent cell cultures are used in industrial setting, suspended cell cultures (e.g. CHO cell- and BKH cell-cultures), and for even more increased yields, extended batch cultures and perfusion processes in phase III- or the production-phase are most abundant.
While biopharmaceuticals requiring relatively little or no PTM’s can be made in platforms ranging from E. coli to mammalian cells, glycoproteins and other pharmaceuticals requiring more complex PTM’s, e.g. glycosylation, mammalian cell lines are the only viable option. This is because the final glycosylation pattern of recombinant proteins is determined by the expression platform; it is well known that there are significant differences in glycan structures between proteins expressed in human, mammalian and yeast cells (see Figure 3). As such, the majority of therapeutic glycoproteins are expressed in mammalian cell lines (e.g. CHO cells)35.
However, the drawbacks to mammalian expression include the number of glycoforms that are expressed and the differences in protein glycosylation between different mammalian cell lines36. For example, glycoproteins expressed in some cell lines, including CHO cells, contain terminal Neu5Gc (N-glycolylneuraminic acid) rather than the human Neu5Ac (N-acetylneuraminic acid). These non-human sialic acids moieties may affect immunogenicity as antibodies against the Neu5Gc have been identified35. Expression in some cell lines, e.g. epoetin delta in human fibrosarcoma cell line HT-1080, can result in glycan chains with no terminal Neu5Gc residues37, however this method is not always a possibility. One way to circumvent this problem, as well as other issues with PTM such as disulfide bond formation is to engineer cell lines to express proteins with the correct PTMs38.
While transient gene expression in mammalian cells is a maturing technology it is not yet approved for pharmaceutical protein production39. Engineering mammalian gene switches and post-transcriptional control (e.g. via RNA aptamer-intramer fusions) will give light to new expression strains: (i) as part of autologous cell therapies, gene circuits encode computational operations that can be programmed by intracellular signals to execute specific tasks, (ii) cell implants consisting of engineered allogeneic or xenogeneic mammalian cells could be plugged into the metabolism of patients to sense and respond to specific biomarkers40.
In addition, the trypanosomatid protozoa Leishmania tarentolae (a nonpathogenic parasite) is under investigations as an alternative production-system for pharmaceutical proteins due to its complex PTMs and significantly easier cultivation requirements than mammalians cells41-45.
Finally, with the first successful commercial production of antibodies in CFPS for the pharmaceutical industry by Sutro Biopharma, CA, USA in a 100 L tank6,7,46 the road is open to start producing more pharmaceutical proteins in not only E. coli lysates, but also eukaryote cell lysates.
Engineering of biopharmaceuticals
Protein engineering can span a wide field of modifications and conjugations including gene manipulations, glycosylation, PEGylation, albumin fusion, PASylation, fatty acid conjugation, amidation, disulfide bond shuffling, and Fc fusion. Here focus on protein engineering methods applied post-expression for improved stability, solubility, potency, reduced immunogenicity, increased proteolytic resistance, and/or improved serum half-life.
N-linked glycosylation is known to mediate a wide spectrum of functions; the most important of which (in terms of protein pharmacokinetics) include activity, stability, solubility, folding, immunogenicity and in-vivo half-life. As such, pharmaceutical glycoproteins that are expressed non-glycosylated (i.e. in E. coli) can exhibit impaired in-vivo activities and in some cases complete loss of in-vivo activity is observed. Recombinant human interferon-β (rhIFN-β) is an example of a naturally glycosylated protein that is active in-vivo when expressed from E. coli (i.e. non-glycosylated)47. However, while both the glycosylated and non-glycosylated variant is available as a biopharmaceutical, the solubility and activity of the non-glycosylated variant is impaired in relation to its glycosylated counterpart. This example highlights the importance of glycosylation in the context of pharmaceutical proteins and the ability to control glycosylation is continually progressing.
Approaches to glycoengineering can vary widely. The most highly cited example of successful glycoengineering is darbepoietin alfa, the long acting erythropoietin derivative that mediates red blood cell production for the treatment of anemia in association with chronic kidney disease (CKD) and/or chemotherapy treatment48,49. Epogen/ Procrit was the first erythropoiesis-stimulating agent (ESA) to be approved in 1989 and the active ingredient, epoetin alfa, is recombinant human erythropoietin expressed in CHO cells. Darbepoietin alfa, marketed as Aranesp by Amgen in 2001, has two additional N-linked glycosylation sites engineered into the peptide backbone. The resulting increase in sialic acid content gives Aranesp a lower affinity for the EPO receptor (Aranesp IC50 of 1.05 compared to epoetin alfa IC50 of 0.54 ng) but an increased serum half-life (Aranesp has a t1/2 of 26 hours compared to 8.5 hours for epoetin alfa)50. As a result, Aranesp can be administered less frequently than epoetin alfa products.
While Aranesp is the only approved drug utilising this methodology, recent examples of ‘hyperglycosylated’ proteins with an improved half-life include coagulation factor IX (four additional glycosylation sites were introduced to yield a variant with a 2.4 fold increase in half-life)51, recombinant human IFN-alpha2 (4- and 5-N-linked IFN variants showed a 25-fold increase in half-life compared to the non-glycosylated hIFN-alpha2)52, human alpha 1-antitrypsin, A1AT (one additional glycosylation site at various positions yielded variants with up to a 3.6-fold longer serum half-life)53, and follicle-stimulating hormone, FSH, (introduction of 4 additional N-linked glycosylation sites yielded a variant with a 2-fold increase in half-life)54. Interestingly, in the case of the FSH, both N- and O-linked glycosylation sites were introduced into the protein. While the N-linked variants showed a significantly increased half-life compared to the O-linked variants, it highlights the use of a relatively less understood and underexploited form of PTM54. Other approaches to glycoengineering include the generation of afucosylated glycoproteins and antibodies, enzymatic cleavage/ addition of glycans and glycoPEGylation55-60.
PEGylation is by far the most prominent conjugation strategy currently used to improve the pharmacokinetics of therapeutic proteins. PEGylation was originally introduced as a method to prevent immune responses in patients; however since then it has emerged as a versatile tool to increase solubility, reduce toxicity and prevent protein aggregation.
The currently available PEGylated biopharmaceuticals span a range of therapeutics with modifications ranging from the addition of a single PEG moiety up to the addition of 9 PEG moieties (see Table 1)61-74. Despite the success of PEGylated proteins in the market, a shortcoming to PEGylation technology is the limited control over the site of ligation; PEG is most often ligated to proteins via accessible amino groups, either the N-terminus or surface lysine residues. Approaches to achieve site specific conjugation revolve around the exploitation of naturally occurring ‘handles’ for ligation such as the modification/introduction of a ‘free’ cysteine75-78 and the use of naturally occurring glycosylation sites, e.g. glycoPEGylation.
In some cases PEGylation can result in lower in-vitro activities as the modification can be within a receptor binding region or active site; however, this loss of in-vitro activity is usually compensated for by an increase in in-vivo activity as a result of increased circulating half-life79-83. This is the case for Mircera, a PEGylated epoetin beta that acts as a continuous erythropoietin receptor activator80. After expression in CHO cells, epoetin beta is chemically modified with a single 30kDa PEG. This modification results in a lower affinity for the EPOreceptor but an increased half-life (t1/2 of 134 hours compared to 8.5 hours for epoetin alfa and 26 hours for Aranesp)82.
GlycoPEGylation, successfully applied to Lonquex (lipegfilgrastim), is the combination of a glycoengineering and PEGylation technology. Lonquex is granulocyte colony stimulating factor (G-CSF) which has been enzymatically modified with a 20-kDa PEG-sialic acid derivative. To achieve this, G-CSF is expressed in E. coli and the 20-kDa PEG-sialic acid derivative is transferred to the unused natural O-linked glycosylation site with a truncated N-acetylgalactosaminyltransferase isoform64. This approach has also been applied to, interferon-alpha2b (IFN-α2b), granulocyte/macrophage colony stimulating factor (GM-CSF), and recombinant activated factors VII, VIII and IX84-87.
Incorporation of unnatural amino acids
Another emerging field in protein engineering for the site-specific conjugation of functional moieties to biopharmaceuticals is the incorporation of unnatural amino acids (uAA). The expansion of the genetic code is essentially the recoding of an antisense codon to biosynthetically incorporate unnatural amino acids into protein scaffolds88,89. This work was first pioneered in an E.coli system by Shultz and co-workers, where the specificity of the Methanococcus jannaschii tyrosyl tRNA synthetase was redirected towards uAAs88,89.
The essential components for the incorporation of uAA’s are an aminoacyl tRNA synthetase (aaRS) that charges a specific tRNA with a uAA and an orthogonal tRNA anticodon mutated to recognise a nonsense codon e.g. the amber stop codon TAG. The aaRS-tRNA must be orthogonal with respect to the expression system – the aaRS must not recognise any host cell tRNA and the tRNA must not be aminoacylated by any host aaRS.
This approach has been utilised in E. coli-, mammalian-, yeast-cells and cell free platforms87-97. Current restrictions associated with uAA incorporation include low overall product yield, the efficiency of the uAA incorporation and the subsequent conjugation efficiency. Despite this, there have been a number of successful examples of conjugated pharmaceutical proteins developed using this methodology, especially for the generation of antibody-drug conjugates93,95-100. The pharmaceutical company Ambrx has recently published the production of Antibody Drug Conjugates in CHO cells using uAA incorporation via their EuCODE technology86. Expression yields for this system were on the 1g/L scale with an overall conjugation yield of 95%.
Over the last 20 years more complex expression systems have come into use. With rapid developments in post-translational engineering and strain development to produce even better defined products, faster approval of biopharmaceuticals can be expected, especially if transient cell expression and CFPS production methods are approved. After the revolution of modern molecular biology to the field of biopharmaceuticals it is time for a PTM revolution via synthetic biology and chemical conjugations.
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Marco Casteleijn is working at the Centre for Drug Research at the Faculty of Pharmacy of the University of Helsinki in Finland. He obtained his Ba in Life Sciences in Utrecht, the Netherlands. Before his PhD, Marco was Test Supervisor and Researcher at the National Institute for Public Health and the Environment at the Dutch drug regulatory offices for biological drug release, and worked as a technician for a fresh water provider (microbiology and analytical chemistry) and in the cosmetics and food industry. Marco obtained his Msc in Biochemistry and his PhD in Bioprocess Engineering at the University of Oulu in Finland where he worked in protein engineering projects on industrial enzymes. He is currently working on pharmaceutical proteins: development and delivery. He has published several peer review articles on protein engineering, bioprocess development, and industrial enzymes. In addition, Marco has a background in quality assurance, was a board member of the Biocenter Oulu graduate school, and is on the scientific advisory board of a German Biotech company. Email: [email protected].
Dominique Richardson graduated with a Master of Chemistry from the University of Durham in 2009. She subsequently undertook her PhD studies in protein biochemistry at the University of Manchester with Prof. Sabine Flitsch. Upon completing this in 2013, she joined the University of Helsinki where she currently works as a postdoctoral researcher with Prof. Arto Urtti on the development of a ‘capture and release’ cell-free platform for the expression and screening of pharmaceutical proteins.