Production Of Recombinant Proteins Biology Essay

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Stable gene expression technologies have been widely employed for the production of recombinant proteins from the mammalian cells. Owing to the drawbacks of these techniques (investment in time, resources) and increasing demand of the recombinant proteins in the market, it has become evident to develop a technique that is faster, cheaper and more reliable. With the advent of the transient systems in 1990s, many works have been done for the production of milligram to gram quantities of protein. Furthermore, the system has been widely developed for the evaluation of different proteins or variants among the same protein {Cho, 2003}.

The main features of the transient expression systems are:

Simple in construction of expression vectors

Time-efficient: generate products in days

Genetically stable and consistent

Multiple processing

Wide applicability among various host cells {Wurm, 1999}.

As 'process is product' is a key factor in the biopharmaceutical industry, the need of better techniques for DNA transfer is essential for the large-scale recombinant protein production in mammalian cells. Many engineered viral vectors such as adenoviruses, alphaviruses, baculoviruses, lentiviruses etc. are used for developing large amounts of recombinant proteins but they conjoin drawbacks such as restricted tropism, low transfection capacity and high cost of production {Pham, 2006}. Non-viral delivery systems have been developed such as calcium phosphate, electroporation, lipid reagents and have shown higher transfection capability with adherent as well as suspension adapted mammalian cells{}. Different cell lines have been transfected using the non-viral delivery systems such as COS (African green monkey kidney) {Blasley, 1996}, HEK-293(Human embryonic kidney) {Jordan, 1998}, CHO (Chinese Hamster Ovary) {Preuss, 2000}, BHK (Baby hamster kidney) {Wurm, 1999}. Non-viral delivery systems has made significant progress in developing better expression vectors, choice of cell lines , culture medium, transfection parameters and process scale-up{Pham,2006}.

Non-viral Gene Delivery/Transfection

The key factors of an ideal gene delivery system are: protect DNA from nucleases, translocate DNA into the nucleus of the target cell and have no adverse effect {Gao, 2007}. Viral delivery systems that have been developed for transfer of naked DNA into the mammalian cells are not very effective as compared to non-viral systems as former generally microinjects plasmid DNA into cytoplasm rather than nucleus and thus proved to be less effective or very little gene expression detected{}. Thus vehicles with nuclear translocation (coupled with DNA and promote entry into the cell) potential must be employed for large-scale production of the recombinant proteins.

There are two types of non-viral gene delivery systems carrier-free gene delivery (physical: needle injection, gene gun, ultrasound, electroporation) and synthetic vector based gene delivery (chemical: Synthetic polymers, lipids and proteins) {Gao, 2007}. Cationic polymers, cationic lipids, nuclear proteins (histones) have shown to be effective vehicles {Colosimo, 2000} {Haberland, 2005} but they are highly expensive for a large-scale production.. Zauner et al. has compared transfection capacity of two liposomal complexes known as Lipofectamine against poly (l-lysine) (PLL) and found polycations have greater transfection capability {Godbey, 2001}. Electroporation of plasmid DNA is also practised but this system requires development of more precision as it conjoins cell death by electrical discharge {Pham, 2006}.Calcium phosphate DNA co-precipitation and polyethyleneimine (PEI) DNA complexes are the most efficient vehicle systems because they are cost-effective and could be used for large-scale production of both adapted and suspension cell lines , they transfer DNA by forming complexes which are taken up by the cells through endocytosis {Wurm,1999}. These systems have shown increase in productivity of the recombinant protein from milligrams to grams with 100L scale-up in process.

Table: Gene Delivery barriers and multicomponent design of Non-viral vectors {Khanna, 2007}

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Calcium-phosphate( CaPi) Precipitation

Graham and Van der Eb developed calcium-phosphate DNA co-precipitation method in 1973 and since then it has been the most efficient transfection system in mammalian cells {Pham, 2006}. Precipitation of calcium-phosphate is formed by nucleation or particle growth and depends on the saturation, at low supersaturation the particle growth exaggerates but due to undersaturation the particle redissolves {Jordan, 2004}. Thus calcium phosphate method is dexterous and dynamic in nature, hence causes the change in characteristics of the particle by the continuous action of DNA on the precipitate.

CaPi transfection generally involves a two-step protocol:

Adding DNA to calcium chloride solution

Furthermore, mixing it with phosphate to form calcium phosphate DNA precipitates

Adding complexes to the culture medium {Graham, 1973}.

The CaPi DNA complexes enter the target cells by the process of phagocytosis and then high calcium concentration causes the rupture of the vesicles. It has been found that plasmid DNA translocate into the nucleus within 1h of transfection {Conrod, 1998}. Also work has been done to show transfer of 1000s of plasmid DNA into individual cell using calcium phosphate transfection. The concentration of the calcium phosphate is the main factor that effects its precipitate formation but other parameters such as temperature, pH and DNA concentration are also important {Jordan, 1996}. The table below describes the critical parameters that affect CaPi precipitate formation.

Table 1: Critical factors effecting calcium phosphate precipitate {Jordan, 2004}.

Polyethylenimine (PEI)

In contrast to the variety of non-viral vectors, polymers have been widely used for the transfection of mammalian cells due to their characteristics of easy preparation, purification and chemical stability {lungwitz, 2004}. PEI is a highly polycationic synthetic polymer that can be derived by condensation of 2-ethyl-2-oxazoline monomers or azridine and is available in linear or branched structures respectively. PEI is extensively used for its characteristics of low cost, potential for operation at scale, minimal cytotoxity and efficient transgene expression.

The branched PEI can be used for plasmid DNA delivery as well as for RNA or oligonucleotides and intact ribozymes {Aigner, 2002}. The efficiency of bPEI- derived vectors depends upon their characteristics of molecular weight, degree of branching, the cationic charge density, buffer capacity, polyplex capacity of DNA binding, content, size and absence or presence serum while transfection {lungwitz, 2004}. The bPEI with higher molecular weight shows higher transfection capability along with stable complex formation as compared to the bPEI with lower molecular weight {Godbey, 1999}. With the increase in N/P ratios increases the net positive charges on the complexes thus help in cell interactions and nuclear uptake {Oh, 2002}.

Fig: 1 Synthesis of branched polyethylenimine by condensation of azridine in aqueous solution {Harpe, 2000}.

Linear PEI (lPEI) distinguishes itself from the branched PEI from the nuclear uptake transfection efficiency and toxicity. It is synthesized by polymerization of either unsubstituted or two substituted 2 oxazolines with acid or base hydrolysis of N-substituted polymer (Fig2) {Brissault, 2003}. It has been observed with increasing quantities of amino groups in the copolymer also effects the buffer capacity and transfection efficiency of the lPEI complexes, also large particles of the lPEI shows higher interaction with cell surfaces {lungwitz, 2005}. In comparison with bPEI , lPEI showed improved cell viability, promote nuclear localization and increased transfection efficiency {Zou, 2000}.

Fig2: Synthesis of linear polyethylenimine {Brissault, 2003}.

The intracellular trafficking pathways have revealed the understanding of the PEI- mediated gene transfer. Plasmid DNA packed with PEI is transferred to the cell by receptor-mediated endocytosis and thus can be used for labelling polyplexes with targeting moieties (Fig3). Cationic polyplexes interact with the negatively charged membrane glycoproteins, proteoglycans, and sulphated proteoglycans and start cell internalization via adsorptive or fluid-phase endocytosis {lungwitz, 2005}. The reactivity between cell and polyplex could be enhanced by increasing the net positive charge on the surface of the polyplex (change in concentration or incubation time) {Bieber, 2002}. The transfection of cell by PEI-DNA polyplexes starts within 3h after their incubation to the medium { Kristensen, 2001}.

The PEI-DNA complexes succeed through the endolysomal pathway and escapes efficiently endosomes/lysosomes by the 'Proton Sponge' effect by which protonable amino nitrogen present at every third atom of PEI buffer the endosomal environment and hence arrest acidification and fusion with the lysosomes and eventually swell the vesicles to release plasmid DNA into the cytoplasm {Akinc, 2005}. PEI-DNA complexes have shown high gene expression resulted in understanding of the fact that it generally transfers plasmid DNA from cytoplasm to the nucleus {Pollard, 1998}. The rate of PEI-mediated transfection and transgene expression depends upon the cell-type and cell culture medium {Kristensen, 2001}.

Experiments have been performed to understand the mechanism of protection of DNA by PEI and it has been stated that it protects DNA from degradation by DNase1 and DNase 2, protecting the DNA from degradation would lower gene expression level so protecting it while delivery is an important parameter {Godbey, 2001}.

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Fig3: Schematic representation of the gene transfer using ligand-decorated non-viral vectors {Kircheis, 2001}.

Transient (Non-Viral) Transfection of Mammalian Cells

Transient gene expression is a rapid method of direct conversion of recombinant DNA into recombinant protein. It has the following advantages over stable gene expression:

Expression over few days/weeks

Transfect in many copies of DNA

Transcription without integration into host genome

No subculturing

No selection for maintenance of expression vector

Quickly generates protein for protein assays, characterization etc.

Rapid for milligram to gram amounts

Repeat transfection for repeat protein batches

Cost effective

For large scale transient production of recombinant protein in mammalian cells, the key factors that should be taken into account are:

The cell line

The expression vector

The plasmid DNA quality

The transfection vehicle

The culture medium {Durocher, 2002}.

Large scale recombinant protein production depends upon the transfection capacity and for the development of such system requires the plasmid DNA expressing reporter proteins like secreted alkaline phosphatase (SEAP) and green fluorescent protein (GFP){Pham, 2006}.

Mammalian cell lines

For the efficient yield of recombinant protein the cell line should be easily transfected. Many different cell lines have been used in past such as COS and CV1 derived from African green monkey for the small scale transient production. The main drawback with these cells is that they show less productivity in contrast with other cell lines. Nowadays, the cell lines that are industrially accepted for the production of recombinant protein are Chinese hamster ovary (CHO), mouse myeloma, baby hamster kidney and human embryonic kidney 293 (HEK293).

CHO

CHO cell line is widely used for the production of recombinant proteins using stable gene expression but less work has been done with transient transfection. Initial work with these cell lines using CaPi-mediated transient transfection has not shown satisfying results as but later work with PEI- mediated transient transfection has given improved productivity of upto 10mg/L for antibodies {Wurm,1999}. CaPi requires serum and osmotic shock for the production of high levels of protein but show low level of gene delivery with suspension-adapted CHO cell lines.

The highest transient yields of 80-100mg/L have been reported by transfecting CHO cells with linear PEI (25 kDa) {Wulhfard, 2008, 2010} {Ye, 2009}. In 2004 Derouazi et al. has worked on the serum free suspension adapted CHO cells and scaled up the process in 20L stirred bioreactor for the production of recombinant protein using PEI-mediated gene delivery (Fig.4) {Derouazi et al., 2004}.Recently Rajendra et al. has reported volumetric productivity of recombinant antibody of upto 300mg/L by optimizing the transient transfection parameters in suspension-adapted CHO cells {Rajendra, 2011}.

Fig4: Transfection of CHO cells in 3L and 20L bioreactor transfected at a DNA: PEI ratio of 1:2 {Derouazi et al., 2004}.

HEK293

Human embryonic kidney 293 cell line is derived from transformed HEK cells with DNA of adenovirus 5 {Graham, 1997}. These cells have been widely used for the production of recombinant protein using transient gene expression as they are highly susceptible to most gene delivery systems and can grow in serum free suspension adapted medium. There are two different types of HEK 293 cells namely 293E (express EBNA1) and 293T (express SV40 large T-Ag) but both support episomal replication. The SV40 ori Tag system in 293 T cells does not promote high levels of transient production in these cell lines because of simultaneous occurrence of two process of gene replication and expression {Pham, 2006}. In contrast 293E cell lines are widely developed for large scale transient production using EBV oriP plasmids {Pham, 2003, 2005}. HKB11 is a new hybrid cell line developed by fusing 293 with Burkitt's lymphoma cell line and reported 20 fold high transient gene expression using oriP/EBNA1 based plasmid {Cho, 2003}.

CaPi and PEI are extensively used gene delivery systems in HEK-293 cell line and former has been utilized for scaled up to 100L in bioreactors {Baldi, 2005}. Durocher et al. used model proteins such as SEAP and GFP to design expression vector and monitor transfection efficiency in 293E cell using 25kDa PEI (Fig5) {Durocher et al., 2002}.

Fig 5: Transfection of different HEK293 cell lines with DNA: PEI ratio at 1:2 for SEAP expression and transfection efficiency {Durocher, 2002}.

Expression Vectors

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