Mammalian Cells in Antibody production

History

Mammalian cells as the name suggests are found in mammals. Unlike plant cells they do not have cell wall and hence they are very sensitive to osmotic pressure [1].

Mammalian cell cultures have been in use for the last 100 years when it was initially used for making human viral vaccines. Only in the last few decades has there been an increase in the use of mammalian cells for recombinant protein technology and monoclonal antibody production [3].

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The first therapeutic protein to be produced was recombinant insulin (Humulin by Genentech) in the year 1982 from E.coli [3]. Since then the technology has improved and this has resulted in an increase in pharmaceutical production.

Due to availability of new techniques new cell lines have been produced or engineered. Each of them has its advantage and disadvantage which will be discussed in the later sections.

Early in the 1970s and 1980s E.coli ( Bacterial expression system) was used for the production of simple pharmaceutical molecule like Insulin which did not require Post Translational Modification (PTM). Later it was realized that complex proteins requiring

PTM required new expression systems for production [3].

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CHO cells

CHO cells or the Chinese Hamster Ovary cells are a type of mammalian cell line which is the most widely preferred for pharmaceutical production. The use of CHO cells dates back to 1919, when it was used to type pneumococci. In 1957 Theodore T. Puck obtained the original CHO cells from a female Chinese hamster [4].

 

The reason for CHO cells being used widely in this sector is:

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• The proteins produced from them has similar glycosylation pattern with respect to humans [6].
• They get easily adapted to the suspension condition and also to protein free media.
• High growth rate and productivity.
• Gene manipulation can be done easily.
• It is the best defined cell line. Its characteristics have been extensively studied [5].

Some of the draw backs of using CHO cells are [7] :

• Costly to setup.
• It can propagate virus and prions.
• Large time scale.
• Inability to control N-glycosylation.
• Glycoprotein’s show glycan heterogeneity.

The monoclonal antibodies produced contain fucosylated complexes( G0,G1,G2). This reduces Antibody Dependent Cell Cytotoxicity (ADCC).

In case of serum free media it is a difficult task to define the chemical media.

1.2) Other expression systems in use [6] [7] :

Expression system: Bacteria

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• Advantages: Cheap, Fast and optimum growth.
• Disadvantages: Used for simple molecules only, minimal PTMs.

Expression system: Yeast

• Advantages: High titres, easily adapted to fermentation process.
• Disadvantages: Lack of tyrosine O-sulfation (a kind of PTM).

Expression system: Insect

• Advantages: High productivity
• Disadvantages: Glycosylation different from humans, demanding culture conditions.

Expression system: Plants

• Advantages: Suitable for edible
• Advantages: Vaccine, more productivity.
• Disadvantages: Proteolytic degradation and gene silencing, field containment, High production costs and public acceptance.

Some of the other mammalian cell line used for production are: Baby Hamster Kidney cells (BHK 21), Hybridoma cells, Mouse myeloma cells(NS0 cells), Human Embryo Kidney cells(HEK 293) [6]. Human cell line: PER C6 (immortalized healthy embryonic retina cells) [3].

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The table above shows the use of new expression systems for production of biopharmaceuticals. Expression systems like bacteria especially Escherichia coli (E.coli) and Bacillus subtilis which is being considered as an alternative to E.coli [8] along with yeasts which includes Saccharomyces cerevisiae are mostly used for production of non glycosylated proteins which are basically simple proteins like: Insulin. Although, attempts are being made to genetically modify yeasts in order to produce human like glycosylated proteins. Here it is important to mention that a new species of yeast Pichia pastoris and Scizosaccharomyces pombe is being currently employed for recombinant production [8].

Recent recombination strategies of producing glycoprotein’s with humanized N-linked glycosylation structures has made yeast an attractive expression system [9].

 

Filamentous fungi are also another excellent expression system for producing recombinant protein [10]. Aspergillus niger (Fig 2) and Aspergillus oryzae (Fig 3) are the commonly used strains. As they have been used for making food eatables like miso, tempeh they are Generally Regarded As Safe (GRAS). Although quantity of protein produced is very less when compared to commercial scale [9].

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Strategies like solid-state culture method and fusion gene system might solve the problem. Also further understanding of the secretory pathways will be of immense help [9].

Insects infected by Baculovirus act as another form of expression system. One of the big advantages it has over especially mammalian cells is that it does not infect vertebrates and hence has no safety issues. It has been shown to produce virus like particles (VLP’s) and vaccine antigens [11]. Cost and different glycosylation pattern makes it less attractive to use. Also the fact that the infected insect cells die after a finite time means that the process of transfection has to be repeated which makes the process time consuming.

Recently transgenic animals have been used as an expression system for producing proteins from animal products like milk [12]. Ethical issues, high cost and low industrial production make this system impractical to use. Also the protein produced lacks some of the important sialic acids associated with their human counterpart. This could lead to fatal immune response [7].

Similarly use of transgenic plants has also become increasingly popular. Protein made is stored in organs like chloroplast. Even there are attempts being made to produce edible vaccines [14]. Being cost effective might make them a threat to mammalian cells in the future as a popular expression system. Having said that, plants differ from human pattern of glycosylation and hence the regulatory issues act as the biggest hurdle for plant based pharmaceutical products reaching the market. The cost of maintenance of the farms is again a problem to deal with.

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Many companies have tried their luck in the sector and have failed, Meristem Therapeutics, Chlorogen Inc are a few to name [14]. This shows that the sector is still underdeveloped and certain areas like glycosylation pattern need to be considered before expecting to make profits from the sector.

Prodi Gene which was considered as one of the biggest players in this sector best illustrates the fact that a small mistake made can be catastrophic for the company itself. Fine of $250,000 was imposed on the company due to contamination issues. Also some 500,000 bushels of harvested were contaminated and had to be thrown away [14]. The case clearly emphasis the point that a lot of research has to be carried on before companies can except plant as an alternative to well established mammalian cells.

Hence there is a need to select the appropriate expression system keeping in mind that the product will have a large impact on consumer’s health.

2) CHO cell a factory for protein production.

Table 2 depicts the different types of biopharmaceuticals produced from CHO cells over the years. Ranging from proteins to monoclonal antibody the CHO cells have come a long way. The thing that makes CHO cells such a remarkable expression system is that it can easily adapt to variety of culture conditions and also the fact that it can be manipulated by genetic engineering with relative ease [15].

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That CHO cells are the workhorses of biopharmaceutical industry has been proved by the fact that out of the 58 products approved between 2006 and June 2010, 32 were produced from mammalian cells predominantly CHO cells. Thus accounting for approximately 55 percent of the products [16].

2.1) CHO cell lines and their production.

From the original CHO cell line obtained in 1957, number of other cell lines have been produced. A glysine-dependent strain (CHO-K1) was derived which was mutagenized to produce CHO-DXB11 [17]. This cell line does not have Dihydro Folate Reductase (DHFR) activity as one of the alleles is deleted and the other mutated. Similarly proline dependent CHO-pro3- strain was mutated to produce CHO-DG44 a cell with both alleles of DHFR deleted [18].

A new host cell line for production of recombinant antibodies has been derived by Lonza and BioWa in collaboration. The cell line is named Potelligent R CHOK1SV. It has been made by using the GS systemTM and the CHOK1SV with PotelligentR Technology of BioWa. So what are the advantages with this cell line [19]:

Grows in chemically defined medium hence the animal free medium can be used. Minimizes regulatory issues.

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Exhibits good growth characteristics:

i) High viable cell concentration.

ii) High viability.

c) High productivity.

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d) Easy to scale up.

e) Process developed and easy to work with.

 

So how are these cell lines derived? The commonly used method is the DHFR selection method but the method is time consuming usually taking around six months and is very laborious.

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a) Transfection

This is the first step in which a vector construct consisting of the gene of interest and the dhfr gene is inserted into the host cell. This is made possible by techniques such as calcium phosphate precipitation, electroporation, lipofection. Once inside, the DNA gets integrated randomly. The random integration though is not quite desired as this leads to variable expression and stability [3]. Hence strategies of target gene insertion are being employed to counter such problems.

b) Selection

Once the DNA is inserted, the transformed cells are selected by growing them in absence of glysine, hypoxanthine and thymidine. This results in the survival of only the transformed cells while killing the no transformed ones.

c) Recovery

The transformed cells are selected from the pool of cells for the process of amplification.

d) Amplification

The selected cells are subjected to high levels of methotrexate (MTX) of around 0.5-1μM. This step helps to increase the copy number which in turn increases the concentration of the recombinant protein hence the name ‘AMPLIFICATION’.

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e) Screening

Now individual clones with maximum productivity are selected. This is necessary because the pool consists of clones with variable production capacity. Series of dilution carried out in multi well plates helps select a group of viable clones.

f) Expansion

The selected clones are undergo several rounds of passaging so as to increase the size of the clones.

g) Growth evaluation

The individual clones are tested under conditions mimicking those found in large scale industrial reactors. The important parameters are compared and a single clone which satisfies them is selected.

h) Cell banking

The selected cell line is than stored in frozen vials in liquid nitrogen for future use in clinical trials or even as production lines.

Another method which is also becoming popular for production of new CHO cell lines is Glutamine SynthetaseR system. This system was developed by Lonza.

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The advantage of using this system over the DHFR system is that it reduces the amount of nitrogenous waste produced in the form of ammonia. Also the process of amplification is absent in this system (Doubt). Hence it requires less time when compared to the other system.

3) Blockbuster products of CHO cells.

In the previous section a list of commercially important biopharmaceuticals produced from CHO cells was mentioned. In this section few of those will be discussed briefly. The market capture by some of the major protein therapeutics is as followed.

3.1) Erythropoietin

It is important for the production of red blood cells and its deficiency causes anaemia. When administrated artificially it is classified as erythropoiesis stimulating agent (ESA). New ways of increasing the quantity of erythropoietin is being researched upon. For instance cytopilot fluidized bed bioreactor has been found to increase epoetin production. Cytopilot bed reactor is a large scale production designed for epoetin production. The cells are grown in microcarriers where they grow without any stress [21].

The total production and cell density attained was double than that of other cultures. This system has industrial implications if further investigated.

There has been an effort to increase the amount of sialylation and galactosylation by using recombinant CHO cells with α2,3-sialyltransferase and β 1,4-galactosyltransferase for sialylation and galactosylation respectively. This enhances the pharmacokinetic properties of the epoetin [22].

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3.2) Coagulation Factor VIII

It is required for the formation of blood clot and its deficiency leads to haemophilia A [23]. Research is being carried out to increase its production from CHO cells by using expression vectors with CMV promoter having CMV Intron A (InA). Similarly X-box binding protein 1 (XBP 1), a transcription factor, was used to increase the productivity but the results were not on the expected lines and there was no net increase in the production amount [24].Combining heavy and light chains to produce recombinant factor VIII from CHO cells has also been successful. The process resulted in an increase of ten folds [25].

3.3) Monoclonal antibodies

The concentration of monoclonal antibodies produced has improved over the last twenty years and now concentrations of 5g/l are achievable. Recent studies aimed at improving the productivity found that the cell lines which had a minimum threshold of all the cellular processes ( Mab translation, assembly and secretion)

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