TABLE 48 Comparison of some key considerations in choosing host cells for recombinant protein expression in pharmaceutical scale

Consideration

Prokaryote

Eukaryote

E. coli

Yeast

Mammalian Cells (CHO, BHK)

DNA size and

4.6Mbp, circular

12.1 Mbp,

2000-3000 Mbp

characteristics

DNA

chromosomal DNA

chromosomal DNA

Post-

None

Capable; but

Capable; similar or identical

translational

different from

to humans

modification

humans

growth rate

3.33/h

0.25/h

0.02/h"

(cycles per

hour)

Cultivation

Fermentation

Fermentation

Fermentation (suspension cells)

method

Roller bottle (adherence cells)

Cost

Less expensive

intermediate

>$1 million/kg

"Based on estimate of antibody producing hybridoma cells.

"Based on estimate of antibody producing hybridoma cells.

performing posttranslational modification. Higher eukaryotic cells produce a degree of glycosylation similar to human cells. Lower eukaryotic cells (such as yeasts) produce glycosylation characterized by branching and terminal glycosyl residues that resemble the products of human cells. While recombinant protein produced in mammalian hosts will ensure almost identical pharmaceutical properties to those of endogenous human protein, it is prudent to consider cost. Prokaryotic cells proliferate more rapidly and are more efficient and less expensive in producing recombinant proteins than eukaryotic cells, especially mammalian cells. Prokaryotic cells are particularly attractive hosts when the disposition and immunogenic properties of the recombinant protein are not critical to safety and efficacy.

4.2.1. Optimization of Product Yield through Manipulation of Genetic Construct and Recombinant Host Cells

The ultimate goal of product optimization is to increase the production of a specific recombinant protein. This is accomplished by improving the large-scale production efficiency of genetically engineered host cells. In the early stage of isolation and characterization, some information regarding the requirements of biologic and phar-macokinetic properties is gathered and used to identify the target indication(s) for which the product will be tested. This information includes consideration of factors to select the best host cells to express the recombinant proteins [1]. For example, if a protein's biologic activity is dependent on fully glycosylated product, the microbial host, E. coli, which is not capable of gly-cosylation, will not be a suitable host. On the other hand, if glycosylation of protein product need not be identical to human protein, a variant of glycosylated protein expressed in yeast is acceptable. The cost to produce the recombinant protein will be much less in yeast than that needed to produce it in mammalian cell culture.

Product yield optimization is a continuous process. It continues through preclini-cal and clinical development because it ultimately translates into the profit margin of the product when it is approved for marketing.

Optimization of DNA sequences in plasmid vectors by molecular biologists and production engineers is often done in collaboration with researchers in the discovery group. Some strategies include construction of elements that will enhance efficiency of protein expression and stability of the plasmids when transfected into the host cells. Once the plasmids are trans-fected into the host cells, these "recombinant host cells" are selected for those that are genetically stable and produce the highest yield of recombinant protein product. As many as 6000 clones of cells are screened to identify the best host cells for production. The chosen cell stock for final production is maintained by means of a master cell-bank. From each master frozen culture, a subculture stock is established for use in large-scale production. The subculture stock becomes the inoculum for every batch of product. In this way each batch of cultured cells is initiated with a common lineage of recombinant host cells. Additional details are found in Figure 4.9.

Many of these processes are not well publicized and are kept as trade secrets for most recombinant proteins. However, the optimization of host cells, such as microbial cells, to produce antibiotics or peptide derivative is well described. The screening process used to isolate and characterize cells with the highest degree of product expression is time consuming and involves multiple optimization steps. Optimization continues even after the marketing of an antibiotic. Production scientists continue to improve efficiency and lower cost per unit of product. It typically takes from 3 to 10 years to achieve a highly efficient production system (Figure 4.10). With high throughput screening, the time required for identification and development of cells that produce the highest yield may be reduced, but it may still take several years to identify a unique host-cell clone that is genetically stable and suitable for large-scale biological synthesis of recombinant products.

Laboratory scale fermentation: typically fed-batch, scaled down production condition

Create

► >

working

cell bank

Construction of plasmid vectors

Optimization and verification of product in host cells, e.g., E. coli

Primary small shake flask evaluation

Secondary shake flask evaluation

Optimize for time and growth media

Laboratory scale fermentation: typically fed-batch, scaled down production condition

Optimization and verification of product in host cells, e.g., E. coli

Primary small shake flask evaluation

Secondary shake flask evaluation

Optimize for time and growth media

Figure 4.9. Optimization of product yield through sequential and systematic screening and selection of genetic construct and recombinant host cells that produce the highest yield and genetic stability. The schematically presented steps are required to develop a master cell-bank, and working stock for the production of biologically active recombinant proteins in pharmaceutical scale.

Figure 4.9. Optimization of product yield through sequential and systematic screening and selection of genetic construct and recombinant host cells that produce the highest yield and genetic stability. The schematically presented steps are required to develop a master cell-bank, and working stock for the production of biologically active recombinant proteins in pharmaceutical scale.

Figure 4.10. Schematic representation of the relationship between unit cost and increased product yield or titer as a function of time.

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