The Science - - BioPharm International


The Science

BioPharm International

When experimentation has produced an optimal version of the cell line, a master cell bank (MCB) is created. It will be maintained as the source of all cells used to produce the company's drug through preclinical and clinical testing and then into commercial sale. Working cell banks (WCBs) are created from the master cell bank for producing batches of product. Each batch will be made by seed stock that came from either another working cell bank or the master cell bank.

Cell lines and cell banking. Many factors are considered in choosing and maintaining a cell line, not just the type of protein being produced. The kinetics of protein production and yield, product stability during the manufacturing processes, and the sensitivity of cells to shear damage are also important. A cell line must produce the desired protein in amounts that warrant the expense and trouble of the manufacturing procedures involved.

Extensive records have to be kept on a master cell bank. The company's quality assurance department must document the origin of the DNA sequence that codes for a protein of interest, including the source from which it was obtained; the method used to prepare the DNA expression construct for genetic engineering; a detailed component map showing the number of copies of the gene of interest, all insertions and deletions, the sites on the DNA molecule where they took place, and the complete annotated sequence of the expression vector used; methods for transferring the expression construct into the host cell line and amplifying it for higher expression levels; and criteria for selecting cell clones used to create a master cell bank.

The company keeps records of all the reagents and media used with the cells, specifying storage conditions, determining the age of cells, and validating all methods and procedures involved. Data derived from the beginning cell line is used to determine the in vitro life span of WCB cells used in production, with additions and adjustments as development progresses through pilot and commercial scales.

Cell banks can be stored frozen (cryo-preserved) or, in the case of microbes, freeze-dried (lyophilized). In the industry, most microbial (bacterial and yeast) cell banks are stored frozen in liquid suspensions. Chemical additives (cryopreservatives) protect cells from ice crystal damage but can present a contamination risk. Most bacteria species used in biotechnology remain viable and productive if lyophilized or kept below –20C. The oldest method of maintaining cell lines is through continuous subculture, in which a population of bacteria or yeast is kept growing in culture. That method is seldom used in biotechnology because of the danger of mutations.

Mutations are inevitable in a living, reproducing system. Perhaps one in a thousand genes is mutated in any given organism. These facts of life help evolution work, but they could wreak havoc in a fermentation process. Cell banking helps biotechnology companies avoid the problem of mutations occurring in their cell lines. There simply isn't enough time between thawing the MCB and running a fermentation batch to allow mutations to occur. But recombinant DNA is less stable than native DNA. Recombinant components can be lost in less time than it would take for a cell line to develop mutations.

On the Cutting Edge

Although fermentation and cell culture are the dominant expression systems in biotechnology, they are not the only methods of producing pharmaceutical proteins. Some people consider transgenics to be the next step in biotechnology. Transgenic mammals produce pharmaceutical proteins in their milk, prompting some devotees of that technology to refer to the mammary gland as "nature's perfect bioreactor." Transgenic hens lay the modern equivalent of the fabled goose's golden eggs, with valuable recombinant proteins inside. Transgenic plants are creating a need for a new kind of farming.

For companies that would rather stick to fermentation than switch to farming, new ideas in fermentation offer exciting possibilities. A Swiss company has developed a way to produce recombinant proteins in a slime mold, Dictyostelium discoideum. A primitive eukaryote that feeds on bacteria, it offers some of the advantages of yeast and some of the advantages of higher animal cells. Like yeast, it grows quickly in simple fermentation equipment and can be stored easily, but its cells do not produce endotoxins or possess cell walls. Like other eukaryotic cells, it can perform complex posttranslational modifications of proteins.

What does the future hold for protein expression systems? If the past is any indication, biopharmaceutical companies can expect to have many choices at hand, each with its own advantages and drawbacks. Bacteria, yeast, and mammalian cell culture may be the dominant choices now, but as therapeutic proteins become more complex and are needed in larger and larger quantities, it's evident that companies will need all these options in the future.

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