3.

1 Overview of Cell Line Development

This week we'll learn how to engineer DNA so that it instructs cells to make a target
biologic that we can harvest and use as a drug. The protein we want is usually not
produced by the host natively. So this process is called heterologous protein
expression. Creating a suitable expression system is typically referred to as cell line
development, and is part of the so-called upstream process development. There are
several steps involved in cell line development. In this video, we'll look at the steps
broadly, and then in later videos consider each step in greater depth. The first step
in generating a cell line for protein expression is to choose both the host cell line
and an expression vector. An expression vector is just an engineered DNA construct
used to put a foreign gene, such as our target biologic, into the cell. Choosing a host
is extremely important because it'll effect almost every aspect of production,
whether we end up with a quality product, how that product is produced and
purified, how quickly a production process can be developed, and of course, how
much our manufacturing process will cost. After we've chosen a host, we have to
get the expression vector that codes for our target molecule into the host cell. This
process is called transfection for eukaryotic cells and transformation for bacterial
cells. Ideally, for eukaryotic cells we'd like our host cell to incorporate the vector
into its genomic DNA rather than just having it floating around inside of the cell.
This stable integration allows continued expression of the target biologic over
millions of cell divisions, and is necessary for commercial manufacturing.
Transfection can also be transient or temporary. In transient transfection, the vector
is not integrated into the host DNA, and the gene of interest only replicates over a
few rounds of host cell divisions. So the target biologic will only be expressed during
a limited number of generations of the host cells. Transient transfection, although
unsuitable for commercial manufacturing, is useful in the early stages of process
development when the manufacturing process itself is still being worked out and the
clinical utility of the biologic is still unknown. Unfortunately, the transfection process
is not efficient. In other words, most cells do not stably integrate the vector into
their DNA. So when we design a manufacturing process for commercial use, we
need to somehow select only the host cells that have integrated the vector and kill
the rest. There are many ways to do this. For example, one is to add a gene to the
vector that allows the host cell to resist a toxin. The gene may code for an enzyme
that neutralizes the toxin, allowing the host cells to survive. Adding that toxin to the
growth media will select for cells that integrated the vector with the gene for our
target. The other cells will die. The selection process ensures that all the surviving
cells have taken up the vector. But some might have more copies than others, or
may have modified the biologic by deleting it or truncating it. So we need to choose
one cell, a single clone that produces our target biologic most effectively and with
the best quality. Once we've chosen that one cell, we'll allow it to reproduce, and
we'll have our master cell line. Typically, we'll evaluate a few candidate clones for
their ability to stably produce the biologic at suitable concentrations, or titers, under
production conditions similar to those that we'll use in the final process. We'll pick
the best clone and allow it to divide until we have billions of cells. Just like you might
make a large batch of dough for bread and freeze portions of it for later use, we'll
split up the cells and freeze hundreds of individual batches. This master cell bank
must provide the cells that will manufacture the biologic over its entire commercial
lifetime. In the remaining segments for this week, we'll look at each of these steps
in more detail, review some of the history related to the types of cells commonly
used in manufacturing today, and consider some of the emerging advances in
technologies in cell lines.

3.2 History of Recombinant DNA

The origins of the multi-billion dollar biotechnology industry and the invention of
many of today's lifesaving biologic drugs can be linked directly to an intersection of
ideas from basic biomedical researchers. Prior to this development, the commercial
isolation of insulin from pigs provided an effective therapy to many individuals, but
the manufacturing process couldn't produce nearly enough insulin for every diabetic
subject. People tried very hard to synthesize insulin chemically by linking together
different segments of amino acids. Yet chemical synthesis was not an optimal
solution, and the question remained, how could one economically produce insulin at
sufficient scale to meet medical needs? The answer turned out to lie in co-opting
cellular machinery so that it would do the synthesis instead. How to do this evolved
from a meeting of two biologists at a coffee shop on Waikiki beach in Hawaii in
1972. Stanley Cohen was a geneticist at Stanford who was interested in the biology
of plasmids. These small circles of DNA are found in many organisms, particularly
microbes. Plasmids replicate naturally during cell division, can be transferred from
one species to another, and had been used to study DNA replication and evolution.
Herbert Boyer was a biologist working at the University of California in San Francisco
in the late 1960s. He had found a way to cut out one gene from a piece of DNA and
paste in another. He used enzymes to cut strands of DNA in specific sequence
locations. These cuts leave an overhanging fragment of base pairs that can be
joined or ligated with another fragment containing the complementary sequence. In
this way, it was possible to selectively insert one portion of a DNA sequence into
another. After their meeting in Hawaii, Cohen and Boyer realized that they could
combine their research to engineer plasmids containing specific genes. Using
enzymes, they could cut plasmids open, insert whatever genes they wanted, and
close them again. This invention, often referred to as recombinant DNA technology,
established the foundation for the heterologous expression of human insulin and
bacteria and led to the formation of Genentech in 1976. In the beginning, most of
the commonly used microbes for the development of recombinant DNA technologies
were strains of the bacteria E. coli. As a result, people learned a lot about this
organism. This breadth of knowledge, and the fact that E. coli is easy to grow and
reproduces quickly, meant that it became the preferred host organism when the
biotechnology industry emerged. As the field exploded, the public expressed
concerns around the potential hazards of recombinant DNA technologies. In 1975, a
group of biologists, lawyers, and physicians met to address this issue at the
Asilomar Conference on the recombinant DNA. The conference established
voluntary guidelines for conducting experiments that ensured the safety of this
emerging technology. This is an excellent example of scientists taking action to
address the social concerns of emerging technologies. Then in '75, we had the
revolution. It was ongoing in some form, but it clearly became possible in 1975 to
link genes from bacteria with genes of virusesand genes of human cells. This was
called recombinant DNA, and it was something that I had contributed to the
development of as I was at Cold Spring Harbor before. But then it became
something that really was doable, and I wanted to do it in '75. But we had a concern
in the scientific community about the safety of this technology, so we had a
moratorium, and then we had a meeting at Asilomar, which I was fortunate enough
to go to as a young investigator, and then we came back to MIT and started working
with the city of Cambridge to get guidelines. And that unfolded in the sort of '76, '77
time period. The convergence of basic insights from biological research made it
possible to combine DNA from one organism with another. Manufacturing biologic
drugs now no longer required isolation of those drugs from animal tissues or human
plasma. Instead you could now make cellular systems that produced the drugs, like
insulin, from scratch at very large scales, safely, and efficiently.

3.3 Design of DNA Vectors for Protein Expression

Stitching a gene into a piece of DNA is critical for producing a protein like insulin.
Let's take a look closely at the elements needed to make a recombinant expression
vector. What cell line we choose to host the production will, of course, influence how
we design our vector, but expression vectors usually share five common features.
These are: an origin of replication, a gene of interest, a promoter sequence, a
terminator sequence, and a gene encoding a selectable marker. These elements are
organized on a circular piece of DNA called a plasmid. This is our vector. Let's look
at each element in more detail. The first is an origin of replication. This sequence is
a region where the process of DNA replication begins. Plasmids are native to many
prokaryotes so the cell's natural machinery will replicate both its genome and the
plasmid. This allows the cells to continue carrying the plasmid through many
generations. It also allows us to produce many copies of the vector in bacteria for
transfection into other hosts. The second element is the gene of interest itself. For a
single protein like insulin, the gene is stitched into the vector using enzymatic
ligation. The gene has to be inserted in frame, so that the gene aligns naturally with
the framework of the plasmid itself. This ensures that the transcription machinery
can read the individual codons correctly. The third element, the promoter sequence,
allows efficient binding of the transcriptional machinery to the plasmid, including an
RNA polymerase. The promoter is positioned at the 5 prime end, or the front of the
gene interest, and will lead to the transcription of the biologic into mRNA, which is
subsequently translated into protein. In the simplest design, the vector will instruct
the cell to produce our protein of interest intracellularly. These elements can be
arranged in many ways. Sometimes additional elements may be inserted in
between them to improve productivity or to make recovering the biologic easier.
The fourth element is a terminator sequence. This section of DNA is designed to
limit transcription to the gene of interest on the plasmid and is positioned at the 3
prime end of the gene. Without a terminator, the transcriptional machinery can
keep going past the end of the gene, leading to variance of your product. The
terminator is transcribed at the end of the mRNA encoding the biologic. It helps
stabilize the mRNA itself by forming secondary loop structures at the 3 prime end.
This additional stability reduces the degradation of the mRNA and ultimately
promotes higher productivity. The fifth element is a gene called a selectable marker.
Not all cells exposed to the vector will take it up. A selectable marker allows you to
select for those that do. This gene confers a survival advantage to cells with the
vector. Exposing all cells to whatever toxin or additive that the gene protects
against will allow the cells to survive. In this way, you can select for only the cells
that took up

the vector and kill the rest. So organizing these five elements into a plasmid make it
possible for us to introduce a heterologous gene for a biologic into a host cell, allow
that host to replicate the DNA sequence and express the protein, and allow us to
select those cells that carry the vector. These transformed cells can now produce
our biologic of interest.