APR January/February 2022 - 36

QC CORNER
Purification Strategies for
Recombinant Therapeutic Proteins
Wayne K. Way, Ph.D.
Head of Protein Preparation and Reagents
MilliporeSigma, Bellefonte, PA,
an affiliate of Merck, KGaA Darmstadt, Germany
George C. Yeh, Ph.D.
Product Manager, Protein Purification
MilliporeSigma, St. Louis, MO,
an affiliate of Merck, KGaA Darmstadt, Germany
Many biopharmaceuticals are recombinant proteins
obtained by biotechnological processes. In various steps
of the pharmaceutical value chain, from drug discovery, to
development and manufacturing, to bioprocessing, these
proteins must be extracted from their complex biological
sources, typically microorganisms or genetically modified
cells. The target proteins are subsequently purified and their
presence confirmed by assay.
However, proteins are more challenging to purify than
macromolecules like DNA or RNA, which can be targeted with
complementary oligonucleotides. The great diversity of natural
protein sequences and the lack of the native complementary
structures that DNA or RNA possess means that scientists must
use a different route to purification.
Affinity tag considerations
A key approach in recombinant protein purification is to
use affinity tags, also known as fusion tags or epitope tags.
These are additional peptide sequences, or in some cases even
full-length protein sequences, that have been engineered
into the recombinant protein at the outset to provide a
way of capturing the expressed target protein by affinity
chromatography. Such tags can be as small as six amino acids,
like the 6-histidine-tag (His-Tag™), or eight amino acids, like the
FLAG® tag, known also as DYKDDDDK. Conversely, an example
of a large molecular weight protein tags is glutathione-Stransferase
(GST), sized about 27 kDa.
Many factors influence the tagging strategy, including the
size and the location of the tag. Larger protein tags are more
likely to interfere with a protein's folding and function, hence
small, peptide-sized tags like FLAG®, the His-Tag™, or the
hemagglutinin (HA) tag, have become very popular. The tag is
usually placed at either the N-terminus or the C-terminus of the
protein, although in principle, it can be engineered anywhere in
a given protein. Large protein tags, on the other hand, are often
easier to detect because they are sterically more accessible to
the antibodies that are supposed to bind them.
Another consideration is protein solubility, especially when
the protein of interest is expressed at very high levels and its
solubility thus becomes an important concern. For this reason
hydrophilic tags, which enhance the solubility of the target
protein upon expression at high levels, tend to be preferred.
The FLAG® tag and the His-Tag™ are prime examples of such
hydrophilic tags, because of their high degree of charge.
The matching chromatography resin
Some tags allow the recombinant protein to be purified
by inexpensive methods, for example poly-histidine by
immobilized metal affinity chromatography, but the purity
levels achieved are often disappointing. Chromatography
resins with bound antibodies that are specific for the protein
tag of interest generally achieve higher levels of protein purity.
There is a cost, though, as antibody resins are expensive to
produce and difficult to reuse. However, the high purity from
using antibody resins often improves cost-effectiveness in the
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| January/February 2022

APR January/February 2022

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