Tosoh
customer magazine
Purification of bispecific antibodies and
antimicrobial active plasma components
04
TOYOPEARL
IN THE LITERATURE
TOYOPEARL chromatography media are applied in a brOAd range of purification schemes of biomolecules ranging
from nucleic acids to proteins. The resins can be applied at various stages of the purification process. Examples of
biotherapeutics purified at large scale with the help of TOYOPEARL are monoclonal antibodies, Epoetin (EPO), growth
factors and blood plasma derived proteins, such as factor VIII. The use of TOYOPEARL in research and method
development has been documented in more than 750 scientific publications. We cite here some recent publications
where TOYOPEARL resins were applied for the purification of bispecific antibodies and of antimicrobial active
fraction of crocodile plasma, respectively.
REFERENCES
1
|
Müller-Späth, Th. et al. Purifying Common Light-Chain Bispecific
Antibodies, Bioprocess International (2013), 11 (5), 36-45
2
|
Kommanee, J. et al., Antibacterial activity of plasma from
crocodile (Crocodylus siamensis) against pathogenic bacteria;
Annals of Clinical Microbiology and Antimicrobials (2012), 11:22
).
Thomas Müller-Späth and colleagues
1
developed a twin-column,
countercurrent chromatography platform process for the purification
of common light-chain (LC) bispecific antibodies. A bispecific anti-
body can bind two different antigens because its variable regions
carry different specificities, which are generated by different heavy
chains (HCs). Recombinant host cells for production of LC bispecific
antibodies carry genes for both HCs, with the different specificities
(A and B), along with one LC gene. A, B, and the light chains are
expressed independently in those host cells, which then assemble
them into three IgG types — AA, AB, and BB — for secretion into
a culture environment. This mixture was captured from the feed-
stock by TOYOPEARL AF-rProtein A-650F which was chosen based
on HCP clearance data. The purified IgGs were separated into the
three different types by cation exchange multicolumn countercurrent
solvent gradient purification (MCSGP), a proprietary chromatographic
process that allows for product isolation with high yield and purity in
difficult separation situations, and further polished by flow-through
anion exchange chromatography on TOYOPEARL SuperQ-650M.
Running the cation exchange step in MCSGP mode enabled isolation
of the bispecific antibody AB at an 87% step yield. Overall yield of the
chromatographic steps of the process was 78%. Thomas Müller-Spath
will give a presentation on this exciting technology on the Forum
Prozesschromatographie in Stuttgart (see page 8).
TOYOPEARL DEAE-650M was used by Jintana Kommanee and
colleagues
2
to fractionate plasma from freshwater crocodiles in order
to examine its antibacterial activity against pathogenic bacteria.
Siamese crocodilians (Crocodylus siamensis) live with opportuni-
stic bacterial infection but normally suffer no adverse effects. They
are not totally immune to microbial infection, but their resistance
thereto is remarkably effective. In this study, crude and purified
plasma extracted from the Siamese crocodile were examined for
antibacterial activity against clinically isolated, human pathogenic
bacterial strains and the related reference strains. The crude plasma
exhibited substantial antibacterial activities of more than 40% growth
inhibition against the six reference strains of Staphylococcus aureus,
Salmonella typhi, Escherichia coli, Vibrio cholerae, Pseudomonas
aeruginosa, and Staphylococcus epidermidis, and the four clinical
isolates of Staphylococcus epidermidis, Pseudomonas aeruginosa,
Salmonella typhi, and Vibrio cholerae. To further localize the anti-
bacterial activity the plasma was fractionated by anion exchange
chromatography on TOYOPEARL DEAE-650M into four fractions
designated as fractions D1-D4. Only fraction D1 showed growth
inhibition in the reference strains and the clinical, human pathogenic
isolates. Subsequent studies are required to focus on the purification
and characterization of the agent responsible for the antimicrobial
activities including the anti-inflammatory activity.
