The method of choice for starting the development of downstream purification processes is the SELDI–TOF technique, using the
sophisticated ProteinChip Array technology from Ciphergen Biosystems (Framingham, CA).5,6 This concept was developed in the 1990s and commercialized by the end of the twentieth century. SELDI–TOF uses coated chips
(ionic, reverse phase, hydrophobic, hydrophilic, or affinity ligand groups) for the binding of proteins, a laser focused beam
to promote gaseous ions from solidstate matter, and a mass spectrometer (MS) as a detection system. This system has already
proven its functionality in clinical research for elucidating new biomarkers in a diverse range of diseases.7 The principle of this system is illustrated in Figure 3. Special ligands covalently attached to the chip are able to bind
proteins. Selection occurs by washing away nonspecific contaminants (proteins) with pH or salt buffers, whereas the target
protein still binds to the chip. Mass spectrometric detection is a powerful analytical method to identify the target protein
and monitor the removal of contaminants during washing. This system is ideal for testing pH and salt conditions by determining
the conditions under which the target protein binds to the covalently attached ligand. For efficient protein ionization, additional
matrix sensitizers (sinapinic acid or alpha cyano-4-hydroxycinnamic acid) are added. These initial screening studies can be
performed in a few hours.
Figure 4 displays the mass spectrometric results of a monoclonal antibody (MAb) binding study. The MAb binds to a strong anionic
exchange (SAX) chip under mild conditions (e.g., TrisCl pH 8.0). Elution takes place at salt concentrations from 0 to 450
mM NaCl. The salt screening results clearly show that the MAb, with a molecular mass of ~147 kD, elutes around 100–150 mM
NaCl, as shown by mass detection. At higher salt concentrations, the ~147 kD signal disappears, indicating that the MAb elutes
from the chip. With these preliminary results (binding or elution conditions on a chip), the second step, resin selection
with the specified ligand, is performed in a more static set-up. However, some proteins do not present an ionization pattern
with MS, therefore detection with the SELDI–TOF system is not always feasible during the initial screening stage.
Robotic Screenings Studies
After initial selection of the preferred ligand (ionic, reverse phase, hydrophilic, hydrophobic, or affinity) chromatographic
resins from various suppliers are selected and screened in a special 96-well filter plate (microtiter plate containing a filter
on the bottom), using a pipetting robot system in a dynamic procedure. This technique, used in few other biopharmaceutical
companies, has only recently been developed following the commercialization of sophisticated new robotic systems (Figure 5).
The sample, containing the target protein, is incubated batchwise with the resin for binding, and elution occurs again by
using different pH or salt concentrations. The supernatant is collected by centrifugation or vacuum filtration in a microtiter
plate, leaving the resin in the filter plate. Protein- or product-specific tests are used for analysis. Figure 6 illustrates
the screening results with different salt conditions of a specific glycoprotein. The results show that most resins depict
a similar binding profile—elution of the glycoprotein occurs around 100–200 mM NaCl. Based on these screening results, the
best resins (with respect to binding capacity, throughput, cost price, etc.) are selected and studied in more detail in the
next step, in which scouting experiments are carried out under dynamic conditions.