Downstream Processing: Platform Technology for Developing Purification Processes - - BioPharm International

ADVERTISEMENT

Downstream Processing: Platform Technology for Developing Purification Processes


BioPharm International
Volume 20, Issue 3

SELDI–TOF STUDIES


Figure 3
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 solid-state 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
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


Figure 5
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.


blog comments powered by Disqus

ADVERTISEMENT

ADVERTISEMENT

Compounding Pharmacy Issues Recall, But Challenges FDA Decision
July 22, 2014
AbbVie's Acquisition of Shire Could Save $8 Billion in Taxes
July 21, 2014
AbbVie to Acquire Shire for $54.7 Billion
July 18, 2014
AstraZeneca Reveals Design for New Global R&D Center and Corporate Headquarters
July 18, 2014
Particulate Matter Prompts Baxter's Recall of IV Solutions
July 17, 2014
Author Guidelines
Source: BioPharm International,
Click here