protein solutionstm - wyatt technology · the same protein that has been pegylated. notice the...

DAWN ® , ASTRA ® , and the Wyatt Technology logo are registered trademarks of Wyatt Technology Corporation. ©1996 Wyatt Technology Corporation 8/30/9 6 6300 Hollister Avenue • Santa Barbara, CA 3117 Phone (805) 681-9009 • FAX (805) 681-0123 E-mail: [email protected] • URL: http://www.wyatt.com CORPORATION Figure 1. 150K Dalton protein before PEGylation. Notice the constant molar mass across the entire peak. Figure 2. The same protein that has been PEGylated. Notice the dramatic change in molar mass across the peak, depending on the number of PEG attachments. Protein PEGylation he rise of biotechnology has spawned differ- ent means of delivering drugs to the human body. Among the many problems encountered is the rapid rejection or clearance of a protein before it has accomplished its task. A variety of methods exist for modifying pro- teins so that they are more effective in the bloodstream. Among them, glycosylation and PEGylation are perhaps the most popular. In the former, sugars are attached to the protein, in the latter, polyethylene gly- col strands are attached as “whiskers” to increase the half-life of the protein and make it less likely to be hydrolized or cleared from the bloodstr eam. In this application note, a monoclonal antibody was measured before and after PEGylation. As illus- trated in Fig. 1, the antibody has a constant molar mass of 150K Daltons across the entire peak. Figure 2 shows what happens when the same antibody has been conjugated with PEG molecules of 5K Daltons each. Using a self-consistent, iterative method, the number of PEGs per protein was calcu- lated, and can be seen on the graph itself. The chromatography set-up consisted of a miniDAWN multi-angle light scattering detector, Op- tilab DSP interferometric refractometer, HP 1050 HPLC system, and a Pharmacia Superose 12 30/10HR column. The results are, perhaps, most remarkable for their specificity. Never before has it been so easy to determine the number of PEG attachments to a protein. Now, using the miniDAWN it has become a straightforward experimental procedure. ~52 PEG/protein ~38 PEG/protein 4-5 PEG/protein 0 PEG/protein Optilab DSP signal Optilab DSP signal Mw = 150,000 g/mol M.W. vs. Volume Elution volume (mL) M w (g/mol) M.W. vs. Volume Elution volume (mL) M w (g/mol) Protein Solutions TM

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Page 1: Protein SolutionsTM - Wyatt Technology · The same protein that has been PEGylated. Notice the dramatic change in molar mass across the peak, depending on the number of PEG attachments

6300 Hollister Avenue • Santa Barbara, CA 3117Phone (805) 681-9009 • FAX (805) 681-0123E-mail: [email protected] • URL: http://www.wyatt.com

CORPORATION

Figure 1. 150K Dalton protein before PEGylation. Notice theconstant molar mass across the entire peak.

Figure 2. The same protein that has been PEGylated. Notice thedramatic change in molar mass across the peak, depending on the

number of PEG attachments.

Protein PEGylation

he rise of biotechnology has spawned di�er-ent means of delivering drugs to the humanbody. Among the many problems encountered

is the rapid rejection or clearance of a protein before ithas accomplished its task.

A variety of methods exist for modifying pro-teins so that they are more e�ective in the bloodstream.Among them, glycosylation and PEGylation are per-haps the most popular. In the former, sugars are at-tached to the protein, in the latter, polyethylene gly-col strands are attached as “whiskers” to increase thehalf-life of the protein and make it less likely to behydrolized or cleared from the bloodstr eam.

In this application note, a monoclonal antibodywas measured before and after PEGylation. As illus-trated in Fig. 1, the antibody has a constant molar massof 150K Daltons across the entire peak.

Figure 2 shows what happens when the sameantibody has been conjugated with PEG molecules of5K Daltons each. Using a self-consistent, iterativemethod, the number of PEGs per protein was calcu-lated, and can be seen on the graph itself.

The chromatography set-up consisted of aminiDAWN multi-angle light scattering detector, Op-tilab DSP interferometric refractometer, HP 1050 HPLCsystem, and a Pharmacia Superose 12 30/10HRcolumn.

The results are, perhaps, most remarkable fortheir speci�city. Never before has it been so easy todetermine the number of PEG attachments to a pro-tein. Now, using the miniDAWN it has become astraightforward experimental procedure.

~52 PEG/protein

~38 PEG/protein

4-5 PEG/protein

0 PEG/protein

Optilab DSPsignal

Mw = 150,000 g/mol

M.W. vs. Volume

Elution volume (mL)

Mw (

g/m

ol)

M.W. vs. Volume