Protein purification

Tomas Bratt tb at
Thu Dec 20 04:42:41 EST 2001

	 Dear discussion group,

	I agree with you that misfolded proteins will probably run with
a MW close to a native structure or they will form aggregates or
polymers. But gelfiltration will give an answer if there are misfolded
proteins because of peak broadening and/or polymeric forms. If the
native protein is dimer or higher -mer it is also worth doing gel
filtration as misfolded variants probably will not form the same -mer
form. For a more general applicable separation see the article below or
others like it that uses RP-HPLC, an efficient and powerful technique
that could be used for many different proteins, just as Rick wanted.
	Good luck

	Preparative isolation of recombinant human insulin-like growth
factor 1 by reversed-phase high-performance liquid chromatography.
	Olson CV, Reifsnyder DH, Canova-Davis E, Ling VT, Builder SE.
	 J Chromatogr A 1994 Jul 22;675(1-2):101-12
	Department of Recovery Process Research and Development,
Genentech Inc., South San Francisco, CA 94080.
	The isolation of recombinant human insulin-like growth factor 1
(rhIGF-1) is complicated by the presence of several rhIGF-1 variants
which co-purify using conventional chromatographic media. These species
consist primarily of a methionine-sulfoxide variant of the properly
folded molecule and a misfolded form and its respective
methionine-sulfoxide variant. An analytical reversed-phase
high-performance liquid chromatography procedure using a 5-micron C18
column, an acetonitrile-trifluoroacetic acid (TFA) isocratic elution,
and elevated temperature gives baseline resolution of the four species.
Using this analytical method as a development tool, a process-scale
chromatography step was established. The 5-micron analytical packing
material was replaced with a larger-size particle to reduce
back-pressure and cost. Since the TFA counter-ion binds tightly to
proteins and is difficult to subsequently dissociate, a combination of
acetic acid and NaCl was substituted. Isocratic separations are not good
process options due to problems with reproducibility and control. A
shallow gradient elution using premixed mobile phase buffers at the same
linear velocity was found to give an equivalent separation at low load
levels and minimized solvent degassing. However, at higher loading there
was a loss of resolution. A matrix of various buffers was evaluated for
their effects on separation. Elevated pH resulted in a significant shift
in both the elution order and relative retention times of the principal
rh-IGF-1 variants, resulting in a substantial increase in effective
capacity. An increase in the ionic strength further improved resolution.
Several different media were evaluated with regard to particle size,
shape and pore diameter using the improved mobile phase. The new
conditions were scaled up 1305-fold and resulted in superimposable
chromatograms, 96% recovery and > 99% purity. Thus, by optimizing the
pH, ionic strength and temperature, a high-capacity preparative
separation of rhIGF-1 from its related fermentation variants was

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