s22 Supplement to BioPharm International August 2014 www.biopharminternational.com

Analytical and Bioanalytical Testing Protein Purification

Using the Purity Quotient Difference to Assess Protein Purification

An automated analytical method determines the purity of chromogenic, fluorescently tagged proteins or metal-bound proteins.

JEFF HABEL

Automation in chromatography has come a long way from strip recorders and peri-staltic pumps. Researchers can now pro-gram their systems to run a two-step purif ication procedure overnight or

monitor a run’s progress via their smart phone or tablet. Technological improvements like these make chromatography less tedious, almost enjoyable.

Analysis, however, is not so simple, especially when it comes to finding which fractions from the column contain the highest purity protein. Things get even more challenging when purifying a new

protein. Here, many conditions, such as chromatog-raphy media, pH, and buffers—and, therefore, many fractions—must be scouted.

Traditionally, 280-nm UV absorbance is used to detect proteins eluted from a column and then a sodium dodecyl sulfate–polyacrylamide gel electro-phoresis (SDS–PAGE) on the fractions shows which contain a given protein of interest (POI). The pre-ferred purification method meets three criteria: it

JEFF HABEL is a senior scient is t at Bio -Rad Laboratories, Inc, Jeff_Habel@bio-rad.com.

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www.biopharminternational.com August 2014 Supplement to BioPharm International s23

Analytical and Bioanalytical Testing Protein Purification

produces the highest amount of POI, demonstrates the highest purity (separation from contami-nants), and elutes with the POI over a small volume.

A NEW INDICATOR OF PROTEIN PURITYA new method can expedite the assessment of purif ication methods against the three crite-ria. The purity quotient differ-ence (PQD), developed for the NGC med ium-pressu re chro -matography system (Bio -Rad Laboratories), is used in circum-stances where the POI and con-taminants absorb at dif ferent wavelengths. Chromogenic pro-teins, f luorescently tagged pro-teins, and metal-bound proteins such as hemoproteins, as well as contaminants such as DNA, absorb light at different wave-lengths (e.g., not at 280 nm). The NGC chromatography system provides the PQD data, which are exported to Microsoft Excel where they can be manipulated as a h istogram cover ing the duration of the purification run (Figure 1). A positive PQD value indicates a fraction with higher POI than contaminants. Thus when looking at a histogram, ta l ler peaks cor respond with purer proteins.

P QD i s c a l c u l a te d u s i n g Equations 1 and 2. The NGC’s multi-wavelength detection mod-ule can distinguish the elution profile of the POI from that of contaminating proteins, which absorb at 280 nm. In this example, the POI is prancer purple (DNA 2.0 reference), a chromogenic pro-tein that absorbs at 525 nm. The area under the absorbance peaks in a chromatogram is propor-tional to the amount of protein eluted in that peak. This num-ber is divided by the total POI to A

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Figure 1: Histogram of the prancer purple purity quotient difference (PQD) (PQ525–PQ280) per fraction from each pH scouting run. pH 7.0 (), 7.5 (), 8.0 (), and 8.5 ().

Figure 2: SDS-PAGE analysis of pH scouting. 20 μl of each fraction was loaded on an 18-well Any kD Criterion TGX Stain-Free SDS-PAGE gel. The prancer purple E.coli lysate (L) was diluted 1:10 before loading 20 μl. Prancer purple molecular weight: 26.4 kDa. S, Precision Plus Protein Unstained standard.

PQfraction 1 = Relative Area fraction 1 (%)/Collected Volume fraction 1 (ml)

PQPOIfraction 1 – PQcontaminantfraction 1 = PQDfraction 1

PQD>0: more of the protein of interest (POI) in the fraction than contaminantsPQD<0: more contaminants in the fraction than POIPQD=0: POI is present in equal amount as contaminants

(Eq.1)

(Eq.2)

s24 Supplement to BioPharm International August 2014 www.biopharminternational.com

Analytical and Bioanalytical Testing Protein Purification

arrive at a relative peak area (per fraction). Dividing the relative peak area by the volume of the fraction normalizes absorbance to fraction size (larger fractions would be expected to contain more protein) and provides an indica-tor of protein concentration called the purity quotient (PQ). The NGC system enables calculation of PQ for both the POI (PQ525) and the total protein (PQ280) by allowing the user to export the data.

Finally, to calculate protein purity, subtract the purity quo-tient of the contaminant (PQ280) from that of the protein of interest (PQ525) for a given fraction. The resulting value is the purity quo-tient difference or PQD per fraction.

A PQD greater than zero indi-cates that the fraction contains more protein of interest than con-taminants, and a PQD less than zero indicates that contaminants are enriched. A PQD of zero indi-cates that contaminants and the POI are present in equal amounts. Researchers can identify the method that produces the highest PQD over the smallest number of fractions (Figure 2).

USE PQD TO SCREEN AND SDS-PAGE TO VERIFYA visual and automated demon-stration of purity is powerful, but researchers should be careful not to rely on PQD analysis alone. While a high PQD indicates enrichment of the POI, it does not mean contaminating proteins are absent. In Figure 1, it would appear that the PQD histograms are similar when the pH of the buffer is at 7.5 or 8.0. SDS-PAGE analysis, however, clearly shows that more contaminants co-elute with prancer purple at pH 8.0 (Figure 2). Researchers should use PQD in conjunction with SDS-PAGE, as PQD analysis can nar-

row down the many, different purification methods/fractions to a few, reducing the number of gels that need to be run to visual-ize contaminants and select the one that best meets their purifica-tion needs.

SDS-PAGE analysis also gives researchers a clue as to the nature of the contaminants. This informa-tion might be useful, for instance, when two columns or conditions demonstrate similar PQD histo-grams but different contaminant profiles via SDS-PAGE. In this case, researchers might achieve even purer protein by using both col-umns in the same workflow.

SUMMARYSDS-PAGE analysis, the traditional method for determining protein yield and purity, can take three to four hours to set up, run, and ana-lyze. Stain-free precast gels, which enable researchers to complete electrophoresis and image the gel within 25 min., combined with PQD analysis, provide a workflow for identifying fractions containing the highest amount of protein of interest and the highest purity. BP

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A visual and auto-mated demonstration of purity is powerful, but researchers should be careful not to rely on PQD analysis alone.