Protein purity

Protein Purity Is Assessed by Polyacrylamide Gel Electrophoresis (PAGE)... 

The primary analytical applications of RPLC in the development of biopharmaceuticals are the determination of protein purity and protein identity. Purity is established by analysis of the intact protein, and RPLC is useful in detecting the presence of protein variants, degradation products, and contaminants. Protein identity is most often established by cleavage of the protein with a site-specific protease followed by resolution of the cleavage products by RPLC. This technique, termed peptide mapping, should yield a unique pattern of product peptides for a protein that is homogeneous with respect to primary sequence. 

Because of the speed and high resolution of CZE separations as well as the small sample volumes required to yield information about complex protein samples, CE is increasingly being used to assess protein purity in multistep purification protocols in laboratory, pilot plant, and process scales. Similarly, it is being considered as a candidate for monitoring fermentation. 

As a result of the problems with protein purity, preparation methods, and sample analysis, there are many discrepancies in the values obtained from various studies. Some of these will be emphasized as we review a few of the more noteworthy proteins specifically. 

Methods for assessing protein purity must quantitate the amount of target protein relative to the amount of contaminant(s) in the sample. This requires two separate analytical methods one for the taiget protein and one for the total protein including contaminants. For example, with enzymatic proteins, assessment of functional purity typically relies on kinetic assays in which the amount of substrate consumed or product produced per unit time is proportional to the amount of enzyme present. The total activity of the sample is calculated and compared to the total amount of protein to give the specific activity of the sample. Methods for determining total protein are discussed in Chapter Bl. An increase in specific activity at a purification step reflects a loss of contaminant proteins. 

The most popular method of electrophoretic separation by gels employs sodium dodecyl sulfate (SDS). This method not only gives an index of protein purity but yields an estimate of the protein subunit molecular weights. The mixture of proteins to be characterized is first completely denatured by adding SDS (a detergent) and mercaptoethanol and by briefly heating the mixture. Denaturation is caused by the association of the apolar tails of the SDS molecules with protein hydrophobic groups. Any cystine disulfide... 

Although electrophoresis is the method of choice for assessing protein purity, it is not frequently used as a purification step, at least not yet. This is because of the small amounts that can usually be analyzed and because of the denaturing conditions that are often used in electrophoretic analysis. 

Potency-infectious titer Strength-particle/genome titer Protein purity... 

Figure 4. Determination of protein purity. Column Hy-Tach micropellicular C-18 silica, 30x4.6mm eluent A, 0.1% (v/v) TFA in water, eluent B, 95% (v/v) ACN in water containing 0.1% (v/v) TFA flow rate, 2.0 ml/min. temp., 25°C. Initial column inlet pressure, 270 bars. Sample, 20 JJ.1 containing 40 ig of carbonic anhydrase (A), L-asparaginase (B) and myoglobin (Q. Elution conditions were,...

Electrophoresis methods are among the most common and potent used in the evaluation of protein purity and homogeneity. They are valuable indicators of protein stability because they detect small molecular or chemical changes in the product caused by denaturation, aggregation, oxidation, deamidation, etc. One of the advantages is that they require only microgram amounts of a sample. 

The purity analysis of a recombinant produced product is difficult because the accuracy of protein purity is method-dependent and is influenced by the shortcomings of the analytical procedures (Chapter 2). Proteins are highly complex molecules therefore, it is generally very desirable to utilize more than one method to define a given protein s purity. To assure the purity of... 

An important fact inherent in the purity analysis of a recombinant pharmaceutical is that the absolute purity of any protein is an elusive, if not an unobtainable, measurement. For biopharmaceuticals, purity is a relative term. Protein purity is method-dependent and is defined by the shortcomings of the analytical procedure. Also, unlike small traditional drugs, proteins are highly complex molecules. For these two reasons, more than one method must be utilized to define a protein s purity. The greater the number of methods used in the purity analysis, the greater the assurance is that the product is pure. Furthermore, the purity determined by an analytical method can only be properly interpreted based on the method s validation. Analytical methods validation is critical to and inseparable from purity determinations. A detailed discussion on analytical methods validation is beyond the scope of this chapter but other sources of information are available for the interested reader.11 13... 

The most unpredictable process in X-ray structure determination is the crystallization of the candidate protein into a form suitable for X-ray diffraction. Each protein requires a unique set of conditions to form crystals. Typically 100 mg of highly purified protein is required to determine the conditions that result in usable crystals of 0.1 to 0.3 mm size, although a size of 0.3 to 0.8 mm is preferred. The occurrence of crystals and the rate of crystallization are influenced by many factors such as protein purity, the solvent, concentration of added precipitants, pH, temperature, and the presence of ions and cofactors. The protein solution at a concentration of typically 5 to 20 mg/ml is allowed to slowly reach supersaturation by the removal of or by changing the composition of the solvent by liquid-liquid diffusion or vapor diffusion methods. Microscale methods have been developed to explore several crystallization conditions simultaneously using minimum amounts of the purified protein sample. Recently, use of the zero gravity atmosphere in space has been explored as a means of facilitating crystallization (Eisenberg and Hill, 1990 Branden and Tooze, 1991 Tomasselli et al, 1991). 

The first, and often most difficult, step in XRC is the production of 3-D crystals of purified protein. It is important to emphasize the importance of protein purity for successful crystallization because, although it is true that a nonhomogenous mixture may crystallize, it has been noted by some researchers that difficulties in crystallizing a protein may be negated by further purification. Crystallization of membrane proteins is a complicated process, and various approaches have been used, including vapor diffusion, microdialysis, batch crystallization... 

---