Protein purity assay

Summary

No method can directly quantify the purity of a protein sample. Typically, protein purity determination involves evaluating the level of specific impurities or simply verifying the presence of impurities in a protein sample. Whether the ultimate g is to interpret analytical data, verify process quality, or ensure the safety of a biopharmaceutical product, the determination of sample purity is a critical component. This chapter will focus on methods for the detection of proteinaceous impurities in protein samples and what their feasibility and limitations are.

Operation method

Protein purity assay

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Before evaluating the purity of a sample, it is necessary to identify the type of impurity to be measured, e.g., nucleic acid, carbohydrate, lipid, irrelevant protein, isoenzyme, inactivated protein, and to determine the physicochemical properties (chemical analysis or physical characteristics) that will distinguish the hypothetical impurity from the target protein under certain solution conditions. Purity is defined as a level of impurity below a specified level. It should be noted that there is no requirement to characterize the nature of the impurity in the above description. The purification process may have reduced the concentration of an impurity below the lower limit of detection, but there is still a residue in the peaks. Undoubtedly, apparent purity depends on the assay method chosen and its sensitivity. Since most separation methods are effective in removing nonprotein impurities, this section will focus on the detection of protein impurities in protein samples. Methods for the detection of nucleic acid, lipid, and carbohydrate impurities are described in other volumes of this series.

With the popularity of protein-based biologics, protein purity has become a major issue in drug management. The International Conference on Harmonisation (ICH) Guidelines for the Quality of Biologics (ICHQ6B, 1998) take into account the impurity content of APIs (matrix materials, bulksubstance) and drug products (dosage forms or finished products). The guidelines (ICHQ6B, 1998) take into account the presence of impurities in APIs (bulksubstance) and drug products (dosage forms or finished products) and recognize the inherent heterogeneity of biomolecules (e.g., proteins). The guidance states, "In addition to evaluating the purity of APIs and drug products, which may consist of the intended product and a variety of product-related substances, manufacturers should also evaluate the possible presence of impurity components. These impurities may be process- or product-related, and they may be structurally known, partially characterized, or uncharacterized." The guidance further distinguishes between process-related impurities (resulting from manufacturing processes, including host cell proteins, host cell DNA, cell culture inducers, antibiotics, and other impurities resulting from downstream manufacturing processes) and product-related impurities, which include molecular variants resulting from manufacturing or storage that do not meet the criteria for activity, efficacy, and safety. and safety characteristics of the intended product." The guidance recognizes these analytical capabilities. For example, the guideline on APIs states that "the absolute purity of bioengineered and biological products is difficult to determine, and the results are often dependent on the measurement method chosen ...... Therefore, a combination of methods is often used to evaluate the purity of an API. The selection and optimization of analytical processes should focus on the separation of the destination product from product-related substances and from impurities." The discussion of drug products is similar to that of APIs, but methods for the determination of product degradation and product-related impurities due to excipient (excipient) interference are also presented.

A number of highly sensitive methods are available for the detection of impurities in samples (Table 38.1). Each of these methods determines a specific physical property of the molecule. The choice of method depends on the following criteria: (i) the amount of protein available for detection; (ii) the nature of the impurity to be measured; (iii) the required precision of detection; (iv) the required sensitivity of detection; and (v) the properties of the protein and its solvent that may interfere with the method. The simplest and most commonly used method is to confirm that only one component can be detected after one or more separations. If the purity criterion is that only one detectable substance can be present, multiple separations are required to detect impurities.

Orthogonal methods are recommended when an unknown impurity cannot be distinguished from the primary analyte by the chosen assay. Finally, careful handling of the sample is important to prevent the impurity profile from being altered during the preparation of the sample for analysis. Also, clean environments, appropriate temperatures, and containers made of suitable materials will assist in the analysis.

Many of the appropriate methods have been described in detail elsewhere in this volume and will not be repeated here. There are a number of molecular mass or molecular size assays that are also outlined in other chapters in this volume, so details from the relevant chapters will sometimes be quoted/referenced in the discussion in this chapter (Rhodesetal., 2009). The focus of this chapter will be on presenting methods that are more applicable to the detection of impurities rather than the quantitative determination of other molecular parameters such as size and mass. In addition, this chapter outlines the principles of method selection, the limitations and advantages of some of the methods, and describes some of the purity determination methods that do not involve separation.

I. Analysis of Protein Composition and Activity

Some methods are able to quantify the number of moles of amino acids, specific cofactor groups, or active sites and can be used to evaluate the purity of a protein sample. If the activity of a pure protein is known, then unit activity measurements can be used to test relative purity. These methods are indirect, since it is always necessary to assume that the analyte is pure from impurities and to use the amount of that assumed pure product as a reference. These methods are mainly suitable for multiphase systems early in the purification process, or for molecules (e.g., membrane proteins) that require a special environment to maintain activity. Two tests are generally required: the first is to analyze the total amount of protein (or raw material) in the sample used; the second is to quantify a known active object or other special analyte. Based on the total amount of protein, the corresponding amount of the desired analyte is calculated. Purity is expressed as the ratio of the measured amount of the analyte to the expected amount. Purity can be quantified very well using terminal group analysis (Chang, 1983), special auxiliary group quantification, and enzyme activity quantification (Biggs, 1976). Details of this method are described in Rhodes et al. (2009).

Problems and limitations

Protein composition and activity analyses only indicate the presence or absence of impurities and usually provide little information about the nature of the impurities (e.g., size and charge). Unless the impurity interferes with the assay process or protein activity, the activity assay may not provide any information about the impurity. As a result, the experimenter is unable to obtain sufficient clues to remove the impurity.

II. Electrophoresis

Electrophoretic methods are most commonly used as they provide the simplest, least expensive, and most sensitive means of determining the number of protein fractions in a sample. Because of their extremely low cost and considerable simplicity, these methods are often used as the first step in protein purity screening, and are even applied to samples that are initially highly heterogeneous. Other chapters in this book describe SDS gel electrophoresis and the use of electrophoresis for the determination of molecular mass and size (Rhodesetal., 2009). Each of these methods can be used individually to determine the purity of a sample, and the choice of method depends on what properties of the protein to be tested are desired (Table 38.1). SDS gel electrophoresis can discriminate impurities if there is a difference between the expected impurity and the molecular mass of the target protein. Molecules of similar molecular mass but different amino acid composition are generally indistinguishable by SDS gel electrophoresis, but they have different electrophoretic mobilities in nondenaturing gel electrophoresis. In addition, proteins of almost any size can be separated by isoelectric focusing. The isoelectric focusing method is described elsewhere in this book (Friedman, 2009) and will not be discussed in detail here.

Depending on the type of electrophoretic assay used, the amount of sample required ranges from nanograms to micrograms. Since each impurity occupies a fixed weightfraction in a given sample and the detection of impurities is dependent on their total amount, gels that are overloaded with sample volume may have a better chance of detecting an impurity. However, the upper limit of sample loading depends on sample solubility and needs to be based on resolution considerations (Lunneyetal., 1971). Generally the latter is more restrictive, as higher sample concentrations or large sample volumes can lead to band broadening and distortion. Band expansion due to overloading will make it difficult to discriminate impurities with similar electrophoretic mobilities. However, electrophoresis, the most sensitive method of band detection (requiring the smallest amount of sample), is not suitable for sample recovery. If the protein sample has been denatured or solubilized under extreme conditions, it is generally difficult to recover the active protein. If non-denaturing electrophoresis is used, the sample can be recovered from the gel by electirophoreticextraction (Friedman, 2009; Garfin, 2009). However, denaturing electrophoresis may not recover natural proteins [see Burgess, (2009) for an exception]. The success of recovery depends greatly on the structure of the protein. Larger or multisubunit proteins are less likely to recover their natural conformation than small monomeric proteins.

Methods

The method of sample preparation depends on the nature of the electrophoresis method chosen. The reader is referred to the discussion of nondenaturing gel electrophoresis and isoelectric focusing electrophoresis elsewhere in this volume. SDS gel electrophoresis is the most commonly used "front-line" method for protein purity testing. Since the purity assessment is usually non-quantitative, there is no need to be concerned with issues such as non-linear migration of extreme sized molecules, migration anomalies due to protein modifications, and uneven SDS binding as discussed by Rhodes et al (2009). In addition, the consistency and homogeneity of the sample handling and preparation process, as well as the environment in which the sample is exposed during the separation, need to be considered. For example, preparation of homogeneous protein samples without complete reduction of disulfide bonds will cause the results to exhibit heterogeneity. Similarly, differences in intragel mobility due to inhomogeneous gel cross-linking can lead to misinterpretation of the results, but appropriate replicates and controls will minimize this possibility.

Often, the experimenter does not know the sizes of all the protein fractions in a sample, so it is difficult to predict the concentration of the gel that will separate a group of protein fractions with optimal results. Gradientgel, also known as gelsofgradedporosity, can cover a very wide range of molecular mass sizes. Although a gradientgel may not produce the best specific separation, it can cover the widest range of molecular masses, thus maximizing the likelihood of identifying an impurity. The ability to discriminate between impurities is largely dependent on the range of gel concentration gradients chosen. The gradient should be designed so that the target protein bands are in the middle of the gel concentration. The acrylamide concentration at the top of the gel (low concentration) should be low enough to allow large molecular mass impurities to enter the gel matrix. The concentration at the bottom of the gel (high concentration) should be high enough to allow small molecule proteins to remain in the gel matrix. Typically, the lowest concentration of precast gradient gels is 4% acrylamide, and the high concentration limit is standardized at 20%. If this range is not suitable, gels can be prepared in concentration ranges as low as 2% and as high as 40%. Proteins with a molecular mass of approximately IMDa should be able to pass through a 2% gel matrix. In general, most gradient gels do not exceed 30% acrylamide concentration. Even in prolonged electrophoresis only a very small number of peptides are able to penetrate 30% gels. The concentration range of a gradient gel determines the resolution of the gel. Therefore, although gradient gels of 2% to 30% are capable of detecting the widest range of molecular sizes, they do not discriminate as well as gradient gels of 8% to 16% when separating two proteins with almost the same molecular mass.

The choice of gradient depends greatly on the researcher's knowledge of the entire system to be tested. For example, if exceptionally small and large molecules are not present, it is preferable to choose a gel with a narrow range of concentration gradients or a gel with a constant concentration.

If a special gradient is required, the process of making a gradient gel differs very little from the process of making a conventional polyacrylamide gel, and is similar to the process of making a sucrose gradient for sedimentation. The difficulty in making gradient gels compared to conventional polyacrylamide gels is that two gels of different acrylamide concentrations must be made in parallel. And the trouble than sucrose gradient preparation is because gradient gels have a time limit for acrylamide polymerization. There are a number of commercially available gradient formers, and the differences in technique can be found in the literature.

The electrophoresis process for gradient gels is similar to conventional electrophoresis [Rhodes et al.

The staining means used for gel analysis is determined by the intended contaminant. Two-dimensional gel electrophoresis analysis can be accomplished by combining other methods with electrophoresis, thereby enhancing the potential sensitivity of the electrophoretic method and the information obtained. Typically the second dimension is gradient SDS~PAGE, whereas the first dimension may be nondenaturing gel electrophoresis, isoelectricfocusinggel, or denaturing electrophoresis under different conditions (e.g., no disulfide bond reduction).

Isoelectric focusing is also capable of detecting a wide range of contaminants and can be used in conjunction with electrophoretic methods. Commercially available precast gels have an isoelectric point coverage of 3 to 10 on the same gel. The isoelectric focusing procedure is described elsewhere in this book and will not be repeated here. The wide range of isoelectric points is a considerable advantage, and for proteins with small differences in isoelectric points, the sensitivity of differentiation can be improved by reducing the isoelectric point range (e.g., 4-5, 5-8). In addition to the ability to separate molecules with different isoelectric points, isoelectric focusing can also discriminate contaminants by other properties.

Problems and limitations

Given its simplicity and low cost, gel electrophoresis is often used as the method of choice for purity evaluation. However, there are some potential problems that need to be kept in mind when employing this method. For denaturing gels, false negatives and false positives may occur.

False negatives can occur if there is co-migration (comigrating) of impurities or if impurities are unable to enter the gel. Therefore, the entire gel, including concentrates and separates, should be stained, and the suitability of the protein dyes should be checked. For example, sample bands that remain in the sample well or between the concentrate and separator gels indicate the presence of high molecular mass impurities or limited solubility of the impurities. Also, commonly used proteinstains, such as Caulmers Blue, bind weakly to fibrous proteins and glycoproteins, which may underestimate the amount of such impurities. False positives may be due to covalent modifications during sample preparation, or to gel inhomogeneity and oxidant residues. Similar problems can occur in nondenaturing gels, and uncertainty about the net charge of the target protein or impurity can also lead to surprises (Rhodesetal., 2009). If the electrostatic charge of the impurity is zero or the opposite of the target protein, the impurity will not appear on the gel unless a center-loaded horizontalgel is used, and the problem can be solved by widening the pH range of nondenaturing gel electrophoresis as much as possible.

Isoelectric focusing-related artifacts (artifacts) occur primarily when the polyamphiphilic electrolyte particles used to generate the pH gradient interfere with the protein, resulting in bands (generally diffuse) on the gel that are independent of the isoelectric point of the target protein. One way to detect this possibility is to isolate the protein by selecting one of the bands and analyzing the isolated protein using the same isoelectric focusing procedure. If the original resultant distribution reflects the actual heterogeneity in the sample, the isolated protein will only form the same points as the original. However, if the original bands are artifacts, then the separated proteins will reproduce the original distribution pattern, including spurious bands.

III. Chromatography 1. Condensation limb filtration chromatography

Gel filtration chromatography is one of the simplest methods for detecting impurities that are different in size from the intended molecule. This method is non-destructive and very fast. Because it is a "zonal method," the sample is diluted as it passes through the gel column, so a starting concentration just above the minimum detection limit is required. The exact amount to be used will depend on the sensitivity of the impurities detected by this method.

Methods

The sample and gel column preparation methods for protein size determination presented in this volume will be used in this chapter to evaluate impurities (Rhodesetal., 2009). The only difference between the two applications is that the assay used in the latter requires high sensitivity not only for the target protein but also for impurities. Although it is not necessary to calibrate the gel column to evaluate the purity of the sample, there are two advantages to doing so. First, by using a calibrated gel column, the experimenter can obtain information about the size of the impurities, and second, by using a calibrated gel column, the molecular mass can be determined at the same time as the presence of impurities is detected. For this purpose, two assays are usually required: the first is a non-specific assay for the protein (e.g., 280 nm, 220 mn absorbance), and the second is a specific assay (enzyme activity assay, immunological assay, mass spectrometry, multi-angle light scattering assay, etc.), which is used to confirm the identity of the protein being analyzed.

Impurities may appear as separate peaks in the chromatogram or may broaden the elution spectrum [e.g. shoulder]. In principle, the elution spectrum should be close to a Gaussian distribution (with small shifts at the edges, see Problems and Limitations). If the analyzed protein peaks are strongly shifted, the peaks are shouldered, or the specific and non-specific measurements do not match, impurities are present in the sample.

Problems and limitations

Gel filtration chromatography is less sensitive than electrophoresis for the detection of molecular size. In general, gel filtration experiments require a larger amount of material than electrophoresis. Since gel filtration chromatography is usually performed in its natural state, the results can reflect sample heterogeneity. If the protein exists as a series of stable oligomers (e.g., fatty acid synthase), it will exhibit heterogeneity in gel filtration chromatography. Similarly, proteins that undergo rapid, reversible self-association will exhibit anomalous concentration-dependent elution profiles. These cases can be further determined to distinguish between heterogeneity and molecular coupling. For example, gel-filtration chromatography is repeated by selecting different fractions from the asymmetric and broadened peaks, or the selected fractions are determined using other methods described in this chapter and this volume. All of these methods can provide useful information about the target molecule.

2. Reversed-phase high-performance liquid chromatography

Another widely used chromatographic separation method is reversed-phase HPLC (reversed-phase HPLC).

This method uses a non-polar matrix, such as modified silica media, as the stationary phase and performs a gradient elution of decreasing polarity. For example, in a buffer containing 0.05% trifluoroacetic acid (TFA), proteins are bound to the stationary phase via hydrophobic amino acid binding to the C-18 modified silica media, and the proteins are eluted by a gradual step up from 0.05% TFA to a certain amount of a suitable organic solvent, e.g., acetonitrile, methanol, or ethanol, usually with a consistent level of trifluoroacetic acid. The organic solvent in the mobile phase reduces the affinity of the proteins for the stationary phase, thus eluting the proteins. As with other gradient elution methods, a slow gradient elution gives the best resolution, but it is preferable to start with a fast gradient elution for the first experiments, so that the optimum solvent conditions for the elution of the main analytes can be obtained and possible impurities can be screened on the basis of the different affinities to the stationary phase.

The detection of proteins is usually based on UV absorbance at 215-220 nm for peptides rather than the aromatic amino acid method using 280 nm detection. Regardless of the preparation time, gradient elution can be completed within Ih, and most optimized elution steps will be completed within 15~20 min.

Because of its simplicity and speed, the availability of suitable equipment, and the flexibility of applying many different properties to the separation of affinity proteins, this method has been commonly used for purity screening of protein samples. If necessary, a number of different mobile phases can be used so that samples can be separated in different gradients and conditions to determine the purity of the primary analyte. The method can also be used in conjunction with other methods [e.g., mass spectrometry (see below)] to determine protein identity and to obtain information about possible impurities. It should be noted that due to the presence of organic solvents, a certain degree of denaturation of the eluted protein can be expected and the natural conformation may not be maintained. The possibility of denaturation can be minimized by using C-4 or C-8 stationary phases due to their weak affinity for proteins, and lower concentrations of organic mobile phases can be used for elution. Many protocols have been published for specific proteins and much product information is available from HPLC equipment manufacturers.

IV. Sedimentation Rate Measurement

The sedimentationvelocitymethod is a simple, rapid and non-destructive method for evaluating the purity of a protein and is sensitive to the ratio of molecular mass to molecular size. The sedimentation velocity method is briefly described in Rhodes et al. (2009). Generally, when using sedimentation rate assays to test protein purity, the experimenter is able to find more than one sedimentation component. This method has the advantage of measuring a very wide range of materials (especially when using refractive optics), but the biggest limitation is that it is not as sensitive as electrophoresis for samples with small differences in molecular mass, which is even unavoidable with bandsedimentation.

Methods

This volume (Rhodesetal., 2009; Coleetal., 2008) describes in detail some of the methods other than rate-zonalsedimentation, such as sample preparation, selection of a suitable optical system, and analysis of the results. Instructions on how to prepare the centrifuge for rate-zonalsedimentation can be found in the equipment manual. Details of the rate-zonalsedimentation method using an ultrafast analytical centrifuge can be found in Eason (1984) or Ralston (1993), and the analysis of the data is similar to that of gel filtration chromatography.

Differentialsedimentationcoefficientdistribution, g(s) (Coleetal., 2008) analysis, is a very effective method for impurity detection. Since this method is model-independent, the presence of multiple components will be in the form of peaks with different company sedimentation coefficient) values or broadening of peaks representing the major analytes. In addition, as with gel filtration chromatography, the sedimentation behavior of self-bound proteins exhibits peak broadening or multiple peaks despite the high purity of the proteins, and the use of equilibrium ultracentrifugation or other methods can help to address this issue.

V. Mass spectrometry

Since mass spectrometry can directly determine the distribution of covalent mass in a sample, this method is able to determine impurities very easily and sensitively. Mass spectrometry not only detects the presence of impurities, but also characterizes the mass of impurities, and is therefore often used to identify the source of impurities. In addition to the ability to directly determine the molecular mass size of proteins, mass spectrometry can also be used to characterize impurities by tandem mass spectrometry (MS,'MS, or MS2) methods such as collisioninduceddissociation (CID), electron transferdissociation (ETD), electron transferdissociation (ETD), and mass spectrometry (MS). (CID), electron transfer dissociation (ETD), electroncaptured dissociation (ECD), and infraredmultiphotondissociation (IRMPD), etc., and can also identify covalent modification features and sites. Among them, CBD and IRMPD can be used for the determination of rather small proteins (<15 kDa), while ETD and ECD can be used for proteins with larger molecular mass (~50 kDa). In addition to this, covalent modification bond modification and its site determination can be done by any of the commonly used protein hydrolysis methods, such as trypsin or cyanogen bromide (CNBr) (Link, 2009). CID can be achieved by quadrupolecollisioncell (Q-toFconfiguration) prior to analysis by time-of-flightmassanalyzer.ETD, ECD and IRMPD are commonly used in ion-trapanalyzers. ETD, ECD, and IRMPD are commonly used in ion-trap analyzers, such as linear iontrap, orbitrap, orbitrap, and fouriertransformioncyclotronresonancemassspectrometer.Q Similar to light scattering, the combination of mass spectrometry and other mass spectrometry methods can be used in a variety of ways. Similar to light scattering, mass spectrometry can be optimized by combining it with other methods (e.g., gel separation chromatography).

Because impurities may have the same mass-to-charge ratio as the primary analyte, combining mass spectrometry with other separation methods can increase the accuracy of sample purity determination. For example, if asymmetric peaks appear in the elution peaks of gel exclusion chromatography, mass detection will help to distinguish whether they are due to impurities of different sizes or to self-associated molecules. Unlike light scattering, which calculates mass based on the measurement of the radius of gyration (rg), mass spectrometry is more accurate in measuring the mass of proteins.

VI. Light Scattering

As described by Rhod= et al. (2009), a number of instruments are now capable of performing static or dynamic light scattering to determine individual samples or to monitor HPLC and field level (FFF) separation processes. The ability to identify eluting proteins is enhanced by the continuous determination of molecular size and apparent molecular mass, and the ability to differentiate between the analyte to be analyzed, aggregates of the analyte to be analyzed in multiple forms, or impurities. The light scattering method is very simple and non-destructive, and current instruments are capable of providing a molecular mass plot as a function of elution time. Thus, if a UV detected peak is asymmetric, the results of multiple angle light scattering (MALS); also known as multiple angle laser light scattering (MALLS) analysis may show that the apparent molecular mass of the main part of the peak is mol/L, while the edge of the peak has a molecular mass of mol/L. The molecular mass of the main part of the peak is mol/L, while the edge of the peak has a molecular mass of mol/L. The results may show that the apparent molecular mass of the main part of the peak is mol/L, while the edge is 2 mol/L, suggesting that a dimer may be present. It should be noted that the presence of a multiplicative molecular mass does not necessarily prove the presence of self-binding, and different methods are needed to verify this result. Although MALS results are not as accurate as mass spectrometry, the equipment required is cheaper and easier to use.

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