Protein concentration and solute removal experiments

Summary

Outstanding growth is expected in novel primary molecules and biosimilar entities. Some of the advances are improved analysis, development and interactions. Numerous methods are now available for the removal of concerns, including lyophilization, reverse extraction, solute precipitation, precipitation, dialysis (solvent exchange) , ultrafiltration, and chromatographic techniques. Notably, great strides have been made in miniaturization and high-throughput protein analysis, supported by numerous micro- and device developments for small-scale sample preparation. Authors: Burgess et al., Translated by Vivian Chen, this experiment is from "Protein Purification Guide".

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Protein concentration and solute removal experiments

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I. Chromatography

The increasing use of proteomics technologies over the past 2 0 years has led to continuous innovation in chromatography for protein concentration and desalting. Proteomics typically involves enrichment of proteins from small amounts of complex mixtures, including selective concentration of specific species of proteins. Due to their high sensitivity and nature of providing detailed biochemical characterization, mass spectrometry (M S ) and antibody-based detection technologies are highly useful in a wide range of applications such as clinical diagnostics, biopharmaceutical drug identification, and fundamental studies of protein structure and function. These applications require the analysis of drug targets at very low levels in biological fluids.
These applications require the analysis of drug targets and biomarkers at low levels in biological fluids, or the collection of detailed information on the structure and post-translational modifications of proteins expressed in heterologous systems. Samples used for proteomics analysis typically come from aqueous solutions, which may contain a range of non-volatile salts, solvents, dyes, detergents, liquid separators, DNA/RNA, and lipids. These contaminants can degrade the performance of highly sensitive analytical techniques, especially for electrospray ionization (ESI) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, where they can have a detrimental effect on resolution and sensitivity by interfering with the ionization of the sample, forming adducts, and fouling the ion source. As a result, many suppliers have developed a wide range of miniature solid phase extraction procedures and equipment for concentrating target proteins and purifying them from contaminating molecules in high yields.

1 Gel Filtration

Gel filtration matrices have been widely used for desalting proteins in non-denaturing situations. Unlike small molecules, large proteins do not enter the small pores of these resins, thus removing the salt during flow. Typically, the resolution of a chromatography column is best achieved when the packing volume is 4 to 20 times the sample volume and the ratio of column length to column diameter is 5 to 10. The sample is diluted at least 1.25 times in the desalting process, but with the right choice of eluent, the loss of sample can be minimal even in the ug/m L range. The polysaccharide-based
Dextran-based gel media from various vendors have been widely used for desalting, and a line of Bio-Gel P acrylamide matrices (Bio-R a d , Hercules, CA) allows the user to tailor the gel exclusion limits of the media to the molecular mass of the target protein. Additional convenience is provided by the ZebaSpin column (Thermo/Pierce, Rockford, IL), which enables rapid desalting by centrifugation of protein samples with molecular masses greater than 7,000 D a without the need for column equilibrium and gravity-flow graded separation . By selecting the appropriate column size, it is possible to provide high recoveries for protein samples as low as 25ug/m L in volumes of 2 to 4000ul, while retaining up to 95% of molecular masses less than 1000 Da. The Zeba SpinMatrix can also be used in 96-well plates to facilitate high-throughput non-denaturing desalting of multiple protein samples simultaneously.

2 Ion Exchange Chromatography

An alternative to gel filtration for protein desalting is the use of mixed-bed ion exchange (IE X) resins. The large particle size and surface modification of the mixed-bed IE X adsorbent effectively capture small anions and cations (<1000 Da), minimizing the retention of high molecular mass protein molecules. Desalting can be accomplished in batch mode or through a matrix-packed bed. However, an undesirable consequence of this technique is that the ion exchange that occurs in a mixed bed of conventional IEX adsorbents can cause acidification and precipitation of the protein sample. In contrast, the
Ion Retention Matrix, salt uptake occurs by absorption rather than ion exchange, and thus it can accomplish protein desalination without significant p H changes. commercially available such as A G ll A 8 resin (Bio-R a d , H e r cules, CA). Similar to gel filtration, the disadvantage of these techniques is that dilution of the protein sample occurs during the desalting process, and therefore concentration of the protein may be required for subsequent processing.

3 Reversed-phase chromatography

Reversed-phase (R P ) chromatography is a popular technique for separating, desalting, and concentrating proteins, due in part to the fact that the sample is concentrated in a small volume of volatile solvent that can be removed by evaporation. With the advent of mass spectrometry (M S) as a routine analytical method, a variety of reversed-phase extraction formats have been commercialized to increase the speed and convenience of the M S process. These formats include microcentrifuge columns, capillaries, microsampler tips, and microtiter plates for rapid desalting of proteins in sample volumes down to the femtomole level and elution in microliter volumes. Typically, optimal binding of proteins to the gels in the purification columns is achieved in the presence of p H < 4 and ion pairing agents (e.g., ○.1 % ~ I.0 % T F A ). Reversible binding of proteins to the gel in the purification column may be facilitated by the presence of an organic component, typically 15 % acetonitrile or methanol, or a liquid-liquid separator (e.g., guanidine hydrochloride). If contaminants such as detergents are present, the sample may need to be diluted to prevent them from interfering with protein binding to the adsorbent. Desalting operations require cleaning of the resin, e.g., with 5 % methanol, 1 % T F A, etc. Selective elution may be achieved by performing experiments with alternative ion-pairing agents. The choice of reversed-phase adsorbent is based on the size of the protein, and aliphatic C 4, C 8, C 12 and C 18 compounds are usually used. An example of a micropipette-based product is ZipTips (Millipore, Billerica, M A ), which are filled with reversed-phase resin particles or N u T i p s (G l y g e n , C o l u m b i a , M D ) , and the inner wall of the tip is encapsulated with a binding material, thus alleviating the backpressure and minimizing nonspecific interactions between the sample and the tip surface. For high throughput desalting of multiple samples simultaneously, it is also possible to place the reversed-phase resin in a 96-well plate, an example of which is the ZipPlate model (Millipore, Billerica, M A ).

4 Hydrophobic and Affinity Analysis

In addition to the commonly used gel filtration, ion exchange, and reversed-phase adsorbents, other materials, such as batch or microdevice resins, are available to support a variety of modes of chromatography for the concentration and desalination of proteins. These include hydrophobic, hydrophilic, metal chelating, and protein affinity resins. Notably, hydrophilic media provide a useful alternative to reversed-phase chromatography when the protein sample is suspended in an organic solvent and contaminating salts or detergents need to be removed. For example, PAGE-P r e p media (T h e r m o /Pierce, Roc k f o r d , I L) can be used for desalting proteins prior to SDS-PAGE analysis when proteins are bound to the resin in the presence of 50 % dimethylsulfoxide. Alternatively, Glygen (Columbia, MD) offers hydrophilic media (N u T i p ) in the form of micropipette tips. Similar to reversed-phase media, a potential disadvantage of hydrophilic resins is that proteins are often recovered in a denatured form. In contrast, the more important immobilized gold
The use of metal affinity chromatography (I M A C ) adsorbents and, more recently, media containing _TiO2, have been selectively used for the concentration and desalting of phosphopeptides prior to M S analysis. This technique has particular application in the field of signal transduction studies, since the detection of low abundance phosphopeptides is essential for understanding cellular responses. A recent report (Hsieh et al., 2007) describes the preparation of T i O 2 nanoparticle matrices capable of selectively binding phosphopeptides in solutions containing 0.兾 % T F A and 50 % acetonitrile ( p H 2. 0) that prevent nonspecific binding. In this case, elution is accomplished by 100 m m o l /L ammonium phosphate at P H 8. 5, which is compatible with assays such as M A L D I -M S for phosphorylated peptides and does not require a further desalting process.

The routine preparation of protein samples for mass spectrometry (M S ) analysis using high performance liquid chromatography (H P L C ) techniques has driven suppliers to offer their absorbents in a range of columns, sleeves, capillaries, etc., which continue to appear and increase in number. This in turn has allowed sample preparation to be streamlined through the use of column switching procedures. This approach starts with rapid concentration and desalting of the protein sample using a short preloaded column, followed by high resolution separation of the protein in the same H P L C system by using a long column. For example, a P e p M a p micro-pre-packed column (Dionex, Sunnyvale, CA) can be used for rapid concentration and desalination of protein samples with a packed bed of only 5 m m in length. Due to the selectivity of the resin used , the analyte trapping efficiency is governed by the choice of packing material, and the reduction in sample volume improves the resolution of subsequent chromatographic steps. In this context, it is also worth commenting that monolithic separation materials are increasingly being used for protein concentration and desalting (Schley et al., 20 0 6). Effective pore sizes of up to 20 um allow rapid solute transfer through the monolithic matrix. In contrast to the slower diffusion-mediated liquid-phase mass transfer achieved within the submicron pores of particle-based adsorbents, the monolithic media allows for efficient separations at high flow rates and low backpressures, which is particularly beneficial for rapid desalination via column switching. However, in a more direct approach, Mass-Prep c o l u m n (Waters, Milford, M A ) has been used for on-line desalting of proteins in hydrophobic matrices, where samples can be injected directly into the E S I mass spectrometer ( W h e a t
etal., 2007). In addition, sample preparation for M A L D I -M S analysis can be performed without the need for a microextraction device. So , numerous adsorbents have been used to desalinate samples in batches before loading the proteins separately onto M A L D I plates. An example of a recent development is the Z n O poly (methyl methacrylate) nanoparticle adsorbent, which eliminates saturated levels of N a C l (6.2 m o l /L ), N H ₄ H C O ₃ (2. 6 m o l /L ), or I m o l /L of urea from protein samples prior to M A L D I -M S (S h e n et al., 2008). Similarly, desalination was achieved by using a hydrophobic matrix added directly to the plate wells of the M A L D I plate (Jia et d . , 2007). In this case, the target protein is captured by the matrix, and the sample is then dried and desalted using a minimal on-plate wash, at which point the sample is ready for MALDI-MS analysis without further purification.

5 Production scale analysis

Similar to small-scale extraction for analytical purposes, preparative scale and production scale protein recovery often includes concentration and desalting. Column chromatography remains the core technology for protein purification, due to the high selectivity that can be achieved, along with the convenience and durability of a packed resin bed in which the feedstock is transported through the resin by flow. Commercial production of enzymes and biopharmaceuticals typically requires the processing of thousands of liters of initial feedstock. In order to minimize operational complexity and reduce processing time while effectively controlling costs, it may be necessary to concentrate the feedstock early in the downstream process to remove most impurities and unnecessary salts. This can be accomplished by conjugate-elution chromatography using a bed of large resin chromatography columns up to 2 m in diameter, which produce
The resulting elution fraction is less than the sample volume. In addition, selective resins can be developed to purify the corresponding protein products. A notable example is the use of Protein A affinity resins to produce antibody proteins expressed by mammalian cells. During the affinity capture process (mediated by specific epitopes in the Fc region of the target molecule), most of the nutrient complexes, salts and impurities pass through the column. Product recovery is usually achieved by elution with a low pH eluent, which typically reduces the volume of the eluted product by 90%. Newer generations of protein A affinity resins have been optimized, primarily in terms of ligand density, particle structure and mass transfer characteristics, to maximize dynamic binding capacity while minimizing column cycling. This in turn minimizes the interstitial volume and effectively concentrates the sample stream in the first step of the process. Typical examples for resins are MabSelect (GE Healthcare, Piscataway, NJ) and ProSep Ultra Plus (Millipore, Billerica, MA), which have a binding capacity of 20-50 g of protein per liter of resin. This binding capacity depends on the nature of the target protein and its contact time with the resin during column loading. Traditional analytical resins, including IE X and hydrophobic chromatography (HIC) absorbents, are similarly optimized in terms of particle size, pore size, and ligand density to achieve the highest binding capacity for a specific range of molecular masses while allowing for efficient elution. For example, ToyoPearl GigaCap S-650M (Tosoh Biosciences, Tokyo, Japan) is a cation exchange resin that has been optimized for capturing Ig Gl molecules with a molecular mass of approximately 150 kDa, and it has a high dynamic binding capacity (>100 g/L resin) while allowing recovery with a minimum elution volume. It has a high dynamic binding capacity (>100 g/L resin) and allows recovery with a minimum elution volume, usually 2 to 3 column volumes. Finally, absorbents have been developed in the hybrid mode, which allows for high-performance binding of IEX layers at relatively high conductivities, and is accomplished by combining equal amounts of IEX and hydrophobic bases to the same ligand. One example is Capto MM C resin (GEHealthcare, Inc.).
One example is Capto MMC Resin (GEHealthcare, Rscataway, NJ), which combines a cationic exchange and a hydrophobic group with a binding capacity for bovine serum albumen of 45 mg per milliliter of resin at a conductivity of 30 mS/cm. This property is particularly useful because optimal purification of proteins is usually achieved by using multiple orthogonal model analyses, which are performed in separate steps; while maintaining the electrical conductivity of the raw material is not a problem. Maintaining the conductivity of the material at a level that is compatible with the traditional IE X step without dilution is very challenging. Therefore, while this type of absorbent may not directly concentrate the material, it may reduce the amount of dilution required to reduce the conductivity in the downstream process. Mixed-mode resins are even more attractive because they offer unique selectivity, which may increase the efficiency of the overall purification process.

II. Electrophoresis

Protein concentration by gel electrophoresis is often used in subsequent analytical techniques to enhance sensitivity. For example, the sensitivity of Western Blot can be enhanced by applying up to 250 uL of protein samples to more than 5 consecutive spikes of 1 to 2 cm of gel on a microgel system (Sheen and AliKhan, 2005). This method allows for effective sample concentration, with vertical band broadening as its only human trace. Prior to Western Blot, similar concentration of diluted samples could be accomplished through a funnel hole. Capillary electroswimming (CE) can be used to concentrate protein samples by stacking (see also
Shihabi, 2002). In addition, a recent report describes the capillary electrophoresis conditions for simultaneous protein concentration and desalting. This is a significant technological advancement because protein usually needs to be desalted before capillary electrophoresis, and the salt ions can lead to band broadening through effects such as Joule heating and electrodispersion. In addition, when the ionic strength of the capillary electrophoresis sample is lower than that of the electrolyte, a desirable stacking effect can be achieved, leading to protein enrichment and increased sensitivity for analytical applications. Desalting of proteins prior to capillary electrophoresis can be accomplished by standard reversed-phase extraction. Although solid-phase microbeds that allow on-line desalting during capillary electrophoresis experiments have been reported, however, this method may still be technically very challenging, so desalting is usually performed as a separation step. To overcome these drawbacks, a new technique known as capillary isoelectric trapping has been developed, which allows for simultaneous concentration and desalting of protein samples during capillary electrophoresis (Booker and Yeung, 2008). This method uses a discontinuous buffer system that creates a stable p H boundary within the capillary. The stationary boundary traps partially ionized proteins at p i while eliminating the
contaminating ionic salts, which continue to migrate due to their constant charge. This technique facilitates the recovery of concentrated and desalted protein samples for use in M S or immediately after switching to another buffer system that allows for capillary zone electrophoretic analysis of concentrated proteins. Finally , microfluidic devices are increasingly finding their utility in sample preparation prior to biochemical analysis, in part due to their ability to rapidly concentrate and desalinate small amounts of protein samples through the use of an electric field [see, e.g., Y u et al. (2008)].

III. Dialysis

Dialysis is a liquid phase separation technique based on molecular mass size, which is achieved by selective diffusion through a semi-permeable membrane. This technique is considered the most popular method for removing small molecule solutes from large proteins. In particular, this non-denaturing desalting technique allows for buffer changes under benign or physiological conditions, where the risk of affecting the properties of the target protein can be minimized.

The principles of dialysis have not changed over the past decade, but the techniques and tools used for dialysis have been improved. In particular, optimized techniques have resulted in high throughput (reduced processing time), increased flexibility in sample size (from 10 uL to 100 mL); improved dialysis membrane morphology has reduced protein adsorption and loss, and increased ease of sample handling.

Typically, the volume of the destination buffer (dialysate) is several orders of magnitude larger than the volume of the protein sample. The sample is transferred into a sealed chamber and exposed to a dialysis membrane that acts as a semi-permeable barrier. The dialysis membrane usually has a specific pore size, the molecular mass cutoff (M W C O ), which allows molecules with a molecular mass less than this limit to pass freely in both directions, while large molecules (e.g., proteins) are retained. The concentration gradient of the buffer on either side of the dialysis membrane drives the diffusion of solutes from regions of high concentration to regions of low concentration (Fig. 9. 1). The flux of diffusive transport can be described by Fick's law of diffusion.

Several aspects can affect the stable recovery of dialysis products, including the time required for buffer exchange, the design of the dialysis system, and the chemical and morphological characteristics of the dialysis membrane. The time required to complete buffer exchange depends on several factors. The time of the dialysis process can be reduced by increasing the intrinsic diffusion of molecules across the semipermeable membrane. Based on the previous equations, substance transport can be improved by increasing the membrane area or the solute flux. When the sample volume is small, a large ratio of sample to dialysate can be easily obtained. However, proteins can adsorb to the dialysis membrane and cause losses. In this context, the effect of increasing the membrane area or increasing the time (for smaller membrane areas) needs to be evaluated. Since the diffusion coefficient of a solute varies with temperature, an increase in temperature will increase the intrinsic diffusion coefficient by accelerating molecular motion, leading to faster transport. However, elevated temperatures can adversely affect the stability of the sample. Since the integrity of the sample is often paramount, it may be necessary to predetermine the maximum temperature at which the protein can be stabilized.

The design of the dialysis system can also determine the time to effective dialysis and the efficiency of product recovery. Solutes diffuse from the sample across the membrane into the boundary layer of the dialysate. The concentration of solutes in the boundary layer is higher than the concentration of solutes in the rest of the dialysate, thus reducing the concentration gradient across the membrane and inhibiting diffusion. However, the boundary layer can be reduced by mediating the convective flow of the dialysate or by constant stirring.

Currently, the mechanical design of dialysis membranes and tubing allows for the processing of a wide range of sample volumes (from 10 uL to 100 mL) with negligible loss of product. Traditional dialysis tubing undergoes complex preparation before it is ready for use. SnakeSkin® dialysis tubing from Pierce Biotech (Rockford, IL) simplifies the dialysis of large sample volumes. The core of this technology is a pleated, regenerated cellulose membrane (3.5 to 10 kDa MW CO), which allows for rapid preparation. For dialysis of small sample volumes, Pierce offers the simple SlideA-Lyzer® Micro Dialysis Tubing.
(10 ~ 100 uL) and dialysis cassettes (0.1 ~ 30 m L) for processing limited, small volumes of biological samples. These dialyzers are disposable cups made of polypropylene and recycled cellulose that allow for easy sample removal using standard laboratory pipettes. The new design improves recovery of valuable small sample volumes.

1 Application of Concentration and Affinity Bonding

The application of protein buffer exchange dominates the use of dialysis membranes. However , dialysis technology is increasingly used in the field of concentration and macromolecular affinity binding interactions.

Concentration of proteins using dialysis membranes is analogous to biphasic extraction, in that a biphasic system is created by the use of a reagent (e.g., polymers such as polyethylene glycol, dextran, etc.), and the dialysis membrane separates the two phases (Waziri etal., 2004). According to the design of the system, proteins are free to pass through the membrane and interact with the precipitant, which is subsequently concentrated and the product is recovered.

This technique is known as equilibrium dialysis, and it is also used for the identification of drug candidates in serum binding assays, the study of antigen-antibody interactions, and the evaluation of low-affinity interactions that cannot be detected using other methods. In equilibrium dialysis, the membrane is selected under the following conditions: the desired protein or ligand can pass freely; the receptor molecule can only be located on one side of the membrane and is not permeable. When proteins diffuse across the membrane, some of them bind to the receptor, while others remain in solution. The diffusion of ligands across the membrane and onto the receptor continues until equilibrium is reached.
The concentration of free ligand in the ligand bath can also be used to assay the binding characteristics of the sample (Waters et al., 2008).

Ultrafiltration

Ultrafiltration (U F ) utilizes a separation mechanism that is essentially equivalent to dialysis. Both techniques utilize a semi-permeable barrier or membrane to separate the sample (containing the desired product or solute) from the solution. The membranes are designed to allow small molecular masses of solutes (e.g., salts) to percolate through, while the desired product is completely retained. Ultrafiltration membranes are well suited for concentrating biologics because they can be operated over a wide temperature range (2 to 26°C) and do not involve phase changes or chemical additives, thus minimizing denaturation and/or degradation of unstable biologics. Ultrafiltration can be distinguished from dialysis in 3 ways: ①)
application; ② mode of operation; and ③ membrane structure. First, dialysis is mainly used for solute or buffer exchange, while ultrafiltration is used for buffer exchange or product concentration. Secondly, dialysis is carried out by diffusive transfer driven by the concentration difference between the two sides of the semi-permeable membrane, whereas ultrafiltration is mainly driven by the pressure difference between the two sides of the membrane and works by conventional mass transfer. Due to the use of different driving forces for solute or solvent transfer, membranes used for ultrafiltration are mechanically stronger than dialysis membranes. Pressure-driven UF membranes are designed to withstand high pressure differentials in excess of 75 psi1 .

In general, three model approaches have been used to describe real mechanisms for solute transfer through ultrafiltration (Denisov, 1994). One of these ultrafiltration transfer mechanisms is shown in Figure 9.2. A pressure-driven flow of fluid causes solute and solvent transport and permeation to the upstream surface of the membrane, and the difference in pressure exerted is called the transmembrane pressure (ΔP _tm ). If the membrane can completely retain a given solute (e.g., protein), as in the case of concentration by ultrafiltration, C _effluent = 0, the retained solute will accumulate on the upstream surface of the membrane and will result in an increase in the concentration of the membrane surface within the thickness δ of the boundary layer. This phenomenon is called concentration polarization. Solute accumulation on the membrane surface affects the solvent filtration flux through several mechanisms (J v). First, the accumulated solutes can create a differential permeability pressure along the membrane that drives the flow from the filtrate toward the feed or seepage residue, thereby reducing the net solvent transport rate. See below and Figure 9.2 for a description of fluxes.

Overall, these models allow the user to better understand the impact of ultrafiltration process parameters on solute and solvent flow, and to optimize conditions to increase membrane flux. Perhaps the most important application of these models is the development and design of ultrafiltration systems for large-scale concentration of expensive products, such as proteins for disease treatment, where both the productivity and recovery of the concentration process are critical. For laboratory-scale ultrafiltration, these factors are not critical; however, they should be considered in the design of ultrafiltration systems for small-scale preparation.

1. Ultrafiltration Membrane

In general, most ultrafiltration membranes contain a strong support structure (e.g., T y v e k ® ) to which a very thin polymer layer is attached. It is this thin layer that actually provides the properties required for a selectively permeable membrane and determines the flow resistance. The technology for producing membranes for ultrafiltration has not changed in the last decades. However, significant improvements have been made in the control of membrane pore size distribution, membrane morphology, and membrane modification. The three main casting techniques for UF membranes are air casting, dip casting and melt casting. They differ mainly in the method and equipment used for desolventization (Zemán and Zydniny, 1996). Current membrane compositions can accommodate a wide range of polymers that can form copolymers with ultrafiltration membranes by joining, fusing, and coating, thereby improving chemical and mechanical stability. Fiber
cellulose-based polymers provide the low protein binding required for many biotechnology applications. However, cellulose-based membranes generally degrade when exposed to alkaline hypochlorite cleaning solutions. New cellulose-based composite membranes, such as Ultracel® from Mllip0r e, offer low contamination and low protein binding, resulting in excellent product retention, recovery properties and higher yields. These Ultracel membranes are constructed by molding renewable cellulose membranes onto a porous material substrate, which provides excellent robustness of these defect-free membranes compared to conventional membranes. Composite technology provides a mechanically strong design that can wit

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