Hydroxyapatite columns for protein chromatography experiments
Hydroxyapatite columns for protein chromatography experiments
Hydroxyapatite (HA) is a calcium phosphate-based hydroxylate that has been used extensively in the chromatographic separation of proteins, mainly in 1991-2009, and initially only for the purification of recombinant proteins.The use of HA is described in Tiselius et al. (1956) and reviewed by Gorbunoff (1985). (1956) and Gorbunoff (1985).
Operation method
Hydroxyapatite columns for protein chromatography experiments Move I. Mechanisms The adsorption and desorption of proteins by HA has been regularly reviewed since 1971 (Bernardi, 1971; Gorbunoff, 1990). A more recent paper (Kandorietal., 2004) cites an earlier mechanism, in which acidic proteins bind via the C (calcium)-site and basic proteins bind via the P (phosphate)-site.The role of the C- and P-sites in monoclonal protein adsorption and desorption has been first proposed by Kawasaki et al. The importance of the C-site in monoclonal antibody purification was further discussed and emphasized by Gagnon (1996)Xt: part of the monoclonal antibody binding is via calciumcoordination complexes interacting with the carboxylcluster in the antibody. The HA-protein interaction can be simplified as follows: the amino group can be adsorbed to the P-site but is repelled by the C-site. are repulsed by the C-site; for the carboxyl group, the situation is reversed and more complex. Although amines can bind to the P-site, their carboxyl groups are initially attracted to the C-site by electrostatic interactions, and binding to the C-site involves chelating ligand bonds that are stronger than anion exchange. For example, as described by Kawasaki (1991), the phosphoryl group is stronger than the carboxyl group for the interaction of proteins and other solutes with the C-site. Further work by Don et al. (2007) centered on bone-protein interactions with the aim of investigating the mechanical interaction of an OH group and an NH2 group with a net charge of o in proteins with the HA surface. Controlled molecular dynamics simulations showed strong interactions between the HA surface and the OH group and the _NH2 group of bonemorphogeneticprotein (a growth factor for bone formation) via water-bridged H-bonds. Shen et al. (2008) used a fibronectin fragment (FN-DIlO) to confirm the observations of Don et al. The above and some similar studies support the C-site and P-site models. HA surfaces have been suggested for the purification of histidine-tagged proteins (e.g., hexameric histidine-tagged proteins) and for the purification of histidine-enriched immunoglobulin G (IgG), respectively (Ngetal., 2007). In this case, based on the co-elution of hexameric histidine-labeled and unlabeled fusion proteins, the trans-histidine interaction would appear to be minor. The protein adsorption mechanism is consistent with that previously described by Gorbunoff (1990), which was improved by the introduction of the water-bridge H-bonding mechanism of action. Some proteins will not adsorb to HA if phosphate is present in the sampling buffer, and Ferguson et al. (1980) suggested that Good's buffer can usually be used in such cases. For example, Schirch et al. (1985) purified E. coli-expressed serine hydroxymethyltransferase by first obtaining a homogeneous sample by anion exchange, then equilibrating the HA chromatography column with pH 7.0, 20 mmol/L JV, JV-2-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) as the buffer, and then eluting it with a small phosphate gradient. Protein desorption can be achieved by reversing P-site interactions, both forms of C-site interactions, and/or H-bonding. Phosphate buffers with linear and stepwise gradients are the most commonly used desorption reagents. In addition, Freitag and Breier (1995) suggested that proteins can be immobilized in chromatographic columns by electrostatic interactions between their net positive charge and the net negative charge of the HA surface, but that proteins can be desorbed by the addition of anions to phosphate buffer or those immobilized proteins can be desorbed by the addition of cations through the interactions of their net negative charge with the net positive charge of the HA surface. The role of Cr, F'ClOr, SCN- and phosphate has been discussed in a series of articles by Gorbunoff (1984a;l984b) and Gorbunoff and Timasheff (1984).Schktterer et al. (2006) used fluoride ions to remove the prostaglandin D synthase from the HA surface. Schktterer et al. (2006) used fluoride ions to desorb prostaglandin D synthase from a ceramicized HA chromatography column. The enzyme, derived from bovine cerebrospinal fluid, was adsorbed and then subjected to a series of desorptions with 10 mmol/LpH6.25 sodium phosphate buffer containing NaF. They used 150~750 mmol/L NaF and 375~750 mmol/L NaF as linear gradients, respectively. The desorption was also significantly affected by the pH of the buffer. According to Ogawa and Hiraide (1996), the elution of proteins at lower phosphate concentrations was more rapid at pH greater than 7.0 than at pH less than 7.0, and the order of elution of porous ceramicized HA was similar for both type I and type n. However, protein desorption required a more potent sodium phosphate buffer for type I columns. Similarly, the pH of the buffer also affects protein desorption on porous ceramicized fluorapatite columns at pH 5.5-6.8. Table 24.1 compares the elution times for the separation of a variety of proteins in the above ranges using an I-ICmX15 cm type I ceramicized HA and ceramicized fluorapatite column. The columns were filled with pH 10, 0.2 mol/L phosphate buffer and equilibrated with 5 mmol/L sodium phosphate buffer at pH 5.5, pH 6.OSpH 6.8. The elution order of proteins does not depend solely on their PI. according to Gorbunoff (1984a; 1984b), the desorption of certain proteins also depends on their structure and the content of the carboxyl group. Calcium and magnesium ions modify the HA surface by forming phosphate-Ca or phosphate-Mg bridges, and although originally proposed more than 20 years ago, it was only recently that Gorbunoff (1984a; 1984b) and Gagnon et al. (2009) used Ca-modified ceramicized HA surfaces to improve the purification of Fab from Mab and Fc antibody fractions. These studies showed that Ca-modified ceramicized HA could be restored to its natural form after treatment with 20 column volumes of a lower concentration of phosphate buffer. HA also has a high affinity for pyrophosphate (PR) and polyphosphate as described by Krane and Glimcher (1962). PH is a contaminant in anhydrous sodium dihydrogen phosphate and disodium hydrosulphate, and perhaps also for the two corresponding potassium salts. The presence of PR in phosphate buffers has both advantages and disadvantages relative to the adsorption and desorption of proteins by the ^^8. As shown in Fig. 24.i, the separation of bovine serum albumin, ovalbumin, pancreatic rennet protease proteins and again cytochrome c was carried out using a phosphate buffer prepared with anhydrous sodium phosphate. The separation of the above proteins using sodium phosphate buffer prepared with Na2 HPO347 s o and NaHJO4 h2o is shown in Fig. 24,2. The sodium phosphate buffer used for the chromatography column was prepared using anhydrous sodium phosphate and was cycled through 4 procedures to minimize the effect of PR (Fig. 24.1). PPi helps ha the chromatographic separation of proteins, especially for some weakly adsorbed proteins. However, care had to be taken to ensure that the levels of higher order phosphates were consistent between all experiments to avoid human error. Accumulation of metals after repeated use can lead to discoloration of chromatography columns. Causes of discoloration can be found in dental researchers studying dental caries, medical researchers studying bone integrity and tissue fluid processing of artificial bones or cadaveric implants (cadaverimplants), and material scientists testing for soil contaminants and metal adsorption. According to Shepard et al. (2000), ceramicized HA chromatography columns undergo discoloration after several cycles of recombinant protein purification. In their chromatography process, ceramicized HA column chromatography is the third step in the chromatography process after cation-exchange and anion-exchange chromatography. They concluded that there are multiple sources of metallic substances that cause ceramicized HA to discolor: process equipment, reagents, water, and fermentation nutrients. However, discoloration of the columns did not affect their ability to purify recombinant proteins. Agronomists have explained this by the preferential adsorption of polyvalent metals by HA (Maetal., 1 i.e., 4) and have demonstrated that HA has a high adsorption capacity for divalent heavy metals. Many of these metals, especially Fe, Al, Zn and Mn, were observed during the discoloration of the ceramicized HA chromatography columns. Large amounts of buffer are used in the chromatographic process, which can create voids at the top of the column or channels in the HA. HA has a solubility constant of 2.4 X KT59, which is equivalent to about 4 ppm. phosphate ions and basic pH inhibit HA solubilization. However, HA can adsorb proteins from very dilute solutions such as Immol/L (pH 6.8) phosphate buffer, and can be bufferless alkaline metal solutions, usually Immol/L NaCl, KCl, or amphoteric buffers.Schirch et al. (W85) demonstrated that some acidic proteins are adsorbed only in water. As explained by Scopes (1993), lower unbuffered loading conditions reduce the control of pH at the HA surface. Researchers (Schroderetal., 2003) have shown that it is possible to use 4-X# oxazepane cyclopropanesulfonic acid (4-morpholinepropanesulfonicacid (MES)) in phosphate buffer to maintain phosphate elution while still maintaining the pH of the sample buffer and elution buffer; however, even though phosphate is used to inhibit HA solubilization, the solubility of HA can be inhibited by the use of phosphate. solubilization of HA, its solubility is still a risk factor. For example, an undefined 0 mmol/L sodium phosphate equilibrated column at pH 6.5 was used and the sample was loaded with equilibrium buffer, followed by sequential elution by increasing the NaCl concentration in 20 mmol/L sodium phosphate buffer. As this cycle continues, voids form in the column and pressure builds up. These voids and the resulting pressure are due to chemical damage to the HA, which is primarily caused by the dissociation of hydrated hydrogen ions accumulated on the surface of the HA. Figure 24.3 shows the change in pH of the effluent and the areas of hydronium hydrate ion adsorption (A and B) and adsorption (C and D). Harding et al. (2005) suggested that the adsorption and desorption of hydronium hydrate ions is a function of the zeropointcharge of the HA. In addition, studies (Skartsila and Spanos, 2007) have demonstrated the relationship between the zeropoint charge, pH, adsorption and desorption of hydronium hydrate ions, and calcium ion content.The zeropoint charges measured by Skartsila and Spanos were inconsistent, being 7.3±0.1 and 6.5±0.2, respectively.The same studies have explained the discrepancy between the pH of the infusion buffer and the pH deviation of the effluent from the column. These studies also explain the difference between the pH of the input buffer and the pH deviation of the post-column effluent. It is reasonable to assume that changing the pH of the input buffer or increasing its buffering capacity would reduce the intensity of the pH change and possibly shorten the duration of the change. However, it has been noted that some proteins do not adsorb to HA when the phosphate concentration is too high, and as some process development engineers have pointed out, even 5 mmol/L phosphate can inhibit protein adsorption in some cases (McCueetal., 2007). Selection of an appropriate buffer can help maintain the integrity of the target protein while facilitating its adsorption to the HA.This step is best accomplished at equilibration, which should be free of any phosphate, and then loading the buffer. However, after a few cycles, the packed column fails. It has been shown that phosphates as low as 2rmnol/L can extend the life of the column while maintaining the purity of the target protein. Phosphate in combination with MES or with 3-(N-morpholino)propanesulfonic acid (MOPS) and a small amount of calcium ions can sufficiently inhibit HA solubilization. These process development engineers did not explicitly mention pH conversion (McCueetal., 2007). The advantage of using MES buffers is shown in Figure 214, which minimizes the pH change and its duration observed in Figure 24.3. Calcium ions were detected in the elution fractions of both system buffers containing high concentrations of NaCl. For the 0~02mol/L sodium phosphate buffer, the calcium ion concentration was 36 ppm; for the 75 mmol/LMES/0.02mol/L mercurial buffer, the depletion ion concentration was 5 ppm. Most proteins can be adsorbed to HA in phosphate buffers with low ionic strengths and concentrations as low as I_ol/L. For adsorbed proteins, high concentrations of phosphate containing NaCl or KCl (salts) are commonly used for elution. Elution of proteins with other types of desorption solutions is not common. If the protein sample contains a large amount of salt, it can be diluted sufficiently, but the phosphate concentration should not be less than Immol/L to ensure that the sample will adsorb to HA. if other buffers are used, they should be at a concentration sufficient to control the pH. the proteins are first eluted in a linear salt gradient to check for desorption. If the protein is still bound to the column, the procedure can be repeated using a linear phosphate gradient as the eluent. The purity of the protein is tested and the salt concentration or phosphate concentration required to elute the protein is determined and used in the elution step of the purification protocol. Compare the purity of the proteins obtained from the stepwise elution and the gradient elution. Adjust the purification protocol accordingly if needed. If other types of bound proteins and biological components need to be desorbed from the chromatography column, wash with 3 column volumes of low ionic strength (LIS) phosphate without other salts, followed by 3 to 5 column volumes of 0.5 mol/L pH 7 phosphate buffer, 3 column volumes of LIS buffer, and 3 to 5 column volumes of 0.5 to 1 mol/ LNaOH. LNaOH for washing. To prepare the column for the next round of separations, 3 column volumes of LIS Buffer can be used, followed by 3 column volumes of 0.5 mol/L Phosphate Buffer at the same pH as the equilibration buffer, and then 3 column volumes of Sample Buffer can be loaded. The packing scheme of laboratory scale HA columns varies depending on the source of HA. The columns used for filling with HAUltrogel adsorbent have an aspect ratio of 1 to 6 and a diameter of 1.6 to 5.0 cm. The adsorbent should be prepared by gently turning the container or by stirring with a plastic stirrer. An appropriate volume of adsorbent slurry should be poured into the vacuum vessel, which should be approximately twice the volume of the column to be filled. Then dilute the slurry by adding about 40% more water. After gentle mixing, a vacuum pump is connected to remove any undissolved air. The degassed adsorbent is slowly stirred to obtain a homogeneous suspension. Pour it into the column in one go, trying not to allow air to enter the slurry stream. Allow the suspension to settle for 5~10 min until a clear liquid layer of Icm is visible at the top of the column. Insert the population adapter into the column and connect it with the liquid chromatography equipment, then run the column with water or equilibrium buffer at a flow rate of 55~250 cm/h, depending on the height of the bed; for a 20 cm high bed, the flow rate of 55 cm/h can be used, for a 5 cm high bed, the flow rate of 250 cm/h can be used. Adjust the position of the population adapter so that it is in contact with the surface of the packed bed to remove trapped air. If air suddenly enters the column, the column must be refilled as described above. CHT is a spherical porous ceramicized medium. Compatible columns include MilliporeVantageL and WatersAP with variable height adapters rated above 3 bar. The weight of CHT required can be calculated based on the size of the packed bed and the density of the CHT. For example, an I.Icm I.D. X 20.0 cm high column with a volume of 19 mL should contain 12 g of CHT, which should be suspended with a plastic stirrer in 3.5 times the volume of filler buffer (0.2 mol/L Na2P047 H20, pH 9~10). The filler buffer was chosen to have an ionic strength of at least 0.Imol/L and to contain at least 20 mmol/L of buffer components at pH 6.8-10. The suspension was allowed to stand so that air enclosed in the filler particles could be separated. Connect the filler extension tube to the column so that all suspended ceramic HA can be processed in one step. fill the column/extension unit with the filler buffer to a height of 1-2 cm. stir the filler particles in the beaker with a plastic stirrer to suspend them, and then pour them into the column/extension unit. Wash the remaining ceramicized HA with Filling Buffer and pour into the column/extension unit. Open the outlet of the column and fill with ceramicized HA by gravity flow until the height is constant. Close the column outlet, remove the extension tube, and insert the inlet adapter so that it is lowered just enough to touch the surface of the gravity-filled ceramicized HA. Connect it to the LC equipment, open the column outlet, and equilibrate the filled ceramicized HA with 3 column volumes of Filling Buffer at a flow rate of 250 cm/h. Adjust the position of the adapter so that it just touches the surface of the ceramicized HA resulting from the flow described above. If air suddenly enters the packed bed, it can usually be removed during the adjustment of the column. Conditioning of the column: Start with 3 column volumes of Equilibration Buffer, then treat the column with 3 column volumes of 0.5~Imol/L NaOH, 3 column volumes of 0.4~0.5mol/L Sodium Phosphate Buffer (pH 6.5~7.2), and 3 column volumes of Equilibration Buffer to acclimatize the column to its new environment. The equilibration buffer is usually a buffer with low ionic strength, such as 2-20 mm0l/L phosphate, and GoocPs buffer should be added when the phosphate concentration is 2-10 mmol/L. The equilibration buffer is usually a buffer with low ionic strength, such as 2-20 mm0l/L phosphate. Microcrystalline HA can be purchased from various companies. Before filling the column with Bio~GelHTP and HT, preparations should be made. After slowly decanting the supernatant of the HT, add an equal volume of Filling Buffer. Suspend the HT by rotating the vessel and pour 2 times the volume of the column to be filled into a beaker. Allow the suspension to settle for 30 min before gently pouring out the supernatant, which may contain fine particles. For HTP, use Ig dry powder for every 2.5 mL of target fill volume. Place the powder in a beaker 2 to 6 times the target fill volume and mix slowly with a plastic stirring bar. Sediment the suspension for 30 min and gently pour out the supernatant, which may contain fine particles. Add fill buffer equal to the target fill volume to obtain a 50% (V/V) suspension. Gently stir with a plastic stirring bar to suspend the HT or HTP, and fill the column with all of the suspension using a funnel or column extension device. after 10 min, open the column drain and fill the column by gravity flow. Do not allow air to suddenly enter the packed bed. When approximately 2 cm of buffer remains at the top of the column, close the outlet, remove the extension tube or funnel, and insert the inlet adapter and lower it to remove air from its access path. Continue to lower the adapter until it just touches the surface of the gravity-filled bed. If DNAGradeBio-GelHTP is used, preparation and filling are similar to HTP. HT and HTP have a maximum flow rate of 100 cm/h; DNAGradeBio-GelHTP has a maximum flow rate of 40 cm/h. For CalbiochemHydroxylapatiteFast; Flow and its High-Resolution For CalbiochemHydroxylapatiteFast; Flow and its high-resolution columns, pretreatment and packing were similar to those of HTP and DNAGradeBkKielHTP, respectively.Clarkson's ceramicized HA and HypatiteC were also similar to HTP and HT in terms of pretreatment and packing. Suppliers of HA do not usually list the full HA product catalog. Table 24.2 lists some of the current suppliers of HA and ceramicized HA. There are several methods of filling CHT columns, the choice of which depends on the type of column and equipment used. Prior to filling a column, you should refer to the instruction manuals for the column, media transfer equipment, and media filling equipment. The maximum packed bed height for open columns should not be greater than 50% of the distance between the surfaces of the media retention plates (glazes or meshes), such as EasyPack (BiCrRadLaboratories), BPG (GEHealthcare), and Moduline2 (Millipore). For example, if the distance is 54 cm, then the maximum fill height is 27 cm. also calculate the volume of the filled column. For each liter of packed bed volume, use 630 g of dry powder and 1.79 L of Filling Buffer to prepare a 50% (V/V) slurry. Close the discharge port of the chromatography column and introduce the Filling Buffer, followed by the dry powder. The CHT-buffer mixture is stirred with a plastic stirrer to hydrate the dry powder, and the two are mixed to form a homogeneous slurry. Reverse the agitation to minimize slurry movement. Install the tip adapter according to the manufacturer's instructions and insert it into the column body. after 5 min to form a resin-free zone, lower the adapter so that the internal air can escape through the tip inlet, and clean the flow collector on the adapter and the inlet wiring with filling buffer. Fill at a flow rate of 200-300 cm/h and run 2 column volumes of Fill Buffer. Once the fill bed is secured, the adapter can be lowered so that there is a 1~5 mm space between the media retention plate and the top of the fill column. Do not lower the adapter into the packed bed to avoid irreversible damage to CHT particles. For closed columns, such as InPlace (Bio-Rad Laboratories) and Bio-ProcessLPLC (GEHealthcare), the slurry should be prepared externally before filling. As with open columns, the maximum packed bed height should not be greater than 50% of the distance between the surfaces of the media retention plates (glazes). Calculate the volume of the filled column. For each liter of packed bed volume, use 630 g of dry powder and 1.79 L of filling buffer to prepare a 50% (V/V) slurry. The filling buffer was poured into the media slurry tank and the dry powder was subsequently added. The CHT-buffer mixture is agitated by a low shear hydrofoil impeller (Model A3) to hydrate the dry powder and mix the two to form a homogeneous slurry. The entire slurry was transferred to the column using a media transfer device. The air bubbles in the slurry are removed at a flow rate of 50 cm/h. The run is stopped and left to form a bubble for 5 min. After stopping the run and letting it sit for 5 min to form a resin-free zone, the adapter is lowered to allow internal air to escape through the top population and the flow collector and inlet lines on the adapter are cleaned with fill buffer. Fill at an axial flow rate of 200-300 cm/h to compress the filled bed. Continue to lower the position of the adapter until the distance between the media retention plate and the top of the packed column is 1-5 mm. When filling stainless steel InPlace or LPLC columns, maintain axial flow until the adapter reaches 2 cm above the target height, then reduce the flow rate to 10 cm/h until the adapter's signal sensor makes contact with the top of the packed bed. Microcrystalline HA is not currently used to fill production scale columns. HAUltrogel is used for large diameter shallow columns. The filling of these columns and media requires the assistance of the appropriate supplier. Evaluation of filled production chromatography columns can be based on recommendations from regulatory agencies, which are often required by end users. The U.S. Food and Drug Administration (FDA) and its counterparts in Europe, Canada, Japan, and Asia have issued guidance on the evaluation of packed chromatography columns. Some suppliers of chromatography media have also proposed simple methods for evaluating both lab-scale and production-scale columns, but it is not necessary to correlate the results of the two. Some biopharmaceutical company innovators (Teeters and Quiftones-Garda, 2005) have been wise enough to propose the use of a residencetimedistribution (RTD) to track the status of filled columns over the life of the column. The use of linear or stepwise phosphate elution remains the dominant desorption strategy in purification schemes for recombinant proteins. For human catalase expressed in Bichiria yeast, a 3-step process-ammonium sulfate precipitation, anion-exchange chromatography, and HA chromatography-was used to achieve 95% purity. A linear gradient of phosphate at 0.05-0.3 mol/L, pH 6.8, allowed the recombinant catalase to be eluted from the HA chromatography column. shi et al. (2007) obtained secreted catalase from the culture medium with a final yield of 60%. A wide range of proteins can be purified using HA chromatography columns with linear or stepwise gradients of phosphate as described by Hsieh et al. (2003), St. La nsk^ et al. (2007), Luellau et al. (1Dau 8) and Nuss et al. (2008). diSalvo et al. (2004) expressed human and two E. coli-derived 卩比咳醛激酶 in E. coli D Cell lysate particles containing the enzyme were first precipitated with ammonium sulphate and then solubilized using phosphate buffer, followed by purification with three types of chromatographic media-hydrophobic interaction gels, anion-exchange resins, and ceramicized HA. For the purification of HA from human pyridoxal kinase, 20 mmol/L sodium N,iV-2-(2-hydroxyethyl)-2-aminoethanesulfonate (BES), pH 7.3, was used as adsorption buffer, and dipotassium phosphate (dipotassium hydrogen phosphate) was used for the elution in a linear gradient up to 100 mmd/L, pH 7.3. Stuhlfelder et al. (2002) performed the purification of tomato kinase by anion-exchange chromatography, gel filtration, and ceramicized HA chromatography. Stuhlfelder et al. (2002) purified methyljasmonate hydrolyzingesterase from tomato (1^noisy^-sz'conescwZe 尬 mot) cell cultures by anion-exchange chromatography, gel filtration, and ceramicized HA chromatography. In the purification step of HA, 20 mmol/L of dipotassium hydrogen phosphate, 20 mmol/L of p-mercaptoethanol, and 0.3 mmol/L of calcium chloride, pH 6.8 were used as the adsorption buffer; the buffer was eluted with a linear gradient up to 500 mmol/L of dipotassium hydrogen phosphate, 20_0l/L of 卩mercaptoethanol, pH 6.8. In a recent utility patent, the inventors used a multi-stage chromatographic process (affinity chromatography, hydrophobic interaction chromatography, HA chromatography and anion-exchange chromatography) for the purification of recombinant erythropoietin (recombinanterythropoietin, rEPO) (Schumannetal., 2007): in the purification step using ceramicized HA, a buffer was eluted with 20 mmol/LTris, 5 minol/LCaCl2, 250 mmol/LNaCl, 9% isopropanol, pH 6.9, was used as adsorption buffer. 10 mmol/LTris, 0.5 mmol/LCaCl2, 10 mmol/LK2 HPO4, pH 6.8, was used as the elution buffer for rEPO. In another purification process regarding rEPO, the inventors equilibrated adsorbed proteins with HAUltrogel adsorbent in 0.05 mol/LTris buffer (pH 7.5) containing Imol/LNaCl and 2 mmol/LCaCl2. The rEPO was eluted with 20_ol/L phosphate buffer (pH 7.5) containing 0.005% (OT/V) polysorbate 80. Purification of immunoglobulins can also be performed using a multi-step chromatographic process. For example, Leibl et al. (1996) further purified human immunoglobulin A (IgA) from human serum CohnFrGI by heparin-affinity chromatography and ceramicized HA chromatography. In the ceramicized HA chromatography step, 10 mmol/L phosphate, 137 mmd/L NaCl, pH 7.4 buffer was used to equilibrate the chromatography, supplemented with IgA heparin fraction. The adsorbed IgA monomers were eluted with 15.3 mmol/L phosphate, 287_ol/L NaCUpH6.8 buffer, and the IgA was further purified by anion-exchange, molecular sieve, and affinity chromatography to remove IgG. in another example, hydrophobic charge induction chromatography (HCH) was used. In another example, hydrophobicchargeinductionchromatography was used to isolate monoclonal IgG (Mab) from murine ascites.Guerrier et al. (2001) used HAgel to isolate the Mab component by equilibrating with 10 mmol/L sodium phosphate, pH 8, and then with 0-5 mol/L KCUOmmol/L (pH 6.8). /L (pH 6.8) of sodium phosphate.Sinacola and Robinson (2002) used HA as a refining step that removes single-chain antibody (scFv) aggregates and inactive monomeric scFvs from active scFvs.HAgel was equilibrated with 200 mmol/L NaCUmmol/L ethylene diamine tetraacetic acid buffer and 100 mmol/mL LTris-HCl, pH 6.8. LTris-HCl, pH 8.3 equilibrated with 200 mmol/LNaCUmmol/L ethylenediaminetetraacetic acid buffer and 100 mmol/ LTris-HCl, pH 8.3, active scFv could not be adsorbed to HA. Clinically, Mab expressed in high titers usually contains large amounts of immunoglobulin aggregates. In phosphate elution systems, the selectivity of the HA surface for monomers and multimers is generally the same. The monomer can be purified from the multimer on top of the HA with NaCl (Sun, 2003). A detailed description can be found in U.S. Patent 200501o7594. Polymers, endotoxin, and DNA can be removed with sodium phosphate at 0.5 mol/L, pH 6.8. When phosphate is used for the elution, Gagnon (2008) has shown that the removal of polymers can be significantly improved by adding PEG to the buffer. If a high concentration of PEG is added to the eluent, the monomers will not be adsorbed on the column and the polymers, endotoxin and DNA will remain adsorbed. For more product details, please visit Aladdin Scientific website.
