Purification and analysis of human alpha1-antitrypsin concentrate by a new immunoaffinity chromatography.

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PURIFICATION AND ANALYSIS OF HUMAN ALPHA1-ANTITRYPSIN CONCENTRATE BY A NEW IMMUNOAFFINITY CHROMATOGRAPHY a

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a

Xuejun Zhang , Yiling Hou , Xiang Ding , Shengliang Ye , Haijun a

a

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Cao , Zongkui Wang , Xi Du , Yi-wu Xie

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& Changqing Li

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Institute of Blood Transfusion , Chinese Academy of Medical Sciences & Peking Union Medical College , Chengdu , Sichuan , China b

Key Laboratory of Southwest China Wildlife Resources Conservation, College of Life Sciences , China West Normal University , Nanchong , Sichuan , China c

China Biologic Products, Inc. , Chengdu , Sichuan , China Accepted author version posted online: 26 Nov 2013.Published online: 06 Jun 2014.

To cite this article: Xuejun Zhang , Yiling Hou , Xiang Ding , Shengliang Ye , Haijun Cao , Zongkui Wang , Xi Du , Yi-wu Xie & Changqing Li (2014) PURIFICATION AND ANALYSIS OF HUMAN ALPHA1ANTITRYPSIN CONCENTRATE BY A NEW IMMUNOAFFINITY CHROMATOGRAPHY, Preparative Biochemistry and Biotechnology, 44:7, 725-737, DOI: 10.1080/10826068.2013.868358 To link to this article: http://dx.doi.org/10.1080/10826068.2013.868358

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Preparative Biochemistry & Biotechnology, 44:725–737, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2013.868358


PURIFICATION AND ANALYSIS OF HUMAN ALPHA1-ANTITRYPSIN CONCENTRATE BY A NEW IMMUNOAFFINITY CHROMATOGRAPHY

Xuejun Zhang,1 Yiling Hou,2 Xiang Ding,2 Shengliang Ye,1 Haijun Cao,1 Zongkui Wang,1 Xi Du,1 Yi-wu Xie,1,3 and Changqing Li1 1 Institute of Blood Transfusion, Chinese Academy of Medical Sciences & Peking Union Medical College, Chengdu, Sichuan, China 2 Key Laboratory of Southwest China Wildlife Resources Conservation, College of Life Sciences, China West Normal University, Nanchong, Sichuan, China 3 China Biologic Products, Inc., Chengdu, Sichuan, China

& Alpha1-antitrypsin is a kind of plasma protein that requires a sequence of different fractionation steps to get generally. To report an effective process for isolating and purifying alpha1-antitrypsin from Cohn Fraction IV based upon a new immunoaffinity chromatography medium, named ‘‘Alpha-1 Antitrypsin Select,’’ characterization of alpha1-antitrypsin (a1-AT) was performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, and tandem mass spectroscopy. Total protein content was determined by the method of Bradford under visible light absorption at 595 nm. Pretreatment process and the immunoaffinity chromatography step achieved a 60.35 1.39% yield. Thus, an overall 71.68 1.32 fold increase in purity and a 41.88 6.98% yield were obtained from plasma. The a1-AT had a specific activity of about 1.00–1.05 PU=mg. This technique will develop an effective process for isolating and purifying, with high purity and specific activity, alpha1-antitrypsin from Cohn Fraction IV or human whole plasma, which could be an efficient and scaled-up method for alpha1-antitrypsin products purification. Keywords alpha1-antitrypsin, chromatography, Cohn fraction IV, mass spectrometry, purification

INTRODUCTION Alpha1-antitrypsin (alpha-1-proteinase inhibitor, a1-antitrypsin, or a1-AT, AAT, a1-PI) is one kind of serine protease inhibitor (serpin) that is synthesized by the hepatic cells. The most important physiological function Address correspondence to Changqing Li, Institute of Blood Transfusion, Chinese Academy of Medical Sciences & Peking Union Medical College, Chengdu, Sichuan 610052, China. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline. com/ lpbb.


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is to inhibit the elastase activity in lung.[1] In 1963, Laurell and Eriksson reported hereditary a1-AT deficiency and its early years with a patient (30– 40 years old) in relationship to the occurrence of emphysema.[2] Epidemiological investigations revealed[3] that the incidence rate of a1-AT deficiency was about 1:2000 to 5000 and liver lesions and early emphysema were the symptoms of a1-AT deficiency, which was usually caused by SERPINA 1 gene mutation. The occurrence of emphysema increased when serum a1-AT was reduced to lower than a threshold of 80 mg=dL (about 35% of normal levels). In 2002, Lomas explained that a1-PI protein secondary structure within the body changes with aggregation of pathological conditions.[4] Clinical manifestations of a1-AT deficiency were mainly lung disease[5] and liver disease,[6] such as emphysema,[7] chronic obstructive pulmonary disease (COPD),[8] hemorrhage,[9] angioedema,[10] cirrhosis,[11] and dementia.[12] Cohn Fraction IV (CF IV) is a by-product currently generated by a process of albumin, but retains approximately 33% of plasma a1-AT. CF IV’s components have been classified as 15% albumin, 55% a1 protein, 28% a1 þ b protein, and 2% c protein.[13] Therefore, CF IV is a kind of comprehensive utilization of cheap raw materials for the isolation of a1-AT. Immunoaffinity chromatography is the use of antigen-specific antibody binding with the multicomponent mixture for the separation of specific antigen (or antibody) in chromatography.[14–16] It is a specific method with a simple and stable ligand used for large-amount protein separation under natural conditions, which is gradually becoming the most effective technology. The fumed silica is mainly used to remove specific alpha1-lipoprotein. Afterward, immunoaffinity chromatography has been used to purify a1-AT further. Most importantly, a1-AT is known to be an important drug for a1-AT deficiency.[17] The importance of these drugs has been demonstrated.[18] We focus on whether the method can isolate this kind of protein on a large scale. There is great practical value if this method can isolate alpha1-antitrypsin on a large scale successfully. In particular, with the new process for isolating and purifying, with high purity and specific activity, a1-AT, biological drug products could be introduced to the market. The present article reports a one-step isolation technique to achieve this purpose.

MATERIALS AND METHODS Materials CF IV was bought from China Biologic Products, Inc. (Beijing, China,). Plasma was bought from China Biologic Products, Inc. (Beijing, China). All these samples were frozen at 60 C until used.

New Immunoaffinity Chromatography

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Immunoaffinity Chromatography Column The AAT ligand was covalently bound to the agarose base matrix via a hydrophilic spacer to enhance accessibility and facilitate effective binding of the target molecule (Figure 1). The ligand was attached via an amide linkage to give a chemically stable covalent bound. Table 1 listed the key characteristics of Alpha-1 Antitrypsin Select. Alpha-1 Antitrypsin Select was poured in a XK26=20 FPLC column (GE Healthcare Bio-Sciences AB Bjo¨rkgatan, Uppsala, Sweden, 26 20 mm), which contained 25 mL of gel equilibrated in the starting buffer. Plasma was thawed at room temperature; CF IV was dissolved with Tris solution in a ratio of 1:12.5 for 0.5 hr at 24 C, and the suspension was centrifuged for 15 min (24 C, 4,750 g). After added treatment with a fumed silica HL-380 and centrifugation, the supernatant was loaded through 1.2-mm filters sequentially. Aliquots of each sample were ready for Alpha-1 Antitrypsin Select purification. The flow-rate was 2.0 mL=min. The column was coupled to an AKTA Explorer 100 fast protein liquid chromatography (FPLC) system (GE Healthcare Bio-Sciences AB Bjo¨rkgatan, Uppsala, Sweden), which allowed the entire automation of the chromatographic steps.

FIGURE 1 Partial structure of Alpha-1 Antitrypsin Select. The spacer arm between the base matrix and ligand facilitates effective binding of the target AAT molecule.

TABLE 1

Characteristics of Alpha-1 Antitrypsin Select

Base Matrix

Highly Cross-Linked Spherical Agarose

Average particle sizea Ligand Ligand density Binding capacityb Storage buffer pH stability

75 mm (d50V) AAT binding ligand Approx. 5.5 mg=ml medium Approx. 10 mg AAT=ml medium 20% Ethanol in water 2 to 11 (Short-termc) 3 to 10 (Long-termd)

a

d50V is the median particle size of the cumulative volume distribution. Determined using a total capacity chromatography method. c Short-term refers to the pH interval for regeneration and cleaning. d Long-term refers to the pH interval where the medium is stable over a long period of time without adverse effects on the subsequent chromatographic performance. b

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Tris (20 mM) and magnesium chloride buffers (2 M) of pH 7.4 were used to optimize the separation of the proteins, and optical density of eluates was monitored at 280 nm. Following the collection of the fractions of interest, which were concentrated to the original volume of the sample applied (1.0 mL) by centrifugation using Centricon Plus-70 10-kD MWCO from Millipore (lot ROHA46075), the columns were stripped with 6 M urea and reequilibrated with the starting buffers. The final a1-AT product was freeze-dried, then stored at 4 C until for further study. Analysis of a1-AT Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in 12% gels according to standard procedures, using a mini-gel apparatus MiniVE (GE Healthcare Bio-Sciences AB, Bjo¨rkgatan, Uppsala, Sweden). Gels were stained with Coomassie blue R-250 (Amersco, Solon, OH, lot 2540C088). Unstained protein molecular weight marker standards SM0431 (7.5 mL, Fermentas, Glen Burnie, MD) were loaded on the sides of the gel for MW estimation. Western Blotting (WB) Total protein were separated by 1-DE and transferred for 1.5 h at 65 mA constant current using a Tris-glycine (25 mM Tris, 192 mM glycine, pH 8.3) buffer in a transfer apparatus TE70 (GE Healthcare Bio-Sciences AB, Bjo¨rkgatan, Uppsala, Sweden). Polyvinylidene fluoride (PVDF) transfer membranes were obtained from GE Healthcare Bio-Sciences AB (0.3 3 m, 0.45 mm, lot NH1177) and positioned on the anodal side of the gel. Blots were incubated overnight at 4 C with a primary antibody (chicken anti-a1-AT, lot 849141, obtained from Abcom Ltd., HKSP, N.T. Hong Kong) and diluted at 1=1000, and then with peroxidase-labeled antibody (rabbit anti-chicken, lot 961379, obtained from Abcom Ltd., HKSP, N.T. Hong Kong), and diluted to 1=2500. The membranes were revealed by chemoluminescence, using the Immun-Star horseradish peroxidase (HRP) substrate kit by Bio-Rad (lot 310008502) and detected by x-ray films. Finally, the x-ray films were scanned. Two-Dimensional Gel Electrophoresis (2D-PAGE) Isoelectric focusing (IEF) was performed in immobilized pH gradient (IEF–IPG) gels obtained from Bio-Rad (lot 400061048), according to the technique originally reported by Eap and Baumann Eap CB, Baumann P.[19] Lyophilized samples were treated by Ready Prep 2-D Cleanup Kit by



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Bio-Rad (lot BK102508). Samples were then loaded onto a PROTEAN IEF cell system (Bio-Rad Laboratories, Hercules, CA) in a total volume of 100 mL of hydration buffer (7 M urea, 2 M thiourea, 10 mM dithiothreitol [DTT], 4% CHAPS, 40 mM Tris, 0.8% Pharmalytes, pH 3–10), overlaid with IPG strips (7 cm, pH 3–10) and incubated for 30 min. Following the addition of mineral oil from Bio-Rad (lot 14907), the IPG strips were rehydrated under active conditions at a constant voltage of 50 V for 14 hr at 20 C. The proteins were prefocused at 250 V for 30 min and then 500 V for 30 min, 1000 V for 1 hr, and finally focused for a total of 35 kV=hr. IPG strips were stored at 80 C until required. Prior to running the second dimension, IPG strips were kept at room temperature for 15 min in the equilibration buffer (375 mM Tris–HCl, pH 8.8, containing 6 M urea, 2% SDS, 20% glycerol, and a trace amount of BPB). The second dimension was performed in 12% precast gels at 10 mA in a PROTEAN xi two-dimensional (2D) cell system (Bio-Rad Laboratories, Hercules, CA). Gels were stained using silver nitrate according to Hochstrasser et al.[20] or with colloidal Coomassie blue R-250 (Amersco, Solon, OH, lot 2540C088). Capillary Electrophoresis (CE) Runs were performed on a Beckman Coulter P=ACE MDQ (Fullerton, CA) automated system in uncoated capillaries obtained from Beckman Coulter (lot M009481) with an inner diameter of 50 mm and a total length of 57 cm (50 cm to the detection window). Samples were injected at 0.5 psi for 10 s (approximately 10 nL injected) and the separation was carried out at a temperature of 25 C applying a voltage of 20 kV for 20 min. The background electrolyte was 40 mM pH 7.0 citrate buffer solution. ESI-Q-ToF MS/MS Electron spray ionization (ESI) qualitiative time-of-flight (Q-ToF) tandem mass spectroscopy (MS=MS) uses a Q-Tof Primer mass spectrometer (Q-TOF, Waters Micromass, Milford, MA) coupled to the ESI ion source. The sample was introduced and the voltage was operated at 3.0 kV. The automatic scan rate is 1.0 s with an interscan delay of 0.02 s. Spectra were accumulated until a satisfactory signal=noise ratio had been obtained. Only consider the double charge peaks or more charge peaks for MS=MS that are in the mass range from 400 to 1600 m=z, which are the intensity of individual ions rising above a threshold of 10 counts=s to produce ion spectra by collision-induced dissociation (CID). When the intensity of ion falls below 2 counts=s or 10 s regardless, switch to MS survey. Trypsin autolysis

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products and keratin-derived precursor ions were automatically excluded. The CID cell was filled with argon at a flow rate of 0.45 mL=min. The collision energy was varied between 18 and 57 eV depending on the mass of the precursor. Standard calibration peptide (Glu-fibrinopeptide) was also applied to the experiment for an external calibration of the instrument before data directed analysis (DDA) acquisition for accurate mass determination. In a survey scan, three MS=MS ions were selected. Consequently, all the information used to extract peak information, which is used to create the MS=MS peak list, must be generated from one combined spectrum. Protein Identification and Database Searching The tandem mass spectrometry (MS=MS) data, ‘‘pkl list’’ (pkl) files acquired by the software of ProteinLynx 2.2.5 (Waters), include the mass values, the intensity, and the charge of the precursor ions (parent ions with þ2 or þ3 charge in this study). The pkl files were analyzed with a licensed copy of the Mascot 2.0 program (MatrixScience Ltd., London) against the Swiss-Prot protein database. Searching parameters were set as follows: enzyme, trypsin; allowance of up to one missed cleavage peptide; mass tolerance, 1.2 Da and MS=MS mass tolerance, 0.3 Da; fixed modification parameter, carbamoylmethylation (C); variable modification parameters, oxidation (at Met) and phosphor (ST); auto hits allowed (only significant hits were reported); results format as peptide summary report. Protein were identified on the basis of two or more peptides whose ions scores both exceeded the threshold, p < .05, which indicates identification at the 95% confidence level for these matched peptides.[21] If proteins were identified by a single peptide, then the spectrum was manually inspected. For a protein to be confirmed, (1) the assignment had to be based on four or more y- or b-series ions and (2) the protein molecular mass had to be consistent with gel migration data. Database searches were performed against the NCBI nonredundant database using the MASCOT (Matrix Science; www.matrixscience.com) search engine. MASCOT scores greater than 65 were significant (pb 0.05) and were subsequently blasted against the Swiss-Prot database. Quantitation and Specific Activity Assay Protein quantitation was performed by the concentration of purified absorbance at 280, using an extinction coefficient of 8.9.[22] The biological specific activity of a1-AT was routinely determined by measuring the inhibition of elastase.[23,24] We diluted 25 mL of a solution containing 60 mg= mL of bovine pancreas (Sigma T1426 lot 050M7020V) with 0.02 M Tris-HCl,



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pH 8.0, and incubated this with 100 mL of the sample at 25 C. Then, 200 mL of substrate (43.5 mg of Na-benzoyl-DL-arginine 4-nitroanilide hydrochloride: BAPNA, Sigma B4875, lo: BVBC5353), dissolved in 1 mL dimethyl sulfoxide and diluted at a ratio of 1:100 in T þ buffer, was added and the increase of absorbency at 405 nm was measured for 5 min. A standard curve was produced using reference plasma (lot 1A3E; Baxter Hyland Immuno, Vienna, Austria), diluted at a ratio of 1:100 to 1:400 in T þ buffer.[25] The test samples were then diluted in T þ buffer to give 20–40% inhibition, and their activity in plasma units (PU) was calculated from the standard curve (1 PU of antitrypsin is equivalent to the antitrypsin activity normally present in 1 ml of fresh citrated plasma).[26]

RESULTS SDS-PAGE As expected, Alpha-1 Antitrypsin Select was able to effectively purify a1-AT and removed many impurities from plasma or CF IV’s treated solutions. Samples obtained from plasma and CF IV’s treated solutions are shown on the SDS-PAGE gels in Figure 2. The first lane loaded standard plasma diluted 50 as a reference; the second lane loaded CF IV’s dissolved solution diluted 10; the third lane loaded CF IV’s dissolved solution treated with a fumed silica HL-380. Most of the impurities of 28-kD lipoprotein are removed in this processing, Finally, even more impurities in the flow-through and wash solution are unbonded in the media (lane 5). When

FIGURE 2 SDS-PAGE of purification processing: (1) standard plasma protein, (2) CF IV’s dissolved solution, (3) CF IV’s dissolved solution treated with a fumed silica HL-380, (4) SM0431 protein molecular weight ladder, (5) impurities in the flow-through and wash solution, (6 and 7) nonreducing a1-AT, and (8 and 9) reducing a1-AT.

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a1-AT concentrate is eluted from the column with Tris and magnesium chloride buffers, which showed two protein bands on gels by non-reducing SDS-PAGE (lanes 6 and 7), one of the molecular mass values was calculated as 54 kD; the others was calculated as 140 kD approximately. After reducing SDS-PAGE, the large-molecular-weight polymer protein band of a1-AT was depolymerized (lanes 8 and 9).


Western Blotting (WB) The result of Western blotting analysis could be related to changes in reaction. A large molecular weight polymer protein band of a1-AT was depolymerized (Figure 3). Photos expose using the Laminated exposure method, exposure time is 3 minutes. The total amount of laminated exposure photographic films was three. The left film was closest to the fluorescent light source and the right was furthest. Add the reducing agent dithiothreitol (or b-mercaptoethanol) conditions, abd lanes 6 and 7 of the high-molecular-weight protein band were depolymerized. 2D-PAGE Purified nonreducing a1-AT’s isoelectric point was 5.5 approximately. Furthermore, ertical electrophoresis were analyzed by nondenaturing polyacrylamide gel electrophoresis; the result is in good agreement with SDS-PAGE (Figure 4).

FIGURE 3 Western blotting of purification processing: (1) standard plasma protein, (2) CF IV’s dissolved solution, (3) CF IV’s dissolved solution treated with a fumed silica HL-380, (4) SM0431 protein molecular weight ladder, (5) impurities in the flow-through and wash solution, (6 and 7) nonreducing a1-AT, and (8 and 9) reducing a1-AT.



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FIGURE 4 2D-PAGE analysis purified nonreducing a1-AT.

Capillary Electrophoresis (CE) Two previous similar curves are A6150 with 4 total peaks, respectively, at 6 min, 9 min, 9.5 min, and 11.5 min, and combined peaks 1, the upper and lower peak for the S-type (Figure 5).

FIGURE 5 CE analysis Sigma A6150 and purified a1-AT.


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FIGURE 6 A representative MS=MS spectrum collected during the experiment shown.

ESI-Q-TOF MS/MS and Protein Identification and Database Searching Through MS=MS tandem mass spectrometry analysis of Alpha-1 Antitrypsin Select gel purified a1-AT concentrates in the protein types, tandem mass spectra were recorded (Figure 6). Masct points and Swiss-Port database search results and shown in Table 2. Through the preceding data analysis, for the purified protein containing human species out of the a1-AT, it also contains a small amount of von Willebrand factor (vWF) and albumin (albumin). Quantitation and Specific Activity Assay Various steps during the purification of a1-AT parameters are shown in Table 3. Three replicates were performed for each sample. The data are expressed as mean SD. Statistical analysis was done by Student’s t-test; p < .05 was considered to be significant. DISCUSSION AND CONCLUSIONS Loss of a1-AT, except for hereditary a1-AT deficiency, but also in cases due to smoking, bacterial infection, and liver injury, is caused by

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New Immunoaffinity Chromatography TABLE 2

Masct Points and Swiss-Port Database Search Results

Protein Name

Score

Sequence Coverage (%)

VWF_HUMAN

von Willebrand factor

185

2%

56.26

317373549

P04275

ALBU_HUMAN

Serum albumin

115

11%

66.99

113576

P02768


Short Name

Unique Peptides

Gi Number

Accession Number

Other

emPAI

OS ¼ Homo sapiens GN ¼ VWF PE ¼ 1 SV ¼ 4 OS ¼ Homo sapiens GN ¼ ALB PE ¼ 1 SV ¼ 2

0.02

0.15

inactivation of the active center of a1-AT or when the body a1-AT was reduced, resulting in a protease–antiprotease imbalance in the system for emphysema, cystic fibrosis, acute respiratory distress syndrome and acute and chronic bronchitis, and in lung disease it sometimes plays a therapeutic role. The U.S. Food and Drug Administration (FDA) has approved four a1-AT products: Prolastin, Zemaira, Aralast, and Glassia, which are separated from the plasma of blood products or blood products of domestic manufacturers of nonrelated products. Of these, Glassia developed by Kamada is the only liquid stable formulation, and inhalation formulations are Zemaira and Aralast, for which the source of raw materials is the production of albumin Cohn waste generated in the process of CF IV. Production processes used in industry for most particular pretreatment processes, such as the polyethylene glycol, zinc chloride-precipitated protein impurities in raw materials, as an intermediate, were used to prepare a1-AT concentrates by a co-chromatography method. We used a new type of immune affinity chromatography media-purified human a1-AT concentrate from CF IV. Purified a1-AT in nonreducing SDS-PAGE shows a 54-kD protein and a 140-kD protein on treatment with the polymer, after reducing SDS-PAGE bands of polymer depolymerization. The Western blot can also be related to changes in reaction; after pretreatment and affinity TABLE 3

CF IV Separation and Purification of a1-AT During Each Step, With the Parameters for a1-AT

Plasma CF IV Pretreatment and affinity chromatography

Total Protein Concentration (mg=ml)

Specific Activity (PU=mg)

Purification Factor (Times)

Steps to Recovery (%)

Cumulative Recovery (%)

68.00 2.58 9.58 0.58 1.08 0.03

0.014 0.04 0.036 0.03 1.03 0.02

1 2.62 0.11 71.68 1.32

1 33.3 1.24 60.35 1.39

100 33.3 1.24 13.96 2.36


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chromatography, a1-AT activity recovery was 60.35 1.39%, a1-AT total activity recovery was 41.88 6.98%, and there was a purification factor of 71.68 1.32 times the specific activity of about 1.00–1.05 PU=mg. This technique will develop an effective process for isolating and purifying high-purity and high-specific-activity alpha1-antitrypsin from Cohn Fraction IV or human whole plasma, which could be an efficient and scaled-up method for alpha1-antitrypsin products purification. The starting material used in the experiment is CF IV, where Cohn is still being used as a procedure in the production of albumin in ‘‘waste.’’ We describe a new type of immune affinity chromatography medium for high-purity and high-specific-activity a1- AT concentrate preparation. At the same time, we analyzed the related a1-AT protein concentrate characteristics. a1- AT may yield the loss of oxidation activity, so we try to avoid using the work in the preparation of dithiothreitol or mercaptoethanol and other reducing agents to reduce the loss of activity; high purity and high specific activity of the purified protein is our goal. For a1-AT in plasma concentration of 1.3 mg=ml in plasma, total protein content of 70 mg=ml, a 54-fold purification factor can be 100% purity. Theoretically for 100% pure a1-AT protein concentrate, the corresponding specific activity would be about 0.77 PU=mg. With 71-fold purification of the results we get 1.00–1.05 PU=mg, so we have the final a1-AT 100% purity higher than the theoretical specific activity. This contradictory finding can be used in terms of nonactivated plasma exist to explain the presence of a1-AT, which has been previously reported.[27,28] We analyze the purity of protein electrophoresis; 140 kD was found to show the existence of protein bands by mass spectrometry analysis of a1-AT concentrate composition, where bands do not meet the albumin 66 kD and VWF factor subunit values of 220 kD, so we restored nonreducing WB and SDS-PAGE for comparison, and analysis of the 140-kD polymer revealed a1-AT protein and the reducing agent under the conditions of joint self-degradation. FUNDING This study was supported by a grant from the P.R. China Sichuan Province Science and Technology Support Program (grant 2009SZ0217). We thank GE Healthcare Bio-Sciences AB for a generous gift of Alpha-1 Antitrypsin Select Media. REFERENCES 1. Carrell, R.W.; Jeppsson, J.O.; Laurell, C.B. Structure and Variation of Human Alpha 1-Antitrypsin. Nature 1982, 298(5872), 329–334. 2. Laurell, C.B.; Eriksson, S. The Electrophoretic Pattern Alpha1-Globulin Pattern of Serum in Alpha1-Antitrypsin Deficiency. Scand. J. Clin. Lab. Invest. 1963, 15, 132–140.



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Purification of human chorionic gonadotropin-receptor complex by immunoaffinity chromatography.

Purification of a brain-specific astroglial protein by immunoaffinity chromatography.

Immunoaffinity co-purification of cytokinins and analysis by high-performance liquid chromatography with ultraviolet-spectrum detection.

Purification of soluble cytokine receptors from normal human urine by ligand-affinity and immunoaffinity chromatography.

Purification of human C-reactive protein by immunoaffinity chromatography using mouse monoclonal antibody.

Purification of an enterotoxin produced by Bacillus cereus by immunoaffinity chromatography using a monoclonal antibody.

Immunoaffinity purification of human thromboxane synthase.

Purification and determination of glutamine synthetase by high-performance immunoaffinity chromatography.

Drug residue analysis using immunoaffinity chromatography.

Purification of transferrin by magnetic immunoaffinity beads.

Purification of the i-antigen 51A from Paramecium tetraurelia by immunoaffinity chromatography.

Preparation of factor VII concentrate using CNBr-activated Sepharose 4B immunoaffinity chromatography.

Immunoaffinity Purification of Proteins.

Purification of soluble guanylyl cyclase from bovine lung by a new immunoaffinity chromatographic method.

protease by immunoaffinity column chromatography using a monoclonal antibody.

Immunoaffinity chromatography using electroelution.

Immunoaffinity purification-chromatographic quantitative analysis of arachidonic acid metabolites.

Isoagglutinin reduction by a dedicated immunoaffinity chromatography step in the manufacturing process of human immunoglobulin products.

Immunoaffinity-chromatography purification of salbutamol in liver and HPLC-fluorometric detection at trace residue level.

Immunoaffinity purification of 11-dehydro-thromboxane B2 from human urine and plasma for quantitative analysis by radioimmunoassay.

Purification of 2',3'-cyclic nucleotide 3'-phosphohydrolase from bovine brain by immunoaffinity chromatography: further biochemical characterization of the protein.

Identification, immunoaffinity purification and partial characterization of a human decidua-associated protein.

Kinetic aspects of membrane-based immunoaffinity chromatography.

Immunoaffinity chromatographic purification of chicken ectoATP diphosphohydrolase.