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1.
Protein A affinity chromatography is a popular purification method for immunoglobulins applied at various scales, ranging from micro-tube up to 1000l column format. Three novel high capacity protein A affinity chromatography media have been subjected to a lifetime study using 50 consecutive purification cycles of a cell culture supernatant (CCS) containing a monoclonal antibody. Chromatographic conditions followed protocols used in industrial antibody processing, including stripping and cleaning-in-place of the resins. For all three media, no significant loss of purification performance (measured by sodium dodecylsulfate polyacrylamide gel electrophoresis and analytical size-exclusion chromatography (SEC)) could be observed over 50 cycles. Eluate samples were analyzed for leaked protein A and host cell protein (HCP) content. MabSelect SuRe, the first protein A affinity medium compatible with alkaline regeneration conditions, exhibited the lowest leakage levels, in the range of 1-3 ppm. For the media MabSelect Xtra and ProSep-vA Ultra, leakage levels were in the range of 30-40 ppm. Host cell protein content of eluates from MabSelect Xtra and SuRe were between 300 and 700 ppm, whereas for ProSep-vA Ultra 3000-4000 ppm was achieved.  相似文献   

2.
An IgG-specific camelid antibody matrix (BAC, Naarden, The Netherlands), developed from an immune phage display library, was characterized regarding engineering properties including mass transfer characteristics. Uptake kinetics and equilibrium binding capacity were determined by a finite bath method. Adsorption kinetic parameters were also determined using a real time biosensor. Slightly different properties to conventional Staphylococcal protein A affinity media were shown; especially a 2–2.5 times lower maximal binding capacity with a value of 26 mg/ml polyclonal IgG was obtained. Mass transfer could be described by using a film and pore diffusion model (De = 5 × 10−8 cm2/s). Determined engineering parameters were used to predict breakthrough behaviour in column mode considering film and pore resistances. The dynamic binding capacity at 10% breakthrough did not change when residence time was at least 6 min.  相似文献   

3.
The robustness of virus clearance with respect to protein A media reuse was demonstrated using media with four matrix chemistries: Protein A immobilized ProSep A, Poros A50, Protein A ceramic Hyper DF and MabSelect SuRe, an alkali resistant protein A ligand. Endogenous retrovirus clearance, step yield, impurity clearance and other performance parameters were evaluated periodically in media cycled up to 300 times. Media lifetime was generally limited by either declining step yield or media fouling. However, clearance of endogenous retrovirus remained in an acceptable range, either increasing or remaining constant. Multiply cycled media were tested for clearance of three viruses (SV40, X-MuLV, and MMV); clearance was comparable to na?ve media. Overall, virus clearance by protein A chromatography appears to be extremely robust with respect to media age.  相似文献   

4.
In the biopharmaceutical industry, column chromatography residuals are routinely assessed by the direct measurement of mock eluates. In this study, we evaluated virus and other impurity carryover between protein A cycles and the feasibility of using a total organic carbon (TOC) analyzer to monitor for column impurity leakage as a correlate for actual measured carryover in mock eluates. Commercial process intermediates were used in scaled down studies of two protein A media, ProSep A (Millipore, Bedford, MA, USA) and MabSelect SuRe (GE Healthcare, Uppsala, Sweden). The chromatography system was programmed to run up to 200 normal load/elution cycles with periodic blank cycles to measure protein and phage carryover, and water flush cycles to measure TOC release. Sustained phage carryover was evident in each study. Carryover and TOC release was lowest in the case where cleaning was most stringent (50 mM NaOH/0.5 M Na2SO4 with MabSelect SuRe). The TOC analysis at this time does not appear to be a viable practical means of measuring impurity carryover; direct measurements in mock eluates appears to be more predictive of column performance.  相似文献   

5.
This work provides the theoretical foundation and a range of practical application examples of a recently developed method to measure protein mass transfer in adsorbent particles using refractive index-based optical microscopy. A ray-theoretic approach is first used to predict the behavior of light traveling through a particle during transient protein adsorption. When the protein concentration gradient in the particle is sharp, resulting in a steep refractive index gradient, the rays bend and intersect, thereby concentrating light in a sharp ring that marks the position of the adsorption front. This behavior is observed when mass transfer is dominated by pore diffusion and the adsorption isotherm is highly favorable. Applications to protein cation-exchange, hydrophobic interaction, and affinity adsorption are then considered using, as examples, the three commercial, agarose-based stationary phases SP-Sepharose-FF, Butyl Sepharose 4FF, and MabSelect. In all three cases, the method provides results that are consistent with measurements based on batch adsorption and previously published data confirming its utility for the determination of protein mass transfer kinetics under a broad range of practically relevant conditions.  相似文献   

6.
A novel protein A affinity chromatography stationary phase has been developed from polypropylene capillary‐channeled polymer fibers modified with a recombinant protein A ligand for the capture and recovery of immunoglobulin G (IgG) with high specificity and yield. An SPE micropipette tip format was employed so that solvent, protein, and antibody consumption was minimized. The adsorption modification of the fiber surfaces with protein A was evaluated as a function of feed concentration and volume. Optimal modification of the fiber surface with protein A yielded a 5.7 mg/mL (bed volume) ligand capacity with the modified fibers showing stability across numerous solvent environments. Performance was evaluated through exposure to human IgG and myoglobin, individually and as a mixture. Myoglobin was used as a surrogate for host cell proteins common to growth media. The efficacy of the selective binding to the ligand is demonstrated by the 2.9:1 (IgG/protein A) binding stoichiometry. Elution with 0.1 M acetic acid yielded an 89% recovery of the captured IgG based on absorption measurements of the collected eluents. Regeneration was possible with 10 mM NaOH. Protein A modified polypropylene capillary‐channeled polymer fibers show promising initial results as an affinity phase for efficient capture and purification of IgG.  相似文献   

7.
Protein A affinity chromatography is a standard method of purifying murine monoclonal antibodies (mabs), primarily because it can be performed easily and achieves high-purity levels. Because of its high concentration capacity, it lends itself particularly well to the isolation of mabs from the supernatants of hybridoma cultures. Unfortunately, murine immunoglobulin (Ig) G1 antibodies, a subclass which occurs frequently in the IgG mabs of mice, binds very poorly to protein A, leading to problems in this isolation procedure. For this reason an attempt was made to increase the effectiveness of protein A affinity chromatography in purifying mabs of this IgG subclass by optimizing the binding conditions. The influence of ionic strength, pH and temperature on the binding capacity of a protein A column was studied. The results show the significance of temperature in the binding of the murine IgG1 mab tested to protein A. Further investigations were carried out to optimize the elution conditions and to study the contamination of mab preparations obtained with non-specific bovine protein A reactive Igs originating from culture medium supplement (10% foetal calf serum). An optimized, automatic single-step procedure to obtain highly purified murine IgG1 mabs from hybridoma culture supernatants was developed.  相似文献   

8.
The present work deals with the development and characterization of a tryptophan based pseudobioaffinity adsorbent for the purification of monoclonal and polyclonal antibodies. Tryptophan as a ligand was selected based on molecular docking and experimental screening studies of the amino acids involved in IgG-Protein A interaction. The ligand was coupled to a polymethacrylate based rigid, porous SEPABEADS beaded matrix to obtain the desired affinity adsorbent, which was named AbSep. Characterization studies with regards to the effect of matrix properties (pore size, particle size, nature of matrix, spacer arm) and the medium properties (pH, conductivity, additives) were performed to elucidate the nature of IgG-AbSep interactions and to determine the optimal conditions for obtaining high binding and purity of IgG. The equilibrium binding capacity of AbSep and dissociation constant was found to be 78 mg/ml and 5.31×10(-6)M respectively. AbSep was able to successfully purify polyclonal human IgG from plasma and monoclonal antibody (chimeric IgG1) from CHO cell culture supernatant. Both binding and elution steps were performed at near neutral pH resulting in a purity and recovery of more than 90% and 85% respectively. Additionally, AbSep was shown to be stable to 0.5M NaOH solutions, the preferred agent for cleaning and sanitization of chromatographic media.  相似文献   

9.
Guo M  Wang J  Wu Y  Xu M  Gao X 《色谱》2012,30(1):86-90
为了获得一种优良的抗体纯化介质,制备了重组金黄色葡萄球菌蛋白A(rProtein A)亲和填料,并考察了所制备的亲和填料的纯化性能。利用自行构建的rProtein A工程菌,经诱导表达、纯化获得rProtein A纯品,将其偶联到经环氧氯丙烷活化的Sepharose 4 Fast Flow凝胶上,得到rProtein A亲和填料,并使用兔抗尿酸氧化酶抗体对该填料的性能进行验证。结果显示,在自制的rProtein A亲和填料上rProtein A浓度为1.5×10~4 mol/L。采用Scatchard模型分析,得到其解离常数和最大表观吸附量分别为2.28×10~7 mol/L和20.697 g/L,说明制得的rProtein A亲和填料对抗体有很好的结合能力。将该填料于0.1 mol/L NaOH溶液中浸泡1 h,其色谱性能未见变化。将该填料用于纯化兔抗体,湿胶结合抗体量可达19 mg/mL;一步柱色谱即可得到电泳纯度的抗体样品,回收率高于96%。本研究为rProtein A亲和填料的国产化奠定了基础。  相似文献   

10.
In process-scale antibody purification, protein-A affinity chromatography is commonly used as the initial purification step. In this paper, two different protein-A media were evaluated. These adsorbents have a porous glass backbone with different pore sizes: 700 A and 1000 A. Adsorption equilibrium data of human immunoglobulins on these media were measured via a batch technique and correlated using the Langmuir isotherm model. A larger static capacity was found for the smaller pore size material, which is probably a result of the larger specific surface area and associated higher ligand concentration. The protein uptake kinetics were also obtained via a stirred tank experiment using different initial protein concentrations. A surface layer model was used to represent the protein uptake by the media and to estimate values of a concentration-independent effective diffusivity within the particle. Experimental breakthrough curves were also obtained from packed beds operated under different conditions. Calculated breakthrough profiles were found to be in good agreement with the experimental results. Experimental breakthrough data were used to determine the dependence of the dynamic capacity of the media as a function of the fluid residence time. A larger dynamic capacity was also found for the smaller pore size media. The permeability of large scale packed beds was also reported and used in conjunction with the dynamic capacity to calculate the process production rate.  相似文献   

11.
贾凌云  杨利  邹汉法  张玉奎 《色谱》1998,16(6):476-480
以木纤维为基质,与甲基丙烯酸环氧丙酯共聚接枝合成了复合膜介质,用复合膜介质制备了proteinA亲合膜色谱柱,考察了proteinA亲合膜色谱柱液相流动特性和吸附性能。实验证明:流速与亲合膜色谱柱柱压呈线性关系,当流速为3mL/min时,柱压为160kPa。免疫球蛋白(IgG)浓度和上样速度是影响proteinA键合容量的重要因素,对其进行了优化研究。用动态吸附法确定了对人IgG动态最大吸附能力可达21.7mg/g(干介质)。  相似文献   

12.
Increased recombinant protein expression yields and a large installed base of manufacturing facilities designed for smaller bulk sizes has led to the need for high capacity chromatographic resins. This work explores the impact of three pore sizes (with dextran distribution coefficients of 0.4, 0.53, and 0.64), dextran surface extender concentration (11–20 mg/mL), and ligand density (77–138 μmol H+/mL resin) of cation exchange resins on the dynamic binding capacity of a therapeutic antibody. An intermediate optimal pore size was identified from three pore sizes examined. Increasing ligand density was shown to increase the critical ionic strength, while increasing dextran content increased dynamic binding capacity mainly at the optimal pore size and lower conductivities. Dynamic binding capacity as high as 200 mg/mL was obtained at the optimum pore size and dextran content.  相似文献   

13.
Bao  Ru-Meng  Yang  Hong-Ming  Yu  Chang-Mei  Tang  Jin-Bao 《Chromatographia》2016,79(19):1271-1276

Protein A affinity chromatography is an important technique that is widely used in purifying polyclonal and monoclonal antibodies. However, improving the IgG loading capacity of protein A affinity materials remains crucial. In this study, a smaller divalent IgG binding molecule derived from the B domain of protein A, i.e., ZZ-domain, was used to develop an affinity adsorbent with high IgG loading capacity by improving the unit area yield of the site-specific immobilization affinity ligand. The engineered ZZ-Cys was tightly immobilized onto Sepharose support via the covalent incorporation of a cysteine handle and a maleimide group, with oriented manner and divalent IgG binding capacity, thereby resulting in homogenous conjugates, namely, Sepharose–ZZSA. Approximately 1.19 mg of ZZ-Cys was coupled onto wet Sepharose g−1 and the maximum saturation binding capacity of Sepharose–ZZSA g−1 was approximately 23.80 mg of IgG. The smaller engineered ZZ-Cys can be produced at a lower cost than protein A and covalently conjugated onto matrix surface with high density and full IgG binding capacity. Thus, the proposed platform may be of general use for IgG purification in an efficient and economical manner.

  相似文献   

14.
A method for fast in situ measurement of adsorption kinetics based on a finite bath was developed. We modified the conventional finite bath by replacing the external loop by a dip probe which enables in situ measurement of the concentration change in the contactor. Deposition of adsorbent particles on the reflection surface of the dip probe compromised measurements. Different membranes, a polyamide, a polypropylene and a nylon membrane were tested to protect the internal reflection surface of the dip probe from fouling with adsorbent particles. The nylon membrane provided efficient protection and high mass transfer evaluated by response time experiments. Unspecific adsorption of the model protein on the membrane could also be excluded. To corroborate the measurements of the dip probe the results were compared to a conventional finite bath and to a shallow-bed. The uptake curves for human polyclonal IgG at different concentrationes (0.1-3 g/l) on rProtein A Sepharose FF and MabSelect were used as model system. The effective diffusion coefficients were determined using a pore diffusion model. These values were in good agreement for all methods.  相似文献   

15.
Human immunoglobulin G (IgG) subclasses each play a unique role in an immune response to foreign antigens. Three of the human IgG subclasses have distinct electrophoretic mobilities and are resolved by capillary zone electrophoresis (CZE). A post-capillary reactor is constructed to allow on-line addition of fragment B (of protein A)-fluorescein to form affinity complexes with separated IgG subclasses. Post-capillary affinity detection provides selective identification of human IgG subclasses and illustrates the effect of affinity binding constant on detection sensitivity. Additionally, post-capillary affinity detection for CZE facilitates rapid and selective heterogeneity analysis of mouse monoclonal anti-(human-1-antitrypsin) and anti-human follicle stimulating hormone in complex sample matrices. A constant mobility difference is observed between the antibody isoforms, likely the result of charge heterogeneity due to deamination, degradation or variation in sialic acid content.  相似文献   

16.
A protein A affinity chromatographic medium based on porous silica modified with phosphorylcholine (PC) groups and amino groups (PNSP) was synthesized. The PC groups functioned as suppressors of non-specific protein adsorption. Recombinant protein A was bound to the amino groups on PNSP with a glutaraldehyde used as a spacer (PNSP-PA). The PC groups and amino groups were immobilized on porous-silica particles using two silane coupling reagents, PC-bound silane, and 3-aminopropyltrimethoxysilane. After optimizing various factors in the synthetic process, the resultant protein A medium showed improvements in non-specific protein adsorption, dynamic binding capacity, and chemical stability under basic conditions compared with conventional protein A affinity media.  相似文献   

17.
Experimental determination and modeling of IgG binding on a new protein A adsorbent based on a macroporous resin were performed. The new adsorbent consists of polymeric beads based on hydrophilic acrylamido and vinyl monomers with a pore structure optimized to allow favorable interactions of IgG with recombinant protein A coupled to the resin. The particles have average diameter of 57 μm and a narrow particle size distribution. The IgG adsorption equilibrium capacity is 46 mg/cm3 and the effective pore diffusivity determined from pulse response experiments for non-binding conditions is 8.0 × 10−8 cm2/s. The IgG adsorption kinetics can be described with the same effective diffusivity by taking into account a heterogeneous binding mechanism with fast binding sites, for which adsorption is completely diffusion controlled, and slow binding sites for which adsorption is controlled by the binding kinetics. As a result of this mechanism, the breakthrough curve exhibits a tailing behavior, which appears to be associated with the slow binding sites. A detailed rate model taking into account intraparticle diffusion and binding kinetics is developed and is found capable of predicting both batch adsorption and breakthrough behavior over an ample range of experimental conditions. The corresponding effective diffusivity is independent of protein concentration in solution over the range 0.2–2 mg/cm3 and of protein binding as a result of the large pore size of the support matrix. Overall, the small particle size and low diffusional hindrance allow capture of IgG with short residence times while attaining substantial dynamic binding capacities.  相似文献   

18.
A high-resolution two-dimensional affinity electrophoresis (2D-AEP) method was developed, using capillary polyacrylamide gel (PAG) isoelectric focusing in the first and slab PAG affinity electrophoresis in the second direction. Using this method, anti-hapten antibodies were separated into a number of monoclonal antibody [immunoglobulin G (IgG)] families, each of which is composed of several IgG spots having an identical affinity to the hapten but different isoelectric points. 2D-AEP may offer a powerful tool for solving fundamental problems in immunochemistry such as antibody heterogeneity, its hapten binding specificity and antigen-dependent somatic mutation.  相似文献   

19.
Anion exchange chromatography (AEC) is a useful and effective tool for DNA purification, but due to average pore sizes between 40 and 100 nm most AEC resins lack truly useful binding capacities for plasmid DNA (pDNA). Equilibrium binding capacities and uptake kinetics of AEC media including conventional media (Source 30 Q, Q Sepharose HP), a polymer grafted medium (Fractogel EMD DEAE (M)), media with large pores (Celbeads DEAE, PL SAX 4000 A 30 microm) and a monolithic medium (CIM-DEAE) were investigated by batch uptake or shallow bed experiments at two salt concentrations. Theoretical and experimental binding capacities suggest that the shape of the pDNA molecule can be described by a rod with a length to diameter ratio of 20:1 and that the molecule binds in upright position. The arrangement of DNA like a brush at the surface can be considered as entropy driven, kind of self-assembly process which is inherent to highly and uniformly charged DNA molecules. The initial phase of adsorption is very fast and levels off, associated with a change in mass transfer mechanism. Feed concentrations higher than 0.1 mg/mL pDNA pronounce this effect. Monolithic media showed the fastest adsorption rate and highest binding capacity with 13 mg pDNA per mL.  相似文献   

20.
The recombinantly produced different forms of protein G, namely monofunctional immunoglobulin G (IgG) binding, monofunctional serum albumin (SA) binding and bifunctional IgG/SA binding proteins G, are compared with respect to their specific affinities to blood IgG and SA. The affinity mode of the recently developed high-performance monolithic disk chromatography has been used for fast quantitative investigations. Using single affinity disks as well as two discs stacked into one separation unit, one order of magnitude in adsorption capacities for IgG and SA were found both for monofunctional and bifunctional protein G forms used as specific affinity ligands. However, despite the adsorption difference observed, the measured dissociation constants of the affinity complexes seemed to be very close. The analytical procedure developed can be realized within a couple of minutes. Up-scaling of the developed technology was carried out using another type of monolithic materials, i.e. CIM affinity tubes.  相似文献   

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