Expanded bed protein A affinity chromatography of a recombinant humanized monoclonal antibody: process development, operation, and comparison with a packed bed method.

We show that expanded bed protein A affinity chromatography using Streamline rProtein A media is an efficient method for purifying a recombinant humanized monoclonal antibody from unclarified Chinese hamster ovary cell culture fluid and that it provides purification performance comparable to using a packed bed. We determined that the dynamic capacity of the expanded bed media is related to flow rate (measured in column volumes per hour) by a power function, which allows a high capacity at a low flow rate. At 250 cm h-1 with a 25 cm bed height (10 column volumes h-1), the dynamic capacity is 30 g l-1. The yield and purity (measured by the amount of host cell proteins, DNA, SDS-PAGE, and turbidity) of the antibody purified by expanded bed is comparable to the yield and purity obtained on a standard packed bed method using Prosep A media.     

Performance of a new protein A affinity membrane for the primary recovery of antibodies

Recovery of antibodies with Protein A affinity chromatography columns has become the standard for the biotechnology industry. Membrane affinity chromatography has not yet experienced extensive application due to the lower capacity of membrane supports compared to chromatographic beads. In this work, new affinity membranes endowed with an interesting binding capacity for human IgG are studied in view of their application in the capturing step of a monoclonal antibody production process. The membranes have been extensively tested with pure IgG solutions and with a cell culture supernatant containing IgG1. The effects of feed flow rate and IgG concentration on the separation performances have been studied in detail, considering in particular binding capacity, selectivity and recovery. These new high capacity affinity membranes appear good candidates to avoid the throughput limitations and other well-known drawbacks of traditional bead-based chromatographic columns.     

Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture

High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the development of a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarified CHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum binding capacity of 65 mg/mL and an adsorption capacity of 25–42 mg IgG/mL bead in suspension for an IgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture step from 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions were obtained in cell broth containing 40 × 10⁶ cells/mL as in clarified supernatant. Two pilot-scale purification runs were then performed on non-clarified cell broth from fed-batch runs of 16 L, where a rapid mAb adsorption ≥96.6% was observed after 1 h. This process using 1 L of magnetic beads had an overall mAb yield of 86% and 16 times concentration factor. After this single protein A capture step, the mAb purity was similar to the one obtained by column chromatography, while the host cell protein content was very low, <10 ppm. Our results showed that this magnetic bead mAb purification process, using a dedicated pilot-scale separation device, was a highly efficient single step, which directly connected the culture to the downstream process without cell clarification. Purification of mAb directly from non-clarified cell broth without cell separation can provide significant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2775, 2019.     

Production and purification of a chimeric monoclonal antibody against botulinum neurotoxin serotype A

Production of recombinant antibodies against botulinum neurotoxin is necessary for the development of a post-exposure treatment. CHO-DG44 cells were transfected with a plasmid encoding the light and heavy chains of a chimeric monoclonal antibody (S25) against botulism neurotoxin serotype A. Stable cell lines were obtained by dilution cloning and clones were shown to produce nearly equivalent levels of light and heavy chain antibody by an enzyme-linked immunosorbent assay (ELISA). In suspension culture, cells produced 35 μg/ml of chimeric antibody after 6 days, corresponding to a specific antibody productivity of 3.1 pg/cell/day. A method for the harvest and recovery of an antibody against botulism neurotoxin serotype A was investigated utilizing ethylenediamine-N,N′-tetra(methylphosphonic) acid (EDTPA) modified zirconia and MEP-hypercel, a hydrophobic charge interaction chromatography resin. Purification of the S25 antibody was compared to that achieved using rProtein A–Sepharose Fast Flow resin. After the direct load of culture supernatant, analysis by ELISA and gel electrophoresis showed that S25 antibody could be recovered at purities of 41 and 44%, from the EDTPA modified zirconia and MEP-hypercel columns, respectively. Although the purity obtained from each of these columns was low, the ability to withstand high column pressures and nearly 90% recovery of the antibody makes EDTPA modified zirconia well suited as an initial capture step. Combining the EDTPA modified zirconia and HCIC columns in series resulted in both purity and final product yield of 72%.     

Development of a polishing step using a hydrophobic interaction membrane adsorber with a PER.C6®‐derived recombinant antibody

Membrane chromatography has already proven to be a powerful alternative to polishing columns in flow‐through mode for contaminant removal. As flow‐through utilization has expanded, membrane chromatography applications have included the capturing of large molecules, including proteins such as IgGs. Such bind‐and‐elute applications imply the demand for high binding capacity and larger membrane surface areas as compared to flow‐through applications. Given these considerations, a new Sartobind Phenyl? membrane adsorber was developed for large‐scale purification of biomolecules based on hydrophobic interaction chromatography (HIC) principles. The new hydrophobic membrane adsorber combines the advantages of membrane chromatography—virtually no diffusion limitation and shorter processing time—with high binding capacity for proteins comparable to that of conventional HIC resins as well as excellent resolution. Results from these studies confirmed the capability of HIC membrane adsorber to purify therapeutic proteins with high dynamic binding capacities in the range of 20 mg‐MAb/cm³‐membrane and excellent impurity reduction. In addition the HIC phenyl membrane adsorber can operate at five‐ to ten‐fold lower residence time when compared to column chromatography. A bind/elute purification step using the HIC membrane adsorber was developed for a recombinant monoclonal antibody produced using the PER.C6® cell line. Loading and elution conditions were optimized using statistical design of experiments. Scale‐up is further discussed, and the performance of the membrane adsorber is compared to a traditional HIC resin used in column chromatography. Biotechnol. Bioeng. 2010; 105: 296–305. © 2009 Wiley Periodicals, Inc.     

增殖细胞核抗原蛋白在Spodoptera frugiperda昆虫细胞中的表达及纯化

目的:利用昆虫细胞表达系统生产重组的人增殖细胞核抗原(proliferating cell nuclear antigen,PCNA),并进行纯化和抗体结合特性鉴定。方法:以HeLa细胞逆转录的cDNA为模板,扩增人PCNA基因,并插入杆状病毒载体AcMNPV。利用昆虫细胞得到PCNA基因的重组杆状病毒。病毒感染细胞表达蛋白,联合镍柱亲和层析和离子交换层析获得高纯度的重组人PCNA蛋白。ELISA法测定抗体结合特异性。结果:以HeLa细胞cDNA为模板得到的基因序列同GenBank的人PCNA基因序列一致。草地贪夜蛾细胞(Spodoptera frugiperda,Sf9)表达重组人PCNA(recombinant human PCNA,rPCNA)的最佳感染值(MOI)和感染时间分别为0.05h和144h。rPCNA的产量高达110mg/L细胞,纯度95%。间接ELISA法检测抗体结合特性,rPCNA的敏感性和特异性分别为93.3%和85.0%。结论:建立了rPCNA的表达和纯化方法,获得了高效表达、高度抗体结合特异性的PCNA蛋白,该蛋白质能进一步开发为PCNA相关疾病的体外诊断试剂盒,具较大的应用价值。     

Effects of temperature, flow rate and composition of binding buffer on adsorption of mouse monoclonal IgG1 antibodies to protein A Sepharose 4 Fast Flow.

The binding capacity of protein A Sepharose 4 Fast Flow for mouse IgG1 monoclonal antibodies (mabs) appears to be highly dependent on the buffer composition with respect to both concentration and ion type. Depending on the particular mab dynamic binding capacities up to 20 mg mab per ml gel could be obtained, when these mabs were isolated from supernatants of protein free hollow fibre cell culture systems. Variation of linear flow rate from 10 up to 300 cm/h and temperature (4 degrees C versus 25 degrees C) had a slight effect on the dynamic binding capacity, when a high ionic strength buffer was used during adsorption. Applying optimum binding conditions, final IgG fractions with a purity of more than 95% monomeric IgG were obtained. However, as side effect of the use of binding buffers with high ionic strength, the binding of acid proteases was also promoted.     

Assessment of the use of cyanogen bromide-activated Sepharose in analytical and preparative immunoadsorption

Procedures are described for the analytical and preparative purification of antigens based on their specific interaction with their complementary antibody immunoadsorbents prepared from cyanogen bromide-derivatized macroporous agarose matrices. In principle, the antigen to be purified in the affinity chromatography/immunoadsorption process should bind specifically and reversibly to the attached antibody, while other proteins pass through unretarded. In the case of tight binding, elution of the antigen is achieved by the use of eluting solutions of very high or very low pH, or with the use of chaotropic solutions such as 3 m KSCN. The performance of immunoadsorbents prepared from Sepharose 4B have been studied with the aim of improving the efficient utilization of immunoadsorption techniques. As a model, human serum was applied serially to several columns of Sepharose 4B sheep anti-human IgG which were then subjected to a number of successive adsorption/desorption cycles. Loading the columns with increasing amounts of serum showed that the performance was best when the antigen load was approximately threefold the ideal binding capacity. By limiting the amount of immobilized protein and carefully controlling the antigen load, significant improvements in yield and purity have been achieved. Antigen loads of threefold the potential binding capacity of the immunoadsorbent column results in the optimal yield of antigen with high purity and significant concomitant reduction in non-specific interference from other serum proteins. The non-specific adsorption which is an inherent problem and which leads to considerable inactivation of the covalently coupled antibody is highlighted. Although the popularity of such matrices is probably unsurpassed, it is clear that use has been made of them very frequently without an examination of quantitative aspects or side reactions.     

Chromatography of human prothrombin from Nitschmann fraction III on Q Sepharose Fast Flow using axial and radial flow column

An axial column (Hitrap Q 5 ml, 2.5 2 1.6 cm) and a radial flow column (3.5 2 5 cm) packed with Q Sepharose Fast Flow media had been evaluated for the separation of human prothrombin. Nitschmann fraction III dissolved in buffered saline (0.10 M sodium chloride buffered with 0.06 M Tris/HCl to pH 7.5) was the starting material. Effects of sample flow rate of the two columns were screened. Under radial flow conditions using the radial column, sample flow rate up to 15 ml/min (i.e. 18 bed volumes/h) was achieved and the operating pressure was below 0.2 MPa eventhough the elution velocity was 30 ml/min. Breakthrough capacity was determined by analyzing the total protein and prothrombin activity of the target protein-containing fraction under subsaturating conditions and both columns had almost the same breakthrough capacity per ml media, indicating that the sample loading was independent of radial column geometry. It was concluded that the radial column is an attractive alternate to traditional axial packed bed column, exhibiting very good potential for use in the separation of human prothrombin.     

Removal of Hexane in Biofilters Packed with Perlite and a Peat–Perlite Mixture

Removal of hexane from air–hexane mixtures in biofilters packed with different solid media under nitrogen supplementation was performed for 70 days. Two columns containing Perlite or a mixture of peat and Perlite, were used. The solid media were supplemented with nitrogen source up to 1 kg/m³ per week for high nutrient supplementation and 0.2 kg/m³ per month for low nutrient supplementation. A high rate of hexane removal: 95 g/m³ h was achieved under high nutrient supplementation, high air flow rate and high hexane concentration. However, the percentage of hexane removal decreased with increasing air flow rate and hexane inlet concentration. For high nutrient supplementation the type of solid medium did not significantly affect the biodegradation capacity. With low nutrient supplementation, the highest removal rate was achieved in the column containing the peat–perlite mixture. The column containing perlite had a significantly lower pressure drop (20 Pa/m) than the 2400–2930 Pa/m observed for the column containing the mixture. Perlite offers an opportunity of running a biofiltration process at a lower and stable pressure drop if the nutrient supplementation is managed properly.     

Screen-less expanded bed column: new approach for the recovery and purification of a malaria transmission blocking vaccine candidate from Pichia pastoris

An experimental malaria transmission blocking vaccine antigen, Pfs25H, expressed and secreted from Pichia pastoris was recovered and purified using a screenless expanded bed column equipped with a rotating fluid distribution system. This column was able to accommodate feed stock, containing 30% biomass, at a flow rate of 300–400 cm/h without affecting column stability. This capability is three times higher than the capability of the expanded bed column currently in use, which is equipped with a perforated plate fluid distribution system; this design could accommodate biomass concentrations of only up to 10%. The screen-less design did not affect the binding capacity, purification level or process yield and, therefore, shorten the process. Purified Pfs25H of 6.4 g were recovered from 37 l of Pichia pastoris culture in one step.     

Antibody purification from CHO cell supernatant using new multimodal membranes

This contribution describes strategies to purify monoclonal antibodies from Chinese hamster ovary (CHO) cell culture supernatant using newly designed multimodal membranes (MMMs). The MMMs were used for the capture step purification of human IgG₁ following a size‐exclusion desalting column to remove chaotropic salts that interfere with IgG binding. The MMM column attained higher dynamic binding capacity than a Protein A resin column at an equivalent residence time of 1 min. The two‐step MMM chromatography process achieved high selectivity for capturing hIgG₁ from the CHO cell culture supernatant, though the desalting step resulted in product dilution. Product purity and host cell protein (HCP) level in the elution pool were analyzed and compared to results from a commercial Protein A column. The product purity was >98% and HCP levels were <20 ppm for both purification methods. In addition, hIgG₁ could be eluted from the MMM chromatography column at neutral pH, which is important for limiting the formation of aggregates; although slow elution dilutes the product. Overall, this paper shows that MMMs are highly effective for capture step purification of proteins and should be considered when Protein A cannot be used, e.g., for pH sensitive mAbs or proteins lacking an Fc binding domain. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:658–665, 2017     

Production of Acetylacetoin by Bacterial Fermentation

Bacillus sp. YUF-4 did not produce acetylacetoin with general culture media, such as bouillon medium containing glucose or acetoin. When diacetyl was added to a medium cultured for 18–20 h in the presence of glucose or acetoin, AAC was produced as culture continued. AAC was assayed by GLC with a Carbowax 20M capillary column. The AAC produced was purified by several steps: the final HPLC using a Shodex E411 column was effective. The yield of AAC was 346 mg per liter of the medium (7.5% recovery) and the purity was 97%. AAC was identified by ¹H-NMR and ¹³C-NMR.     

Paper-PEG-based membranes for hydrophobic interaction chromatography: purification of monoclonal antibody

This article discusses the preparation of novel Paper-PEG interpenetrating polymer network-based membranes as inexpensive alternative to currently available adsorptive membranes. The Paper-PEG membranes were developed for carrying out hydrophobic interaction membrane chromatography (HIMC). PEG is normally very hydrophilic but can undergo phase separation and become hydrophobic in the presence of high antichaotropic salt concentrations. Two variants of the Paper-PEG membranes, Paper-PEG 1 and Paper-PEG 2 were prepared by grafting different amounts of the polymer on filter paper and these were tested for their hydraulic properties and antibody binding capacity. The better of the two membranes (Paper-PEG 1) was then used for purifying the monoclonal antibody hIgG1-CD4 from simulated mammalian cell culture supernatant. The processing conditions required for purification were systematically optimized. The dynamic antibody binding capacity of the Paper-PEG 1 membrane was about 9 mg/mL of bed volume. A single step membrane chromatographic process using Paper-PEG 1 membrane gave high monoclonal antibody purity and recovery. The hydraulic permeability of the paper-based membrane was high and was maintained even after many runs, indicating that membrane fouling was negligible and the membrane was largely incompressible.     

Miniaturised expanded-bed column with low dispersion suitable for fast flow-ELISA analyses

Flow-ELISA measurements of the monoclonal antibody concentration in cultivation broth containing murine hybriboma cells were carried out using a small expanded-bed column (0.5×2.5 cm) charged with protein A. A new specialised pellicular agarose/stainless steel matrix designed for high flow rates with fast mass transport properties was used. Special care was taken to get an efficient flow distribution. The axial dispersion coefficient was very low (2×10^–6 m² s^–1 for latex particles at a linear velocity of 10 cm min^–1). Breakthrough curves for polyclonal IgG on the protein A-derivatised support (at 2–11 cm min^–1) further emphasised its advantageous properties. No significant change in dynamic capacity was found over the entire speed range.     

Evaluation of commercial chromatographic adsorbents for the direct capture of polyclonal rabbit antibodies from clarified antiserum

We have carried out a rigorous evaluation of eight commercially available packed bed chromatography adsorbents for direct capture and purification of immunoglobulins from clarified rabbit antiserum. Three of these materials featured rProtein A (rProtein A Sepharose Fast Flow, Mabselect, Prosep rProtein A) as the affinity ligand, and differed from one another primarily with respect to the underlying base matrix. The remaining five matrices comprised various synthetic low molecular weight ligands immobilised on hydrophilic porous supports and these included: MEP HyperCel, MabSorbent A1P, MabSorbent A2P, FastMabsA and Kaptiv-GY. The general experimental approach taken was to sequentially challenge packed beds of each matrix with a series of different strengths of a clarified antiserum; beginning with the weakest and ending with the strongest. Marked differences in performance (principally evaluated on the basis of dynamic binding capacity, recovery, and purity) were obtained, which allowed clear recommendations concerning the choice of adsorbents best suited for antibody capture from rabbit antisera, to be made.     

Pilot scale recovery of monoclonal antibodies by expanded bed ion exchange adsorption

The aim of the investigations was to estimate the scale up properties of an efficient chromatographic first capture step for the recovery of murine IgG₁ from undiluted and unclarified hybridoma cell culture broth using an ion exchange matrix in expanded bed mode. The tested new sulfopropyl-based ion exchange matrix (Streamline^TM SP XL, Amersham Pharmacia Biotech) stands out due to its enhanced capacity compared to its precursor (Streamline^TM SP). Defining the working pH in preliminary electrophoretic analyses (titration curve, SDS-PAGE) and small-scaled chromatographic binding studies showed, that the optimal value for the IgG purification was pH 4.6, where a co-chromatography of the medium supplement albumin (500 mg l^-1, pI = 4.8) could not be avoided. Further scouting experiments dealt with the dynamic capacity of the matrix, which was evaluated by frontal adsorption analysis. In packed bed mode no break-through of the target protein was achieved even after 6.5 mg IgG per ml matrix were applied. These results could not be reproduced in expanded bed mode with cell-free supernatant, where the dynamic capacity was found to be only 1.5 mg IgG/ml SP XL. Processing cell-containing broth resulted in an additional decrease of the value down to 0.5 mg ml^-1, presumably caused by the remarkable biomass adsorption to the matrix. The search for the reasons led to the examination of the hydrodynamic conditions. Buffer experiments with a tracer substance (acetone) pointed out, that the flow in expanded bed was significantly more influenced by back-mixing effects and channel formations than in packed bed. These effects could be compensated with an enhanced viscosity of the liquid phase, which was achieved by the addition of glucose. As a result of the improved hydrodynamic conditions in the expanded bed, the dynamic capacity could be increased from 0.5 to more than 4.5 mg IgG/ml matrix for the processing of cell culture broth with 400 mM glucose. Finally, the scale up from a Streamline^TM 25 to a Streamline^TM 200 column was performed under conditions, which proved to be optimal: 100 L of unclarified hybridoma broth were concentrated with a binding rate of 95% in less than 3.5 hours. Loading the column no break-through of the target protein was achieved. However, the eluate still contained debris and cells, which points out the major disadvantage of the method: the biomass attachment to the matrix.     

Characterization of interactions between Escherichia coli molecular chaperones and immobilized caseins

The molecular chaperones were affinity purified with immobilized alpha-casein (45mg protein/g beads) and beta-casein columns (30 mg protein/g beads) from two heat-induced E. coli strains, NM522 and BL21. After removing nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water, 1 mM Mg-ATP, or 6 M urea. The eluates from affinity columns were analyzed by SDS-PAGE and Western analysis. Western analysis identified five E. coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES in eluates. Among samples, ATP eluates showed the highest chaperone purity of 80-87% followed by cold water eluates with 62-68% purity. The beta-casein column showed a higher chaperone binding capacity than the alpha-casein column. A higher concentration of chaperones was purified from strain BL21 than strain NM522 which may have been due to the lack of lon protease in the BL21 strain.     

Characterization of cryogel monoliths for extraction of minor proteins from milk by cation exchange

Extraction and purification of high‐value minor proteins directly from milk without pre‐treatment is a challenge for the dairy industry. Pre‐treatment of milk before extraction of proteins by conventional packed‐bed chromatography is usually necessary to prevent column blockage but it requires several steps that result in significant loss of yield and activity for many minor proteins. In this paper, we demonstrate that it is possible to pass 40–50 column volumes of various milk samples (raw whole milk, homogenized milk, skim milk and acid whey) through a 5 mL cryogel chromatographic column at 550 cm/h without exceeding its pressure limits if the processing temperature is maintained above 35°C. The dynamic binding capacity obtained for the cryogel matrix (2.1 mg/mL) was similar to that of the binding capacity (2.01 mg/mL) at equilibrium with 0.1 mg/mL of lactoferrin in the feed samples. The cryogel column selectively binds lactoferrin and lactoperoxidase with only minor leakage in flowthrough fractions. Lactoferrin was recovered from elution fractions with a yield of over 85% and a purity of more than 90%. These results, together with the ease of manufacture, low cost and versatile surface chemistry of cryogels suggest that they may be a good alternative to packed‐bed chromatography for direct capture of proteins from milk. Biotechnol. Bioeng. 2009;103: 1155–1163. © 2009 Wiley Periodicals, Inc.     

Capture of a monoclonal antibody and prediction of separation conditions using a synthetic multimodal ligand attached on chips and beads

A synthetic ligand called 2-mercapto-5-benzimidazolesulfonic acid has been successfully used for the specific chromatographic capture of antibodies from a cell culture supernatant. Adsorption occurred at physiological ionic strength and pH range between 5.0 and 6.0, with some binding capacity variations within this pH range: antibody uptake increased when the pH decreased. With very dilute feedstocks, as was the case with the cell culture supernatant under investigation, it was found that the pH had to be slightly lowered to get a good antibody sorption capacity. To optimize separation conditions, a preliminary study was made using ProteinChip Arrays that displayed the same chemical functionalities as the resin. Arrays were analyzed using SELDI-MS. By this mean, it was possible to cross-over simultaneously different pH conditions at the adsorption and the desorption steps. Best conditions were implemented for preparative separation using regular lab-scale columns. At pH 5.2, antibody adsorption was not complete, while at pH 5.0 the antibody was entirely captured. pH 9 was selected at elution, rather than pH 8.0 or 10.0, and resulted in a complete desorption of antibodies from the column. Benefits of the prediction of separation conditions of antibodies on MBI beads using SELDI-MS were a significant reduction in analysis time and in sample volume. This was possible because the separation of IgG on the chip surface did mimic very well the separation on beads.