Impact of antibody aggregation on a flowthrough anion‐exchange membrane process

The impact of typical anion‐exchange flowthrough conditions on the IgG mass loading of an anion‐exchange membrane scale‐down unit (Mustang® Q coin) was investigated. High performance size‐exclusion chromatography and multiangle laser light scattering results suggested the presence of a small fraction of IgG aggregates with average radius >100 nm under anion‐exchange flowthrough conditions. The small filtration area presented by the 0.35 mL membrane volume Mustang® Q coin limited the membrane throughput due to fouling from the aggregates at higher antibody loading. Data in this report indicated that a 0.2 μm hybrid polyethersulfone and polyvinylidene fluoride membrane in‐line prefilter with a minimum filtration area of 20 sq cm alleviated the Mustang® Q coin fouling. The combined cake filtration and intermediate blocking model was proposed as the most likely membrane pore blocking mechanism. Increasing the filtration area in the in‐line prefilter resulted in higher IgG mass throughput. Thus, using an appropriately sized in‐line prefilter could provide more robust antibody throughput performance on scale‐down membrane anion‐exchange units. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Sulfated membrane adsorbers for economic pseudo‐affinity capture of influenza virus particles

Strategies to control outbreaks of influenza, a contagious respiratory tract disease, are focused mainly on prophylactic vaccinations in conjunction with antiviral medications. Currently, several mammalian cell culture‐based influenza vaccine production processes are being established, such as the technologies introduced by Novartis Behring (Optaflu®) or Baxter International Inc. (Celvapan). Downstream processing of influenza virus vaccines from cell culture supernatant can be performed by adsorbing virions onto sulfated column chromatography beads, such as Cellufine® sulfate. This study focused on the development of a sulfated cellulose membrane (SCM) chromatography unit operation to capture cell culture‐derived influenza viruses. The advantages of the novel method were demonstrated for the Madin Darby canine kidney (MDCK) cell‐derived influenza virus A/Puerto Rico/8/34 (H1N1). Furthermore, the SCM‐adsorbers were compared directly to column‐based Cellufine® sulfate and commercially available cation‐exchange membrane adsorbers. Sulfated cellulose membrane adsorbers showed high viral product recoveries. In addition, the SCM‐capture step resulted in a higher reduction of dsDNA compared to the tested cation‐exchange membrane adsorbers. The productivity of the SCM‐based unit operation could be significantly improved by a 30‐fold increase in volumetric flow rate during adsorption compared to the bead‐based capture method. The higher flow rate even further reduced the level of contaminating dsDNA by about twofold. The reproducibility and general applicability of the developed unit operation were demonstrated for two further MDCK cell‐derived influenza virus strains: A/Wisconsin/67/2005 (H3N2) and B/Malaysia/2506/2004. Overall, SCM‐adsorbers represent a powerful and economically favorable alternative for influenza virus capture over conventional methods using Cellufine® sulfate. Biotechnol. Bioeng. 2009;103: 1144–1154. © 2009 Wiley Periodicals, Inc.     

Functionalized micro-capillary film for the rapid at-line analysis of IgG aggregates in a cell culture bioreactor

A micro-capillary film has been developed that offers the potential for an at-line analytical tool for rapid aggregate analysis during biopharmaceutical antibody production. A non-porous walled micro-capillary film (NMCF) with cation exchange functionality was demonstrated to act as a chromatography medium that could be operated with high linear fluid velocities and was highly resistant to blockage by entrained particulates, including cells. The NMCF containing 19 parallel microcapillaries was prepared using a melt extrusion process from poly(ethylene-vinyl alcohol) copolymer (EVOH). The NMCF-EVOH was modified to have cation-exchange functionality (NMCF-EVOH-SP) and shown to differentially bind monomer and aggregated species of IgG antibody directly from a bioreactor. The use of NMCF-EVOH-SP to quantify aggregate concentrations in monoclonal antibody preparations in less than 20 minutes was demonstrated.     

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography. While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process. Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.). Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment. To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available. To demonstrate this protocol, representative results are presented in which chimeric murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber. Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.     

Production of monoclonal antibodies against a cell surface concanavalin a binding glycoprotein

Concanavalin A-binding (Con A)-binding cell surface glycoproteins were isolated, via Con A-affinity chromatography, from Triton X-100-solubilized Chinese hamster ovary (CHO) cell plasma membranes. The Con A binding glycoproteins isolated in this manner displayed a significantly different profile on sodium dodecyl sulfate-polyacrylamide gels than did the Tritonsoluble surface components, which were not retarded by the Con A-Sepharose column. [¹²⁵I]-Con A overlays of the pooled column fractions displayed on sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) demonstrated that there were virtually no Con A receptors associated with the unretarded peak released by the Con A-Sepharose column, whereas the material which was bound and specifically eluted from the Con A-Sepharose column with the sugar hapten α-methyl-D-mannopyranoside contained at least 15 prominent bands which bound [¹²⁵I]-Con A. In order to produce monoclonal antibodies against various cell surface Con A receptors, Balb/c mice were immunized with the pooled Con A receptor fraction. Following immunization spleens were excised from the animals and single spleen cell suspensions were fused with mouse myeloma P3/X63-Ag8 cells. Numerous hybridoma clones were subsequently picked on the basis of their ability to secrete antibody which could bind to both live and glutaraldehyde-fixed CHO cells as well as to the Triton-soluble fraction isolated from the CHO plasma membrane fraction. Antibody from two of these clones was able to precipitate a single [¹²⁵I]-labeled CHO surface component of ～265,000 daltons.     

膜渗滤亲和层析法纯化腹水单克隆抗体

用膜渗滤亲和层析法纯化腹水单克隆抗体（McAb）.采用硝酸纤维素膜（NCM）作固相支持物吸附抗原,用负压使小鼠腹水渗滤NCM,在滤过的同时腹水中的McAb不断结合于NCM上吸附的抗原,再将McAb从NCM上解离,从而得到高纯度的McAb.用此法纯化抗白蛋白（Alb）McAb.结果提纯的Alb McAb纯度达PAGE电泳单条带,将此McAb点样NCM用于斑点免疫渗滤法（DIFA）检测Alb,其灵敏度比用腹水点样时高20倍.该法快速简便,可代替亲和层析柱用于纯化McAb.     

生物功能化色谱技术研究胰岛素与其受体间的相互作用

生物功能化色谱技术是通过模拟细胞膜表面环境,在色谱系统内实现研究生物分子间相互作用过程的新技术。利用生物功能化色谱方法研究了胰岛素与其受体间的相互作用。将提取出的胰岛素受体混合物固定化在人工膜(Immobilized artificial membrane, IAM)的表面,并确定了固定化的最佳pH值为7.2,最佳离子强度为20 mmol,最佳有机溶剂浓度为2% (v/v)。通过区带洗脱法确定了胰岛素受体的存在及其与胰岛素间存在相互作用;通过前沿分析法确定了胰岛素受体与胰岛素间相互作用的结合常数大小约为0.701 nmol/L。     

Salt tolerant membrane adsorbers for robust impurity clearance

Clearance of impurities such as viruses, host cell protein (HCP), and DNA is a critical purification design consideration for manufacture of monoclonal antibody therapeutics. Anion exchange chromatography has frequently been utilized to accomplish this goal; however, anion exchange adsorbents based on the traditional quaternary amine (Q) ligand are sensitive to salt concentration, leading to reduced clearance levels of impurities at moderate salt concentrations (50–150 mM). In this report, membrane adsorbers incorporating four alternative salt tolerant anion exchange ligands were examined for impurity clearance: agmatine, tris‐2‐aminoethyl amine, polyhexamethylene biguanide (PHMB), and polyethyleneimine. Each of these ligands provided greater than 5 log reduction value (LRV) for viral clearance of phage ?X174 (pI ～ 6.7) at pH 7.5 and phage PR772 (pI ～ 4) at pH 4.2 in the presence of salt. Under these same conditions, the commercial Q membrane adsorber provided no clearance (zero LRV). Clearance of host‐cell protein at pH 7.5 was the most challenging test case; only PHMB maintained 1.5 LRV in 150 mM salt. The salt tolerance of PHMB was attributed to its large positive net charge through the presence of multiple biguanide groups that participated in electrostatic and hydrogen bonding interactions with the impurity molecules. On the basis of the results of this study, membrane adsorbers that incorporate salt tolerant anion exchange ligands provide a robust approach to impurity clearance during the purification of monoclonal antibodies. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009