Technology transfer of a monitoring system to predict product concentration and purity of biopharmaceuticals in real-time during chromatographic separation.

Technological developments require the transfer to their location of application to make use of them. We describe the transfer of a real-time monitoring system for lab-scale preparative chromatography to two new sites where it will be used and developed further. Equivalent equipment was used.

Ethanol precipitation for purification of recombinant antibodies.

Currently, the golden standard for the purification of recombinant humanized antibodies (rhAbs) from CHO cell culture is protein A chromatography. However, due to increasing rhAbs titers alternative methods have come into focus. A new strategy for purification of recombinant human antibodies from CHO cell culture supernatant based on cold ethanol precipitation (CEP) and CaCl2 precipitation has been developed.

Economics of recombinant antibody production processes at various scales: Industry-standard compared to continuous precipitation.

Standard industry processes for recombinant antibody production employ protein A affinity chromatography in combination with other chromatography steps and ultra-/diafiltration. This study compares a generic antibody production process with a recently developed purification process based on a series of selective precipitation steps. The new process makes two of the usual three chromatographic steps obsolete and can be performed in a continuous fashion.

Host cell protein analysis in therapeutic protein bioprocessing - methods and applications.

The analysis of host cell proteins (HCPs) is one of the most important analytical requirements during bioprocess development of therapeutic moieties. In this review, we focus on the comparison of different methods for the analysis of HCPs and how cell lines, fermentation conditions, and unit operations influence HCP distribution during the process chain. Current guidelines typically require reduction of HCPs to the ppm level, depending on the intended use, the route of administration of the product, and the production system.

Branched polyethylene glycol for protein precipitation.

The use of linear PEGs for protein precipitation raises the issues of high viscosity and limited selectivity. This paper explores PEG branching as a way to alleviate the first problem, by using 3-arm star as the model branched structure. 3-arm star PEGs of 4,000 to 9,000 Da were synthesized and characterized.

Protein precipitation by polyethylene glycol: a generalized model based on hydrodynamic radius.

PEGs for protein precipitation are usually classified by molecular weight. The higher molecular weight precipitants are more efficient but result in higher viscosity. Following empirical evidence that the precipitation efficiency is more comprehensively characterized by PEG hydrodynamic radius (r(h,PEG)) than molecular weight, this paper proposes a model to explicate the significance of r(h,PEG).

Two-dimensional difference fluorescence gel electrophoresis to verify the scale-up of a non-affinity-based downstream process for isolation of a therapeutic recombinant antibody.

For therapeutic antibody production Protein A chromatography is often replaced by non-affinity-based purification sequences, which are considered as more economical. 2-D DIGE was applied for evaluation of scale-up of non-affinity based process of a humanized monoclonal antibody, anti-Rh(D) IgG(1), in comparison with other conventional analytical methods, like SDS-PAGE, Western blot, or SEC. Due to a high sensitivity of this technique (125 pg protein/spot) and high dynamic range of five orders of magnitude, low molecular weight impurities were detected in purified samples.

Stem cell separation: a bottleneck in stem cell therapy.

The substantial progress in embryonic stem cell (ESC) research could lead to new possibilities in the treatment of various diseases. Currently, applications of ESC for cell therapy are impeded by the presence of potentially teratoma-forming undifferentiated ESC. Thus, a selective and quantitative removal of undifferentiated ESC from a pool of differentiated and undifferentiated cells is essential before cell therapy.

Engineering of a two-step purification strategy for a panel of monoclonal immunoglobulin M directed against undifferentiated human embryonic stem cells.

A two-step purification strategy comprising of polyethylene glycol (PEG) precipitation and anion-exchange chromatography was developed for a panel of monoclonal immunoglobulin M (IgM) (pI 5.5-7.7) produced from hybridoma cultures.

Two-dimensional fluorescence difference gel electrophoresis for comparison of affinity and non-affinity based downstream processing of recombinant monoclonal antibody.

Although Staphylococcus Protein A (SpA) affinity chromatography is the state of the art capture step for antibody purification, non-affinity methods are more economical. We used two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) to evaluate the purification of a recombinant IgG(1) antibody from cultured cells, with two different processes: (1) SpA capture followed by cation-exchange chromatography (CEX); and (2) CEX capture, followed by anion exchanger, then hydrophobic interaction chromatography. Efficiencies were similar in sodium dodecylsulphate polyacrylamide gel electrophoresis and size-exclusion chromatography; however, 2-D DIGE revealed higher efficiency with SpA than with CEX capture.

2-D DIGE to expedite downstream process development for human monoclonal antibody purification.

Two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) is an established method for assessing protein expression strategies, understanding pathogenesis mechanisms, characterizing biomarkers, and controlling therapeutic processes. We applied 2-D DIGE to facilitate the development of a purification process for a recombinant IgG1 antibody against Rhesus D antigen expressed by Chinese hamster ovary cells. The variability of two expression clones as well as the influence of cell viability on the host-cell protein pattern was assessed quantitatively.

Adaptation of hybridomas to protein-free media results in a simplified two-step immunoglobulin M purification process.

An IgM antibody was purified from hybridoma supernatant containing serum using a three-step purification process comprising of tangential flow filtration, anion-exchange chromatography and size-exclusion chromatography. Recovery and purity were significantly improved upon adaptation of the hybridoma to serum-free media. The process could even be simplified by omitting the initial tangential flow filtration step.

High-performance monolith affinity chromatography for fast quantitation of immunoglobulin G.

High-performance monolith affinity chromatography employing protein A resins has been introduced previously for the fast purification of IgG from different sources. Here we describe the design and evaluation of a fast and specific method for quantitation of IgG from purified samples as well as crude supernatant from Chinese hamster ovary (CHO) cells. We used a commercially available affinity monolith with protein A as affinity ligand (CIM protein A HLD disk).

Hydrophobic interaction chromatography of proteins IV. Kinetics of protein spreading.

Adsorption of proteins on surfaces of hydrophobic interaction chromatography media is at least a two-stage process. Application of pure protein pulses (bovine serum albumin and beta-lactoglobulin) to hydrophobic interaction chromatography media yielded two chromatographic peaks at low salt concentrations. At these salt concentrations, the adsorption process is affected by a second reaction, which can be interpreted as protein spreading or partial unfolding of the protein.

Dispersion effects in preparative polymethacrylate monoliths operated in radial-flow columns.

Monolithic media have found widespread use as excellent tools for fast analytical separations of small molecules, proteins, pDNA and viruses. Polymethacrylate monoliths with large channels are attractive for capturing large molecules, like immunoglobulins, DNA, and viruses. For preparative purposes, these monoliths are operated in radial flow mode.

Comparison of protein A affinity sorbents II. Mass transfer properties.

Protein A affinity chromatography is the standard purification method for isolation of therapeutic antibodies. Due to improvements in expression technology and optimization of fermentation, culture supernatants with high antibody content must be processed. Recently protein A affinity media with improved adsorption characteristics have been developed.

In situ determination of adsorption kinetics of proteins in a finite bath.

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.