Anne Tscheliessnig

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. Cost of Goods (CoGs) analyses were done for: (i) a generic chromatography-based antibody standard purification; (ii) the continuous precipitation-based purification process coupled to a continuous perfusion production system; and (iii) a hybrid process, coupling the continuous purification process to an upstream batch process. The results of this economic analysis show that the precipitation-based process offers cost reductions at all stages of the life cycle of a therapeutic antibody, (i.e. clinical phase I, II and III, as well as full commercial production). The savings in clinical phase production are largely attributed to the fact that expensive chromatographic resins are omitted. These economic analyses will help to determine the strategies that are best suited for small-scale production in parallel fashion, which is of importance for antibody production in non-privileged countries and for personalized medicine.

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. A range of immunospecific and non‐specific methods are available that have been globally accepted by regulatory bodies. Immunospecific methods, such as ELISA, are simple to use in routine analysis and can quantify low levels of HCPs when specific antibodies are available. Non‐specific methods are more complex; however, they provide a holistic view of the HCP profile and qualitative information of the composition of HCP in the sample. Different methods for the comparison of bioprocessing strategies during scale‐up and purification development are compared herein. The methods include immunospecific methods, such as ELISA, western blot, and threshold, and non‐specific methods, such as 2D‐DIGE and 2D‐HPLC combined with MS.

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). Interferences of CHO host cell proteins with the quantitation of IgG from CHO supernatant were eliminated by a careful choice of the equilibration buffer. With this method developed, it is possible to quantify IgG within 5 min in a concentration range of 23-250 microg/ml. The calibration range of the method could be extended from 4 to 1000 microg/ml by adjusting the injection volume. The method was successfully validated by measuring the low limit of detection and quantification, inter- and intra-day precision and selectivity.

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. We evaluated the highly selective magnetic activated cell sorting (MACS) method for the quantitative removal of undifferentiated ESC. We found that the clearance rates for undifferentiated ESC decreased with decreasing amount of undifferentiated ESC in the cell pool. Using a simplified model calculation we could predict that, assuming an initial purity of 60%, an estimated 31 steps are required to achieve less than 10–1 cell per 109 cells. Thus, a log clearance rate of 10, which would be necessary for a therapeutically application, is hard to achieve. Our work clearly indicates that the current MACS technology is insufficient to meet the purification needs for cell therapy.

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). A general expression was formulated to characterize the PEG effect exclusively by r(h,PEG). The coefficients of a linearized form were then fitted using empirical solubility data. The result is a simple numerical relation that models the efficiency of general-shaped PEG precipitants as a function of r(h,PEG) and protein hydrodynamic radius (r(h,prot)). This equation also explains the effects of environmental conditions and PEG branching. While predictions by the proposed correlation agree reasonably well with independent solubility data, its simplicity gives rise to potential quantitative deviations when involving small proteins, large proteins and protein mixtures. Nonetheless, the model offers a new insight into the precipitation mechanism by clarifying the significance of r(h,PEG). This in turn helps to refine the selection criterion for PEG precipitants.

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. This method is based on the cold ethanol precipitation, the process used for purification of antibodies and other components from blood plasma. We proof the applicability of the developed process for four different antibodies resulting in similar yield and purity as a protein A chromatography based process. This process can be further improved using an anion-exchange chromatography in flowthrough mode e.g. a monolith as last step so that residual host cell protein is reduced to a minimum. Beside the ethanol based process, our data also suggest that ethanol could be replaced with methanol or isopropanol. The process is suited for continuous operation.

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. PEG precipitation was optimized with regards to concentration, pH and mixing. For anion-exchange chromatography, different resins were screened of which Fractogel EMD, a polymer grafted porous resin had the highest capacity. Despite its significantly slower mass transfer, the binding capacity was still higher compared to a convection driven resin (monolith). This purification strategy was successfully demonstrated for all 9 IgMs in the panel. In small scale most antibodies could be purified to >95% purity with the exception of two which gave a lower final purity (46% and 85%). The yield was dependent on the different antibodies ranging from 28% to 84%. Further improvement of recovery and purity was obtained by the digestion of DNA present in the hybridoma supernatant using an endonuclease, benzonase. So far this strategy has been applied for the purification of up to 2l hybridoma supernatants.

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. The effects of PEG branching were then elucidated by comparing the branched PEG precipitants to linear versions of equivalent molecular weights, in terms of IgG recovery from CHO cell culture supernatant, precipitation selectivity, solubility of different purified proteins, and precipitation kinetics. Two distinct effects were observed: PEG branching reduced dynamic viscosity; secondly, the branched PEGs precipitated less proteins and did so more slowly. Precipitation selectivity was largely unaffected. When the branched PEGs were used at concentrations higher than their linear counterparts to give similar precipitation yields, the dynamic viscosity of the branched PEGs were noticeably lower. Interestingly, the precipitation outcome was found to be a strong function of PEG hydrodynamic radius, regardless of PEG shape and molecular weight. These observations are consistent with steric mechanisms such as volume exclusion and attractive depletion. Biotechnol. Bioeng. 2012; 109:736–746.

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) IgG1, 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. Cation exchange chromatography was efficient capture step for IgG1 purification in laboratory and pilot scale. The differences between samples after first purification step in laboratory and pilot scale were compensated with second purification step where almost the same protein pattern was observed. 2‐D DIGE is a helpful tool for monitoring of purification effects and for scale‐up verification of downstream processes.

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. Thus, 2-D DIGE is a valuable tool for downstream process development.