Biopharmaceutical production: Applications of surface plasmon resonance biosensors.

Surface plasmon resonance (SPR) permits the quantitative analysis of therapeutic antibody concentrations and impurities including bacteria, Protein A, Protein G and small molecule ligands leached from chromatography media. The use of surface plasmon resonance has gained popularity within the biopharmaceutical industry due to the automated, label free, real time interaction that may be exploited when using this method. The application areas to assess protein interactions and develop analytical methods for biopharmaceutical downstream process development, quality control, and in-process monitoring are reviewed.

Decision-support software for the industrial-scale chromatographic purification of antibodies.

The high therapeutic and financial value offered by polyclonal antibodies and their fragments has prompted extensive commercialization for the treatment of a wide range of acute clinical indications. Large-scale manufacture typically includes antibody-specific chromatography steps that employ custom-made affinity matrices to separate product-specific IgG from the remainder of the contaminating antibody repertoire. The high cost of such matrices necessitates efficient process design in order to maximize their economic potential. Techniques that identify the most suitable operating conditions for achieving desired levels of manufacturing performance are therefore of significant utility. This paper describes the development of a computer model that incorporates the effects of capacity changes over consecutive chromatographic operational cycles in order to identify combinations of protein load and loading flowrate that satisfy preset constraints of product yield and throughput. The method is illustrated by application to the manufacture of DigiFab, an FDA-approved polyclonal antibody fragment purified from ovine serum, which is used to treat digoxin toxicity (Protherics U.K. Limited). The model was populated with data obtained from scale-down experimental studies of the commercial-scale affinity purification step, which correlated measured changes in matrix capacity with the total protein load and number of resin re-uses. To enable a tradeoff between yield and throughput, output values were integrated together into a single metric by multi-attribute decision-making techniques to identify the most suitable flowrate and feed concentration required for achieving target levels of DigiFab yield and throughput. Results indicated that reducing the flowrate by 70% (from the current level) and using a protein load at the midpoint of the range currently employed at production scale (approximately 200-500 g/L) would provide the most satisfactory tradeoff between yield and throughput.

Detection and quantification of affinity ligand leaching and specific antibody fragment concentration within chromatographic fractions using surface plasmon resonance.

Rapid analyses of chromatographic steps within a biopharmaceutical manufacturing process are often desirable to evaluate column performance, provide mass balance data and to permit accurate calculations of yields and recoveries. Using SPR (surface plasmon resonance) biosensor (Biacore) technology, we have developed a sandwich immunoassay to quantify polyclonal anti-digoxin Fab fragments used for the production of the FDA (Food and Drug Administration)-approved biotherapeutic DigiFab. The results show that specific Fab may be quantified in all affinity process streams and accurate yield and mass balance data calculated. Control experiments using sheep Fab and Fc indicate that the assay is specific to DigiFab. The quantification of potential leached ligand within chromatographic fractions may also be technically challenging, particularly when low-molecular-mass ligands are covalently coupled with an affinity absorbent. Typical methods to assess ligand leakage such as DDMA (digoxin-dicarboxymethoxylamine; digoxin analogue) often involve the use of labelled ligands and relatively complex and labour-intensive analytical techniques. Using the same analytical methodologies, an assay to detect leached or eluted ligand off the column was developed. The results indicate minimal levels of leached ligand in all chromatographic fractions, with total levels of leached DDMA calculated to be 1.52 microg. This is less than 0.01% of the total amount of DDMA coupled with the laboratory-scale affinity column. The SPR methods described in the present study may be applicable for the rapid in-process analysis of specific polyclonal Fab fragments (within a polyclonal mixture) and to rapidly assess leakage of small molecule ligands covalently attached to chromatographic supports.

Characterisation of an industrial affinity process used in the manufacturing of digoxin-specific polyclonal Fab fragments.

This paper describes the effect of several variables on the affinity process for the production of the FDA approved biotherapeutic product Digoxin Immune Fab (Ovine) (DigiFab, Protherics Inc., TN, USA). The study considers the effects of column re-use on matrix capacity and on the subsequent recovery of the antibody product, and the impact of varying column loading on matrix performance. The methodology used could be equally applied to assess the feasibility of using an affinity matrix for commercial scale purification of alternative antibody derived biotherapeutics. The capacity and specific Fab recovery were calculated through 24h equilibrium and mass balance studies. Results were assessed against data obtained through confocal scanning laser microscopy. Scale-down experiments produced specific Fab recoveries and purities that were comparable with those at production scale. The matrix capacity was found to be 45+/-15 mg of Fab/ml of matrix. Through the use of fluorescent DigiFab and confocal scanning techniques, Fab uptake onto single affinity bead was evaluated. Average intensity values calculated for each sample provided direct real-time, measure of Fab binding and matrix capacity. The results suggest that the affinity matrix had a limited reuse life as a drop in recovery is observed following the completion of a small number of process cycles (30% after three runs). The findings support that which is seen at the current manufacturing scale, where the affinity column is used for a limited number of runs. Results from this study can be used as a basis for future optimisation of this purification process.