Robert L. Fahrner

Industrial Purification of Pharmaceutical Antibodies: Development, Operation, and Validation of Chromatography Processes

Recombinant monoclonal antibodies are becoming a great success for the biotechnology industry. They are currently being studied in many clinical trials for treating a variety of diseases, and recently several have been approved for treating cancer (Carter et al., 1992; Anderson et al., 1996; Baselga et al~, 1996; Bodey et al., 1996; Longo~ 1996). Although there are several types of antibodies produced in different types ofcel1lines, the most clinically significant antibodies are full-length humanized IgG. produced in CHO cells. This review describes the methods used to purify these antibodies at industrial scale, focusing on chromatography processes~ and with particular reference to recent work at Genentech. Routine laboratory purification ofantibodies has been well described (for example see Scott et aL, 1987), but the considerations for large-scale production of pharmaceutical-grade antibodies are much different than those for laboratory scale. There are extreme purity requirements for pharmaceutical antibodies~ and routine large-scale production requires high yield and process reliability. To gain regulatory approval, the process must be completely validated to run consistently within specified limits, so the process should be designed to facilitate validation, Large-scale production of antibodies as pharmaceutical products is a complex

Membrane ion-exchange chromatography for process-scale antibody purification

The large-scale production of recombinant monoclonal antibodies demands economical purification processes with high throughputs. The potential for ion-exchange membrane adsorbers to replace traditional ion-exchange columns was evaluated. Breakthrough capacities of commercially available cation-exchange membranes were determined as a function of flow-rate and layer number. Due to economic and process restrictions, cation-exchange membranes may not currently be advantageous for process-scale antibody purification in a bind and elute mode. However, anion-exchange membranes in a flow-through mode may provide a reasonable alternative to columns for the removal of low levels of impurities such as DNA, host cell protein, and virus.

Performance comparison of Protein A affinity‐chromatography sorbents for purifying recombinant monoclonal antibodies

We describe the performance characteristics of five Protein A affinity‐chromatography sorbents (Sepharose Fast Flow, Poros 50, Poros LP, Prosep and Streamline) for purifying a recombinant humanized monoclonal antibody from clarified Chinese hamster ovary cell culture fluid. We measured the dynamic capacity at varying flow rates, maximum capacity, pressure drop and production rate. For purified antibody, we measured yield and purity (by SDS/PAGE, the amount of DNA, the amount of host‐cell proteins and the amount of Protein A). We found that, whereas all sorbents provided significant and essentially equivalent antibody purification, there were differences in capacity and pressure drop, which affected the production rate and had implications for process applications.

Selective Antibody Precipitation Using Polyelectrolytes : A Novel Approach to the Purification of Monoclonal Antibodies

We evaluated the potential for polyelectrolyte induced precipitation of antibodies to replace traditional chromatography purification. We investigated the impact of solution pH, solution ionic strength and polyelectrolyte molecular weight on the degree of precipitation using the anionic polyelectrolytes polyvinylsulfonic acid (PVS), polyacrylic acid (PAA), and polystyrenesulfonic acid (PSS). As we approached the pI of the antibody, charge neutralization of the antibody reduced the antibody–polyelectrolyte interaction, reducing antibody precipitation. At a given pH, increasing solution ionic strength prevented the ionic interaction between the polyelectrolyte and the antibody, reducing antibody precipitation. With increasing pH of precipitation, there was an increase in impurity clearance. Increasing polyelectrolyte molecular weight allowed the precipitation to be performed under conditions of higher ionic strength. PVS was selected as the preferred polyelectrolyte based on step yield following resolubilization, purification performance, as well as the nature of the precipitate. We evaluated PVS precipitation as a replacement for the initial capture step, as well as an intermediate polishing step in the purification of a humanized monoclonal antibody. PVS precipitation separated the antibody from host cell impurities such as host cell proteins (HCP) and DNA, process impurities such as leached protein A, insulin and gentamicin, as well as antibody fragments and aggregates. PVS was subsequently removed from antibody pools to <1 µg/mg using anion exchange chromatography. PVS precipitation did not impact the biological activity of the resolubilized antibody. Biotechnol. Bioeng. 2009;102: 1141–1151.

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.

Monoclonal antibody purification using cationic polyelectrolytes: An alternative to column chromatography

The potential of cationic polyelectrolytes to precipitate host cell and process related impurities was investigated, to replace one or more chromatography steps in monoclonal antibody purification. The impact of antibody isoelectric point, solution properties (pH and ionic strength), and polyelectrolyte properties (structure, molecular weight and pKa) on the degree of precipitation was studied. At neutral pH, increasing solution ionic strength impeded the ionic interaction between the polyelectrolyte and impurities, reducing impurity precipitation. Increasing polyelectrolyte molecular weight and pKa enabled precipitation of impurities at higher ionic strength. PoIy(arginine) was selected as the preferred polyelectrolyte in unconditioned cell culture fluid. PoIy(arginine) precipitation achieved consistent host cell protein clearance and antibody recovery for multiple antibodies across a wider range of polyelectrolyte concentrations. Poly(arginine) precipitation was evaluated as a flocculant and as a functional replacement for anion exchange chromatography in an antibody purification process. Upstream treatment of cell culture fluid with poly(arginine) resulted in flocculation of solids (cells and cell debris), and antibody recovery and impurity clearance (host cell proteins, DNA and insulin) comparable to the downstream anion exchange chromatography step.

Expanded bed adsorption in the purification of monoclonal antibodies: a comparison of process alternatives.

Expanded bed adsorption (EBA) was examined as the initial capture/purification step in the purification of monoclonal antibodies from Chinese hamster ovary (CHO) cultures. Two process alternatives each using EBA were compared to a conventional Protein A process without EBA. One alternative used Protein A affinity EBA followed by packed-bed cation and anion-exchange steps. The other alternative used cation-exchange EBA as the capture step followed by packed-bed Protein A and anion-exchange steps. The process using Protein A EBA produced comparable purity (host cell protein, DNA, Protein A, antibody aggregate) to the conventional process. However, the Protein A EBA column showed a significant decrease in dynamic capacity with a limited number of cycles. The process using cation EBA achieved comparable levels of host cell proteins (HCP) and DNA but not antibody aggregate or leached Protein A compared to the conventional process.

Using precipitation by polyamines as an alternative to chromatographic separation in antibody purification processes

Polyamine precipitation conditions for removing host cell protein impurities from the cell culture fluid containing monoclonal antibody were studied. We examined the impact of polyamine concentration, size, structure, cell culture fluid pH and ionic strength. A 96-well microtiter plate based high throughput screening method was developed and used for evaluating different polyamines. Polyallylamine, polyvinylamine, branched polyethyleneimine and poly(dimethylamine-co-epichlorohydrin-ethylenediamine) were identified as efficient precipitants in removing host cell protein impurities. Leveraging from the screening results, we incorporated a polyamine precipitation step into a monoclonal antibody purification process to replace the Protein A chromatography step. The optimization of the overall purification process was performed by taking the mechanisms of both precipitation and chromatographic separation into account. The precipitation-containing process removed a similar amount of process-related impurities, including host cell proteins, DNA, insulin and gentamicin and maintained similar product quality in respect of size and charge variants to chromatography based purification. Overall recovery yield was comparable to the typical Protein A affinity chromatography based antibody purification process.

Real-time control of antibody loading during protein A affinity chromatography using an on-line assay.

We show that an on-line chromatographic assay can reliably control antibody loading in real-time during protein A affinity chromatography purification of a recombinant antibody from clarified Chinese hamster ovary cell culture fluid. The on-line assay directly sampled preparative column effluent and provided real-time measurement of antibody breakthrough during loading. The on-line assay used protein A immobilized on perfusion chromatography media, equilibrated with phosphate-buffered saline at pH 7.2 and eluted with phosphate-buffered saline at pH 2.2. The assay reliably ended loading at 1% breakthrough with minimal yield loss. Reproducible yield and purity were obtained over 23 consecutive cycles. Yield remained constant while breakthrough capacity and the antibody concentration in the load changed.

Real-time control of purified product collection during chromatography of recombinant human insulin-like growth factor-I using an on-line assay

During preparative reversed-phase chromatography of recombinant human insulin-like growth factor-I (IGF), the separation of IGF from IGF aggregates cannot be determined using UV absorbance. An on-line reversed-phase chromatographic assay was developed that provides a quantitative measurement of IGF and IGF aggregates every 4 min, allowing real-time control of purified IGF collection. Process control using the on-line assay is a reliable and accurate method to collect purified IGF.