Journal of Chromatography B (v.848, #1)

Foreword by Anthony R. Newcombe; Keith Watson (1).

Antibody production: Polyclonal-derived biotherapeutics by Claire Newcombe; Anthony R. Newcombe (2-7).
Antibody based therapies using monoclonal or polyclonal antibodies are emerging as an important therapeutic approach for the treatment of a number of diseases. With increasing emphasis on new technologies associated with monoclonal antibody expression and purification, the clinical need of polyclonal therapeutics for treatment of a variety of specific illnesses and infections is often overlooked. Despite being largely abandoned in the early twentieth century due to the development of antibiotics, polyclonal antibody therapeutics are today widely used in medicine for viral and toxin neutralization and for replacement therapy in patients with immunoglobulin deficiencies. Over the past 20 years, intravenous immunoglobulins have shown beneficial immunomodulatory and anti-inflammatory effects in many illnesses. Hyperimmune antibody preparations have been used over the past century for the treatment of a variety of infectious agents and medical emergencies, including digoxin toxicity, snake envenomation and spider bites. Here, we examine the contemporary techniques and applications, and assess the future therapeutic potential, for polyclonal-derived antibody therapeutics.
Keywords: Polyclonal; Antibody; Hyperimmune; Serum; Immunization; Therapeutic;

Pressures for cost-effective manufacture of antibodies are growing given their high doses and increasing market potential that have resulted in significant increases in total site capacities of up to 200,000 L. This paper focuses on the process economic issues associated with manufacturing antibodies and reviews the cost studies published in the literature; many of the issues highlighted are not only specific to antibodies but also apply to recombinant proteins. Data collated at UCL suggest current benchmark investment costs of $660–$1580/ft2 ($7130–$17,000/m2) and $1765–$4220/L for antibody manufacturing facilities with total site capacities in the range of 20,000–200,000 L; the limitations of the data are highlighted. The complications with deriving benchmark cost of goods per gram (COG/g) values are discussed, stressing the importance of stating the annual production rate and either titre or fermentation capacity with the cost so as to allow comparisons. The uses and limitations of the methods for cost analysis and the available software tools for process economics are presented. Specific examples found in the literature of process economic studies related to antibody manufacture for different expression systems are reviewed. The key economic drivers are identified; factors such as fermentation titre and overall yield are critical determinants of economic success. Future trends in antibody manufacture that are driven by economic pressures are discussed, such as the use of alternative expression systems (e.g. transgenics, E. coli and yeast), disposables, and improvements to downstream technology. The hidden costs and the challenges in each case are highlighted.
Keywords: Process economics; Cost of goods (COG); Benchmark; Monoclonal antibody; Production/manufacture; Mammalian cell culture; Transgenic; E. coli;

Downstream purification processes for monoclonal antibody production typically involve multiple steps; some of them are conventionally performed by bead-based column chromatography. Affinity chromatography with Protein A is the most selective method for protein purification and is conventionally used for the initial capturing step to facilitate rapid volume reduction as well as separation of the antibody. However, conventional affinity chromatography has some limitations that are inherent with the method, it exhibits slow intraparticle diffusion and high pressure drop within the column. Membrane-based separation processes can be used in order to overcome these mass transfer limitations. The ligand is immobilized in the membrane pores and the convective flow brings the solute molecules very close to the ligand and hence minimizes the diffusional limitations associated with the beads. Nonetheless, the adoption of this technology has been slow because membrane chromatography has been limited by a lower binding capacity than that of conventional columns, even though the high flux advantages provided by membrane adsorbers would lead to higher productivity. This review considers the use of membrane adsorbers as an alternative technology for capture and polishing steps for the purification of monoclonal antibodies. Promising industrial applications as well as new trends in research will be addressed.
Keywords: Membrane adsorbers; Antibodies; Immunoglobulins; Affinity; Ion exchange;

Downstream processing of monoclonal antibodies—Application of platform approaches by Abhinav A. Shukla; Brian Hubbard; Tim Tressel; Sam Guhan; Duncan Low (28-39).
This paper presents an overview of large-scale downstream processing of monoclonal antibodies and Fc fusion proteins (mAbs). This therapeutic modality has become increasingly important with the recent approval of several drugs from this product class for a range of critical illnesses. Taking advantage of the biochemical similarities in this product class, several templated purification schemes have emerged in the literature. In our experience, significant biochemical differences and the variety of challenges to downstream purification make the use of a completely generic downstream process impractical. Here, we describe the key elements of a flexible, generic downstream process platform for mAbs that we have adopted at Amgen. This platform consists of a well-defined sequence of unit operations with most operating parameters being pre-defined and a small subset of parameters requiring development effort. The platform hinges on the successful use of Protein A chromatography as a highly selective capture step for the process. Key elements of each type of unit operation are discussed along with data from 14 mAbs that have undergone process development. Aspects that can be readily templated as well as those that require focused development effort are identified for each unit operation. A brief description of process characterization and validation activities for these molecules is also provided. Finally, future directions in mAb processing are summarized.
Keywords: Monoclonal antibodies; Fc fusion proteins; Cell culture harvest; Protein A chromatography; Viral inactivation; Viral filtration; Ultrafiltration/diafiltration; Process characterization; Process validation;

Protein A chromatography for antibody purification by Sophia Hober; Karin Nord; Martin Linhult (40-47).
Staphylococcal protein A (SPA) is one of the first discovered immunoglobulin binding molecules and has been extensively studied during the past decades. Due to its affinity to immunoglobulins, SPA has found widespread use as a tool in the detection and purification of antibodies and the molecule has been further developed to one of the most employed affinity purification systems. Interestingly, a minimized SPA derivative has been constructed and a domain originating from SPA has been improved to withstand the harsh environment employed in industrial purifications. This review will focus on the development of different affinity molecules and matrices for usage in antibody purification.
Keywords: Affinity chromatography; Cleaning-in-place (CIP); Deamidation; Protein A; Protein engineering; Stabilization;

Future of antibody purification by Duncan Low; Rhona O’Leary; Narahari S. Pujar (48-63).
Antibody purification seems to be safely ensconced in a platform, now well-established by way of multiple commercialized antibody processes. However, natural evolution compels us to peer into the future. This is driven not only by a large, projected increase in the number of antibody therapies, but also by dramatic improvements in upstream productivity, and process economics. Although disruptive technologies have yet escaped downstream processes, evolution of the so-called platform is already evident in antibody processes in late-stage development. Here we perform a wide survey of technologies that are competing to be part of that platform, and provide our [inherently dangerous] assessment of those that have the most promise.
Keywords: Monoclonal; Antibody; Purification; Protein A; Alternatives to protein A; Large scale; Industrial; Protein precipitation; Depth filtration; Membrane filtration; Chromatography; Membrane chromatography; Viral clearance; Future; Centrifugation; Aqueous two-phase separation; Protein crystallization; Manufacturing facilities; PAT; Disposable technology;

Scale-up of monoclonal antibody purification processes by Suzanne Aldington; Julian Bonnerjea (64-78).
Mammalian cell culture technology has improved so rapidly over the last few years that it is now commonplace to produce multi-kilogram quantities of therapeutic monoclonal antibodies in a single batch. Purification processes need to be scaled-up to match the improved upstream productivity. In this chapter key practical issues and approaches to the scale-up of monoclonal antibody purification processes are discussed. Specific purification operations are addressed including buffer preparation, chromatography column sizing, aggregate removal, filtration and volume handling with examples given.
Keywords: Monoclonal antibodies; Antibody purification; Scale-up; GMP antibody production;

Process analytics for purification of monoclonal antibodies by Stephen Flatman; Imtiaz Alam; Jeffery Gerard; Nesredin Mussa (79-87).
The application of appropriate analytical methods is an essential requirement for the purification of therapeutic antibodies. A range of analytical methods need to be employed to effectively determine the purity, identity, integrity and activity of these important class of pharmaceuticals. These include notably electrophoresis, high performance liquid chromatography and immunoassays. Regulatory and industry demands in recent years have brought the need for improvements and many have been successfully implemented. This article reviews the current analytical methods applied to support the purification of monoclonal antibodies.
Keywords: Antibodies; Purification; Analytical methods; Process analytical technology;

Characterisation of an industrial affinity process used in the manufacturing of digoxin-specific polyclonal Fab fragments by Pranavan Thillaivinayagalingam; Kieran O’Donovan; Anthony R. Newcombe; Eli Keshavarz-Moore (88-96).
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 24 h 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.
Keywords: Chromatography; Affinity purification; Antibody; Confocal scanning laser microscopy;

Performance comparison of protein A affinity resins for the purification of monoclonal antibodies by K. Swinnen; A. Krul; I. Van Goidsenhoven; N. Van Tichelt; A. Roosen; K. Van Houdt (97-107).
During the selection of protein A affinity resin for the purification of monoclonal antibodies, dynamic binding capacity (Q dyn10%), volumetric production rate (Prvol) and ‘process robustness’ are essential parameters to be evaluated. In this article, empirical mathematical models describe these parameters as a function of antibody concentration in load (C 0), load flow rate (u load) and bed height (L). These models allow us to select optimal process conditions for each of the evaluated protein A affinity resins. C 0, u load and L largely affect dynamic binding capacity (Q dyn10%) and volumetric production rate (Prvol). Maximum Q dyn10% is generally obtained at high C 0 and at low u load. Maximum Prvol is obtained at high C 0 and at lowest L, run at high u load. All evaluated resins have a relatively high robustness against variations in C 0. |δQ dyn10%/δC 0| ranges from 0.0 to 7.8. It is clear that Q dyn10%, Prvol and ‘process robustness’ cannot be maximized all at the same time. Furthermore, some other aspects like IgG recovery, protein A leaching, easiness to pack, easiness to clean, number of re-uses and cost of production might be important to be taken into the equation. Certain evaluation parameters may be more important than others, depending on the specific situation. Therefore, a case-by-case evaluation is recommended.
Keywords: Protein A; Affinity chromatography; Monoclonal antibody; Purification; Dynamic binding capacity; Process robustness; Breakthrough curve; Production rate; Design of experiments; DOE; Packed bed absorption; PBA;

A potential generic downstream process using Cibracon Blue resin at very high loading capacity produces a highly purified monoclonal antibody preparation from cell culture harvest by F. Riske; M. Smith; M. Menon; S. Goetschalck; I.V. Goidsenhoven; A. Krul; V. Pimpaneau; I. Renaers; N. Van Tichelt; K. Van Houdt; M. Hayes; C. Lawrence; R. Bigelow; J. Schroeder (108-115).
The use of a dye-ligand chromatography for the purification of monoclonal antibody (MAb) from cell culture and other feed streams has been largely overlooked in large scale production. Cibracon Blue dye (CB), a polycyclic anionic ligand, interacts with protein through a specific interaction between the dye, acting as a mimic of NAD+ and NADP+, or through non-specific electrostatic, hydrophobic, and other forces. In this paper, a CB resin was used to effectively and efficiently separate an IgG4 MAb from host and process impurities following the capture of the MAb on a Protein-A (PA) column. The CB unit operation, challenged at ≤180 g MAb/L of resin with the PA eluate, reduced BSA (1–2 log), host cell protein (HCP; 2–3 log), MAb oligomer (31–85%), fragment (from ∼0.8% to <0.1%), and other undesired MAb species. Purity, as measured by non-reducing (NR) SDS-PAGE, was improved 33–85%, to 92–99.5% overall (>99% by reducing SDS-PAGE). A facile three column scalable production scheme, employing CB as the second column in the process was used to generate highly purified MAb from cell culture harvest derived from two media of very different compositions. Free CB dye was ≤1 ng/mg in MAb preparations purified through the three column process and then concentrated and buffer exchanged into the appropriate buffer using tangential flow filtration (TFF).
Keywords: Generic downstream process; Cibracon Blue resin; Monoclonal antibody; Purification of cell culture harvest;

We have carried out a rigorous evaluation of eight commercially available packed bed chromatography adsorbents for direct capture and purification of immunoglobulins from clarified rabbit antiserum. Three of these materials featured rProtein A (rProtein A Sepharose Fast Flow, Mabselect, Prosep rProtein A) as the affinity ligand, and differed from one another primarily with respect to the underlying base matrix. The remaining five matrices comprised various synthetic low molecular weight ligands immobilised on hydrophilic porous supports and these included: MEP HyperCel, MabSorbent A1P, MabSorbent A2P, FastMabsA and Kaptiv-GY. The general experimental approach taken was to sequentially challenge packed beds of each matrix with a series of different strengths of a clarified antiserum; beginning with the weakest and ending with the strongest. Marked differences in performance (principally evaluated on the basis of dynamic binding capacity, recovery, and purity) were obtained, which allowed clear recommendations concerning the choice of adsorbents best suited for antibody capture from rabbit antisera, to be made.
Keywords: Biomimetic ligands; Hydrophobic charge induction; Immunoglobulin purification; Mixed mode chromatography; Protein A; Lipoproteins;

A simple mathematical model to predict initial breakthrough profiles from preparative chromatographic separations of biological macromolecules has been developed. A lumped parameter approach was applied, employing Langmuirian adsorption kinetics to describe the rate of mass transfer (MT) from the bulk liquid in the column to the bound state. Equilibrium and kinetic adsorption data were determined for six different packed bed chromatographic adsorbents: two derivatised with rProtein A; and four functionalised with synthetic low molecular weight ligands. All adsorption isotherms were well described by the Langmuir model, whereas the data fitting to kinetic batch experiments showed that the model was inadequate after the first ∼25 min of adsorption for four of the six adsorbents. The model underestimated the dynamic Ig breakthough on packed beds of rProtein A Sepharose FF, MabSelect, MBI HyperCel, and MabSorbent A1P, applying a feedstock of 20–100% (v/v) clarified rabbit antiserum. However, when employing a maximum adsorption capacity 25% greater than that determined in batch binding studies, excellent agreement was obtained at all antiserum strengths for most adsorbents. Useful insights into scale-up and process design can be obtained by applying the model, without determining tentative parameters specific for each adsorbent and target protein concentration. However, the model parameters are solvent dependent so a prerequisite for its true applicability is that binding is both Langmuirian and essentially independent of the ionic strength of the feedstock applied.
Keywords: Biomimetic ligands; Hydrophobic charge induction; Immunoglobulin purification; Mixed mode adsorbents; Modelling; Protein A affinity adsorption;

We present fast, simple immunoturbidimetric assays suitable for direct determination of antibody ‘concentration’ and ‘functionality’ in crude samples, such as in-process samples taken at various stages during antibody purification. Both assays display excellent linearity and analytical recovery. Possible influences from commonly employed buffers and salts (present in samples at various concentrations), and of pH variations, were studied for both assays. Interference effects were shown to be negligible for the ‘concentration’ assay, such that sample pre-treatment prior to assay is unnecessary. The ‘functionality’ assay displayed concentration dependent sensitivity to interference for ammonium sulphate and Tris-(hydroxymethyl)-amino-methane, but was essentially unaffected by all other salts and buffer combinations tested. The immunoturbidimetric assays described here are generically applicable to polyclonal antibodies, require only basic laboratory equipment, are robust, fast, cheap, easy to perform, and readily adapted to automation.
Keywords: Immunoglobulins; Nephelometry; Precipitin reaction; Titre; Turbidimetry;

The production of pharmaceutical antibodies requires reliable and rapid processes with high purity and yield. Although protein A gels selectively and efficiently bind antibodies in the capture step, intense research is going on to find alternatives that can abolish the drawbacks of protein A chromatography. Ion exchangers e.g. are more robust, considerably cheaper and can eliminate ligand leaching. For the strong cation exchangers Fractogel® EMD SO3 (M) and Fractogel® EMD SE Hicap (M) we have evaluated the influence of pH for optimised binding and removal of host cell protein (HCP). In a fast initial screening we measured batch binding capacities. Subsequent scale-down to 96-well plate format proved that assay miniaturisation still provided reliable data. We demonstrated with the principle of residence time that scout columns are suitable for dynamic studies. The optimum pH range from batch binding was transferred to scout columns which were then used to screen for maximum dynamic capacities. In addition IEF titration curve analysis was employed to define a final operational pH. With this pH we ran labscale columns to purify monoclonal antibody. The cation exchangers showed high step yields and host cell proteins in the pools from gradient elution were reduced very effectively.
Keywords: Cation exchange chromatography; Residence time; Resin screening; Comparative study; Monoclonal antibodies; Purification; Host cell protein removal;