The delicate nature of biopharmaceutical species, such as MABs, can make traditional separation and characterisation techniques inappropriate. Dr Thorsten Klein, of Postnova Analytics, Germany, examines the forms and applications of Field Flow Fractionation (FFF) as an alternative.
The nature of drug development has changed immeasurably over the past couple of decades, with an increased focus on biopharmaceutical solutions and, consequently, the behaviour of biological molecules in formulation.1 In particular, monoclonal antibodies (MABs) are a relatively new biopharmaceutical development that has stimulated a variety of academic, medical and commercial interest.2
Monoclonal antibodies represent an ingenious manipulation of the body’s immune system for targeted therapeutic purposes. Within the body, beta cells produce individual antibodies targeted at a specific foreign species, such as a virus or cancerous cells. Antibodies attach to specific epitopes, short amino acid sequences on the surface of the undesirable species, acting as beacons for phagocytes, which locate and destroy the antigen. This can be used in the form of a doseable therapeutic treatment through the creation of a monoclonal population of beta cells producing antibodies targeted to a specific antigen.2
Antibody-based treatment has emerged as a broadly applicable technique with innumerable applications for a wide range of academic, commercial and medical uses and significant revenue potential.3 One of the most widely known applications for MABs is within cancer treatment, ranging from use in traditional radio immunotherapy techniques to directly target the walls of cancerous cells. Use of MABs has even been suggested for ‘untreatable’ diseases, with some research suggesting a potential treatment for HIV/AIDS.4
One issue that arises during the production of MAB species is the aggregation of the protein species during formulation. Naturally occurring protein aggregation within the body can lead to significant neurodegenerative issues, such as Alzheimer’s or Parkinson’s disease. Similarly, the presence of aggregated species within therapeutic proteins can have a serious effect on drug performance, influencing key characteristics such as drug activity and bioavailability, potentially causing severely negative immune reactions. Separating and analysing the presence of these species are key to the formulation, stability and bioavailability.5
However, traditional separation techniques, such as GPC/SEC, are often inappropriate for this type of material. This is primarily due to the relatively delicate nature of biopharmaceutical species, such as MABs, which are particularly susceptible to damaging shear effects encountered during interactive chromatographic separation.
In response there has developed a genuine need for gentle, non-destructive characterisation techniques to support the understanding of the formulation process and development of biopharmaceutical technology. In particular there is a need to analyse and monitor the aggregation of proteins during formulation of monoclonal antibodies, with an emphasis on the macromolecular 100nm – 1µm range.1
Field Flow Fractionation (FFF) is increasingly being turned to as a highly sensitive technique that, unlike its chromatographic counterparts, can gently and non-destructively separate aggregate protein species in MAB formulation.
Field Flow Fractionation principles
FFF was created by American chemist Professor J. Calvin Giddings in 1966 as a gentler, higher resolution alternative to traditional chromatographic methods. Twice nominated for a Nobel Prize, Professor Giddings pioneered the technique producing the first commercially available fractometer and founding the US-based FFFractionation, later to become Postnova Analytics following his death.
Today it is FFF’s ability to separate large macromolecular structures from solution with minimal sample preparation and without sample matrix interaction that has made it such an appealing technique to the biopharmaceutical industry.
FFF’s appeal comes not only from its breadth of analysis but also its operational simplicity. There are two components that make up the FFF system: first, the laminar flow that carries the sample through the separation chamber; and second, the separation field applied perpendicular to the channel, against the sample flow (see Figure 1).6
As particles flow along the chamber the cross flow separation field pushes the molecules towards the bottom of the channel. As they pass by the bottom they diffuse back into the channel against the carrier flow (see Figure 2). The extent to which the molecules can diffuse back into the channel is dictated by their natural Brownian motion, a characteristic based on size that is unique to each individual species. Smaller particles have a higher Brownian motion than larger ones and are able to diffuse higher into the channel against the cross flow.
Figure 2. Flow Field Flow Fractionation (AF4) channel cross section, where the rate of laminar flow within the channel is not uniform. It travels in a parabolic pattern with the speed of the flow, increasing towards the centre of the channel and decreasing towards the sides
The rate of laminar flow within the channel is not uniform. It travels in a parabolic pattern with the speed of the flow increasing towards the centre of the channel and decreasing towards the sides (see Figure 2). Therefore, the rate at which particles will be carried through will depend on their position within the channel. Those with a greater diffusion located higher in the channel will be transported with a greater velocity. The larger particles in the shallow, slower moving stream are transported with lower flow velocity and elute later than smaller particles. This results in a gentle separation of particles based on mass with the elution order of smallest to largest.
Around this basic principle numerous FFF techniques have been developed that are eminently suited to particular applications (see Figure 3). These vary both in the nature of the separative applied field and the size of particle undergoing separation. They include Flow FFF, Centrifugal FFF, Thermal FFF and Gravitational FFF, all named for the form of the separation field applied.
Figure 3. Schematic of the different applications that FFF technology would be aptly used in the separation phase
Recent developments have led to the newer technique of Centrifugal FFF, wherein the separation field is supplied via a centrifugal force. The channel takes the form of a ring that spins at 4,900rpm as illustrated in Figure 4. The flow and sample are pumped in and the mixture centrifuged, allowing the operator to resolve the particles by size and density. The advantage of Centrifugal FFF lies in its ease of use in particle separation and in the high resolution that can be achieved by varying the speed and force applied.
In contrast to chromatographic techniques, no stationary phase interactions occur, eliminating particle interaction and shearing. Another advantage of the technique is that molecules can be separated by particle density, rather than just particle size. This can be particularly useful for novel new products such as composite materials and coated polymer containing nanoparticles, samples that may not vary in size but do vary in density. In this way two identically sized particles can still be separated into two peaks, providing the density is different.
The new CF2000 instrument from Postnova Analytics has been developed as a modular FFF system that can be easily interfaced with other existing detection systems such as UV, DLS, MALS, SAXS or ICP-MS.
Historically, the separation and characterisation of aggregate species has been performed by chromatographic techniques, popularly GPC/SEC. However, there are severe limitations in using this technique for separation of relatively delicate macromolecular species.
Figure 4. Recent developments have led to the newer technique of Centrifugal FFF, where the separation field is applied via a centrifugal force. The channel takes the form of a ring, which spins at 4900rpm, with separation based on size and density
The advantages of FFF are borne out of the limitations of chromatography using a stationary phase within the column. Unpredictable sample-matrix interactions can result in low recovery due to absorption of the analyte onto the column material, while additional shear effects can potentially change the sample’s composition, size or shape. Filtering aggregates via a column matrix in this way can lead to inaccurate results at any time and often cannot offer the resolution needed to differentiate between the complex macromolecular mixture. In response FFF has emerged as one of the few techniques that can provide the level of resolution and sensitivity needed for monoclonal antibody analysis.
Additionally, GPC/SEC has limitations in the size exclusion limit of the column, which restricts the upper molar mass and particle size range of the technique. As a consequence the analysis of monoclonal antibodies by GPC/SEC tends to observe significantly lower volumes of aggregates and smaller particle sizes that for the same separation performed with Field Flow Fractionation.
Case study
Comparison of AF4, AUC and SEC for monoclonal antibody aggregate quantitation
An experiment was carried out to illustrate the performance of FFF for the separation and characterisation of monoclonal therapeutic antibody sample containing aggregates. This analysis was performed using a Postnova AF2000 Focus Asymmetric Flow FFF coupled with PN3211 UV detection.5
UV is typically used as a standard detector for aggregated identification and quantitation. Additionally, Multi-Angle Light Scattering (MALS) and Dynamic Light Scattering are advanced detectors that can be used for further characterisation to provide an absolute molar mass and particle size. So effective is this technique that the US Food and Drug Administration (FDA) has recommended the use of AF4-MALS for the characterisation of antibody formulations.5 The fractogram of an antibody obtained with AF4-UV (see Figure 5) indicates the presence of different sized species, caused by protein aggregation assumedly formed during the formulation process.
Figure 5. Fractogram of antibody obtained from AF4-UV illustrating the ability of the technique to separate aggregated species of different size from a monoclonal antibody solution
To illustrate FFF’s potential to supplant traditional separation techniques the same sample also underwent characterisation with GPC/SEC. FFF recorded a percentage of protein aggregates at 28%. GPC/SEC on the other hand measures an aggregate content of only 1%. This variation in results relates to the number of aggregates that have been filtered off and degraded by the column material, or those that have been adsorbed onto the column itself. Without an interactive stationary phase FFF provides a significantly less destructive, more representative technique that offers significant improvement in accuracy.
Subjecting the sample to additional separation using AUC (Analytical Ultracentrifugation), which measured a 24% aggregate concentration, corroborated this conclusion.
Equally, this separation could have been undertaken on the CF2000, providing even higher resolution and separation. The Centrifugal FFF CF2000 instrument has the unique feature of separating particles by Dynamic Diffusion on the basis of both particle size and density, which allows for the separation of particles with only a 5% difference in size. In AF4 separation it is based on a 1:1 ratio of mass to time. The addition of the third parameter of density to Centrifugal Fractionation produces a ratio more akin to mass to time to the power of three. This produces significantly larger distinction between peaks and results in a greatly improved resolution.
FFF presents the gentlest separative technique available, and is the only one that provides the delicate separations required within the biopharmaceutical industry.
References
1. www.malvern.com/labeng/products/iwtm/protein-aggregation.htm – accessed 13/03/13.
2. www.bio.davidson.edu/courses/molbio/molstudents/01rakarnik/mab.html – accessed 13/03/13.
3. Therapeutic Antibodies in Review. K. John Morrow Jr, Rathin C. Das, BioPharm International, February 2013.
4. Reassessing Antibodies as Treatment for HIV Infection, by Jon Cohen on 24 October 2012.
5. Comparison of AF4, AUC and SEC for Monoclonal Antibody Aggregate Quantitation – Postnova Application note.
6. New separation concept based on a coupling of concentration and flow non-uniformities. J.C. Giddings, Sep. Sci., 1, 123–125 (1966).
