Chromatography Focus


Facing the Challenges in Bio-Pharmaceutical Production: Developments in Ion Exchange Media to Bring Down Cost of Goods


As the bio-pharmaceutical industry matures, terms like ‘cost of goods’ are becoming more and more important. Up to now, strain optimisation for high productivity and upstream purification were the bottleneck for most bio-processes. However, with the progress made in recent years, titers in fermentation processes have increased significantly. Obviously, this increased volumetric productivity will help reducing the cost of goods, but it also has an impact on the downstream processing. Therefore, improved downstream processing media are required to process the increased product load in the same timeframe. Recently, new materials, based on fully synthetic polymer based matrices became available and show important advantages over traditional polysaccharide-derived media. In the following article the focus is on ion exchange chromatography (IEX) as an important step in the biopharmaceutical process.


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BASICS OF ION EXCHANGE CHROMATOGRAPHY


IEX has been used for many years for analysis and purification of bio-molecules [1]. Its simple concept of charge induced reversible binding has several important advantages, two of which are: binding is fast and media show a high capacity. Also, compared with other chromatographic methods, such as hydrophobic interaction chromatography (HIC) or mixed mode resins, method development is straightforward. The binding/elution behaviour can be described by a simple ‘on/off’ mechanism. The molecules will bind to the chromatographic support at low ionic strength at a pH below (for cation exchange) or above (for anion exchange) its isoelectric point. Release will take place at increased ionic strength or by pH shift.


In both cases, there is a distinct and narrow zone of pH/salt concentration, which determines whether there is binding or not. This also means that isocratic elution is not possible with IEX. Simple salt (step) gradients are most commonly used for elution. Stationary phases are generally resistant to a wide range of pH. All these characteristics make the technique ideal for the two main process steps of capture and (intermediate) purification. In addition final polishing steps can also be performed with IEX. The differences between these three steps are summarised in Table 1.


the range from 1-10g/L fermentation broth for e.g. monoclonal antibodies and up to more than 20g/L for e.g. interferon. Together with a range of other methods (e.g. precipitation, ultrafiltration, protein A affinity chromatography), IEX is frequently used for this task, where up to several thousand litres of fermentation broth are being processed. The advantage of using IEX over less discriminating methods, such as ultrafiltration, is that the amount of fermentation impurities (e.g. host cell proteins, unwanted proteins, proteases) present in the captured material can often be reduced significantly


Due to the fast adsorption/desorption mechanism it is possible to process the large volumes of fermentation broth in an acceptable amount of time. Traditionally cross- linked dextran and agarose gels (originally developed by scientists at Pharmacia [2]) have been used. New fully synthetic polymer-based materials, including YMC’s BioPro IEX bulk materials, have favourable properties compared to the traditional media. The newer materials feature higher binding capacities, better pressure stability, lower non- specific binding and higher reproducibility due to their fully synthetic origin.


Because of the large volumes involved in a capture step, the flow rates should be as high as possible in order to get to acceptable process times. As a result, the most important factors impacting on the efficiency of a capturing step are the dynamic binding capacity (DBC) and the maximum flow rate achievable. The latter point is


Because of the large volumes involved in a capture step, the flow rates should be as high as possible in order to get to acceptable process times.


Table 1: Media Characteristics for Typical Steps in Bioprocessing. Process step Capture


sometimes higher


- High dynamic binding capacity at high flow rates (up to 1000cm/h and more) - Good flow characteristics.


(Intermediate) Purification


Polishing


- Particle size ca. 30-75µm - Low non-specific binding


-- Narrow particle size distribution Particle size between <10 – 30µm


In a capture step the target compound is extracted and concentrated from the (homogenised) fermentation broth where it is present in low concentrations. The main aim in this step is to concentrate the target compound, achieve complete recovery of the target compound and the removal of bulk impurities (including protease). In this step, high purity of the resulting concentrates is an advantage, but is not essential. During (intermediate) purification the separation of the target compound from the main impurities is a key factor and purity aspects become more important.


Author Details:


Noriko Shoji, Akiko Matsui, Masakatsu Omote and Naohiro Kuriyama, YMC Co. Ltd, Ishikawa, Japan; and Britta Blödorn, Daniel Kune and Charles A. White, YMC Europe GmbH, Dinslaken, Germany.


Even though IEX is a comparatively simple method, there are still several parameters to keep in mind when developing a cost effective large-scale production process. In the following section, some key factors having an impact on the efficiency of an IEX-process step are discussed.


CAPTURE STEP:


In a capture step, the target compound is extracted and concentrated from the (homogenised) fermentation broth, where it is present in comparatively low concentrations in


- Purification of material up to 90+% purity,


- Reduction of endotoxins Purification of up to 99+%


controlled by the fluid dynamic properties of the chromatographic column; DBC will be discussed in the next section.


As for every chromatographic application, particle size and its distribution, together with the mechanical stability of the chromatographic medium, limit the maximum flow. If the mechanical stability is inadequate, the stationary phase will collapse under the backpressure resulting from high flow rates. Modern fully synthetic polymer based materials, are generally sufficiently pressure stable. Obviously, the backpressure generated is influenced by the particle size and distribution. For new synthetic polymer based materials both parameters can be controlled more easily. However, the most efficient particle size for high flow rates will always be a trade off between binding capacity and backpressure. In principle, the binding capacity increases with decreasing particle size. At the same time backpressure increases. There are big differences between different materials with regards to flow properties and DBC at high flow rates.


Important material characteristics - Particle sizes between 45-200µm,


Typical Application


- Harvest of fermentation supernatants (capturing)


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