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Clarification through Millistak+ media graded density depth filters

Tathagata Ray, Priyabrata Pattnaik, MS Mahadevan

Clarification stands in the critical junction of fermentation and further downstream processing. Though there are several stages of clarification, all satisfy the same objective—reduction of unwanted contaminant to the lowest possible limit. Centrifugation, tangential flow filtration, depth filtration and surface filtration are common technologies of post fermentation clarification. Combination of different technologies based on process need, is critical for effective clarification. In biopharmaceutical industry depth filtration is getting more and more importance due to its high efficiency, cost effectiveness and minimum complexity. The introduction of today's graded density charged depth filters has made clarification much more efficient than before, but process optimisation still remains a 'not so easy zone to play around'. Moreover traditional lenticular format remains the main bottleneck in high scale production due to safety concern, excessive validation requirement and considerable handling time. Disposable pod formats of recent time addresses these issues successfully.

Clarification

One of the most critical steps today in biopharmaceutical production is clarification. The primary objective of clarification is separation of very low concentration of product from high percentage of contaminants. It directly affects product yield, consistency and reproducibility. Depth filtration is one of the most cost and process effective tool in clarification. This article covers in brief depth filtration technology in comparison with other methods of clarification, recent industry trends, bottlenecks and addresses the challenges faced in the industry.

Broadly, cell culture clarification can be divided into three stages—primary clarification of extracellular product deals with removal of whole cells, cell fragments and large particles whereas intracellular expression requires a concentration, lysis and release step followed by clarification of cell debris. Dissolution and refolding followed by removal of aggregates in products like inclusion bodies, is also a type of primary clarification. Typical methods used in primary clarification are tangential flow filtration, centrifugation and depth filtration.

Removal of colloidal proteins and smaller particles are taken care in the secondary clarification step. In general, it protects the downstream columns or normal flow filtration. Combination depth filters are the best methods or techniques for this step.

Tertiary clarification refers to bioburden reduction/sterile filtration. Generally 0.45µm or 0.22µm sterilising grade filters are used for the same. This step is critical in controlling bioburden to a pre-qualified limit for protection of downstream operation.

Figure 1: Performance of Millistak+C0HC filter, 23 cm2 mini capsule (mAb, primary clarification of CHO cell culture harvest)

Technologies for clarification

Centrifugation is the most commonly used tool in primary clarification due to its cost and space advantage over the other methods. It can handle considerably high concentrations of insoluble material in the feed. Certain critical drawbacks of using centrifuge are that it attributes to cell disruption due to shear, resulting into generation of smaller particles which cannot be separated by centrifuge efficiently. The precipitation efficiency of centrifuge decreases with increasing deposition of solid sludge in the bowl resulting into frequent discharge which in turn, decreases product yield and increase process time. Buffer or water used for bowl flush can create osmotic difference between culture broth and flush fluid resulting in cell lysis and further downstream complication.

Filtration, one of the most effective techniques in down stream processing, can be classified into two parts based on application (normal flow filtration and tangential flow filtration). In normal flow filtration (NFF) pressure acts on the perpendicular direction of the membrane whereas in tangential flow filtration (TFF) pressure acts in both parallel (differential pressure or delta P) and perpendicular (trans membrane pressure or, TMP) direction, thus plugging of membrane can be controlled to a significant extent.

In clarification, tangential flow filtration has the advantage of reducing additional filtration steps due to its ability of specific contaminant cut off. But handling of high contaminant load and high concentration of host cell protein sometimes results into concentration polarisation, is a limitation.

Depth filtration—An effective tool of clarification

Depth filtration, a type of normal flow filtration, refers to porous medium capable of retaining particles, colloids throughout its width rather than just on the surface (i.e. surface, membrane filtration). The major advantages of depth filters are single use (reduction of process validation requirements), effective removal of contaminants, cost effectiveness (no CIP and SIP), scaleable. In general, depth filtration operates on two different separation mechanism, mechanical sieving and adsorption. A depth filter can have only work on mechanical sieving mode or dual mode of mechanical sieving and adsorption. In mechanical sieving or size exclusion, particles that are bigger than the large pore size present in the filter are retained. Due to even distribution of pore size, a large spectrum of particles can be retained on the surface and throughout the depth of the filter media. Adsorption works for relatively smaller particles, colloids, lipids, proteins, lipopolysaccharides and nucleic acids which bear charge on its external surface. Molecular interactions other than electrical forces may also come into picture. These molecular and electrical forces causes attraction between contaminants and inorganics filter aids of depth filter resulting into retention. Flux (flow rate per unit area) has a significant impact on capacity (throughput) of a depth filter not in terms of time but due to the fact that higher flux results into lower capacity incase of depth filtration. Hence maximum throughput of a depth filter could be attained, if constant flow rate is maintained (generally at the lower end).

In today's biopharmaceutical processes, increased cell count and longer culture times (fermentation) result into higher product expression. However, proportionately it results into reduced cell viability and increased cell debris and other contaminants, which maybe difficult to handle in further downstream processing. A proper combination of clarification tools at the post fermentation harvest step is a big challenge. In general, centrifugation and TFF are widely accepted as the primary clarification methods; whereas, these two take care of the key portion of contaminants, following depth filtration confers the polishing step. In a typical monoclonal antibody process (expressed in CHO cell line), the process flow is as follows:

In most of the cell culture based biotherapeutics, the clarification follows almost the same steps. In some of the vaccines where product is extracellular the trend is similar with slight modification in either the centrifugation or the filtration step due to product binding (generally polysaccharide).

In some of the biotherapeutics, tangential flow filtration replaces the centrifuge step. The process flow is as follows:

Replacement of centrifuge due to its inherent limitations with another depth filter is becoming more and more common nowadays. The steps are as follows:

Some of the filter manufacturers, e.g. Millipore provides two graded density depth filter media layers followed by one cellulosic 0.1 micron membrane (Millistak+ HC). In this case, 0.22µm/0.45µm filtration may also be avoided, but, however due to batch variability and safety concerns, biopharmaceutical manufacturers still prefer to keep a 0.22µm/0.45µm filtration (bioburden reduction) step prior to any column or ultrafiltration operation.

Clarification goal

To devise a clarification train, the following process inputs are needed as they are having considerable impact on the performance of depth filter directly or indirectly:

a) Step—i.e. direct harvest clarification, post centrifuge clarification etc.
b) Cell count
c) Cell viability
d) Cell type
e) Media type
f) Contaminant profile—charge, shape, nature, concentration etc
g) Product behavior—charge, shape, concentration etc
h) Product to contaminant size ratio
i) Turbidity
j) Fermentation batch variability limits

Due to all of the above mentioned variables, sizing trials is an absolutely essential (Pmax, constant flow test) for sizing of depth filters. Generally, a safety factor needs to be taken into account while deciding the required filtration area from experimental sizing data due to different combinations of above mentioned variability.

Figure 1a captures the pressure and resistance (pressure normalised for flow rate) curves as a function of amount of fluid processed for Millistak+C0HC filter and figure 1b captures the NTU breakthrough curves as a function of amount of fluid processed for Millistak+C0HC filter.

Generally plugging study at a constant flow rate is carried out till the pressure differential of 20-30 psi or 10 percent NTU (nephalometric turbidity unit)/OD600 breakthrough, which ever is earlier was considered as end point for calculation on filtration area. Intermittently cumulative filtrate volume under real time and NTU/OD600 reading of samples monitored to understand behavior of filter with respect to filtrate quality and volume of product processed. Monitoring of OD280 is a standard practice to check for protein recovery.

Figure 1 defines relationship between cell count/cell viability (both together attribute to turbidity) and filtration capacity. There are several interesting facts that can be derived from the above graphs. High cell count with high cell viability (eg cell count—3.3million/ml, viability-85 percent, NTU—787) results into pressure breakthrough (refer to figure 1a), whereas low cell count with low cell viability (eg cell count—1.3million/ml, viability-47 percent, NTU—853) results into NTU breakthrough rather than pressure breakthrough (refer to figure 1b). This is because of the fact that high cell count with high viability results into more entrapment by sieving rather than adsorption where as low cell count with low viability results into just the opposite phenomena.

Red colored line graph in figure 1b shows sharp NTU breakthrough at the very beginning of the process which is due to presence of high salt concentrations. High salt concentration is known to interact with the charged sites of the depth filter matrix and interfere in adsorption mechanism leading to faster turbidity breakthrough.

Millistak+ offers consistently high capacity with variable feed characteristics which is an indication of process robustness. NTU/pressure breakthrough and colloidal load in secondary clarification is critical in evaluating charged depth filter performance. Never the less, process developers need to watch out for presence of salt concentration (high conductivity) in feed which can affect NTU breakthrough there by filter performance.

Scaling up

Scaling up of experimental data is another challenge in depth filter clarification. Following important points should be considered when scaling up from lab scale.

1. Process compression possibility
2. Consideration of enough safety factor due to batch variability
3. Reduced piping consideration and floor space
4. Ease of use and safety
5. Scalability
6. Less validation requirement

There are two available options for scaled up clarification step—conventional lenticular disc and pod format. There are distinct advantages of pod format over lenticular discs.

Like any other manufacturing industry, today's biopharmaceutical industry also is driven by quality, quantity, time and cost. Clarification is a step where all four comes into picture with equal importance. Large quantities to be processed to get good quality filtrate with minimum cost and time. Graded density depth filter in disposable Pod format addresses all the requirements of this step successfully.

(Tathagata Ray is Process Development Specialist, Priyabrata Pattnaik is Group Leader-Process Development Sciences Group, and MS Mahadevan is Vice President of Biopharmaceutical Division at Millipore India, Bangalore-560058 India. They can be contacted at p_pattnaik@millipore.com. The authors acknowledge the support of Valencio Salema and Dr Subhasis Banerjee for their valuable inputs.)