Column chromatography has been used for many years to successfully characterize, purify and manufacture products for the food and drug industries. Scaling up from laboratory-scale to process-scale purification is one of the most important manufacturing activities for the pharmaceutical industry. However, many challenges exist in this activity. Some issues start with column selection and transitioning the workflow protocol smoothly. Columns range in size from a few millimeters in diameter for laboratory-scale to two meters or more for process-scale. With larger diameter columns, issues with irregularities in media packing, pressure and flow can occur. Here we discuss a few key considerations when transitioning from small to larger-sized columns.
Optimize procedures
When scaling up, although the chemistry usually stays consistent, procedural differences are often overlooked. The differences in the size of equipment used will require changes to some aspects of the method. For example, a small-scale column of 0.5-5 cm in diameter may include an open system where the operator takes the column top off to add media. The purification process will likely be by flow packing with manual compression. When performing at a pilot scale, the column might be 10-45 cm diameter but the packing procedure can still be comparable to the laboratory scale. Moving to process-scale, a column 2 meters in diameter generally utilizes pack-in-place technology where media is added without requiring removal of the top flow adapter (closed system): at these larger sizes, the piston is often very heavy and can require a hoist to lift it. Also cGMP manufacturing spaces often use a closed system to avoid contamination. Although the chemistry remains the same, going from discovery to process-scale conditions can be quite challenging procedurally.
Packing process-scale columns
Packing a process-scale column can become more of an art than a science. Often, each manufacturing step has different physical constraints (space considerations, ceiling height and pump size to name a few) and requires alterations in vendor-recommended procedures. In addition, the large number of available media precludes a “one-size-fits- all” procedure for varying experimental situations. On top of all this, tight deadlines often require the packing to be “right the first time.”
So, how does one achieve a well-packed column? Although the details often vary, several important principles remain the same. For example, the relationship between pressure and flow and knowing the resins critical velocity (the point at which the pressure-flow relationship is no longer linear) is very important during scale-up. Understanding column wall support is also important. As the diameter of the column increases, wall support decreases. Small columns have a higher surface area to media ratio meaning a larger fraction of the media is touching the wall helping to give support to the bed as it resists the compressive effects of flow. This ratio decreases in larger columns, so the critical velocity decreases as well. A column bed which is stable to varying flow rates with a minimum of back pressure also requires packing to the proper compression factor. Although an over-compressed bed may not further compress at high flow rates, the added compression leads to poor asymmetries and a higher overall backpressure. Often it’s the little things that may be overlooked that often create the most issues when switching to manufacturing scale.
Tip: Before use, the column should be clean and the components should be inspected for damage. Follow the manufacturer's instructions to assemble the column and perform hydrostatic integrity testing as recommended.
Well-packed resin
A well-packed, homogeneous bed delivers the best separations. The goal is to consolidate the media so that it is homogeneous between the top and bottom and from center to the column wall. The reason for having a well-packed bed is to keep product yield and reproducible consistent between runs to minimize lot-to-lot variations and maximize bed stability over time. Irregularities in packing the media can cause uneven flow within the bed and changes in pressure leading to increased backpressure. All of this can change peak retention time and/or cause peak broadening, leading to a loss in product yield and product quality. Consistency in column packing leads to a successful manufacturing workflow.
Tip: Assemble and pack your column in the same room you intend to run it if possible. If the column is assembled in ambient temperature and subsequently moved to a cold room, or vice versa, check the fittings and seals because differential expansion of the component materials can occur. This usually affects the seals or induces stress or constriction in the structure.
Reproducibility is critical
At the process-scale, you will probably perform 10-100 runs before changing the column bed. Because of the repetitive use, the reproducibility that comes along with a homogeneously packed column is critical. With that said, it’s not trivial to pack a two-meter column well. There are different in-place packing methods to consider, such as flow packing, stall packing and Dynamic Axial Compression (DAC). In DAC, the piston can be driven up or down with an external motor coupled to the central piston shaft. While all of these methods have pro’s and con’s, packing by pressure or flow can lead to variance in the bed compression and column volume that will affect packing reproducibility and bed stability. Using DAC, high flow or pressure to pack the column is not needed. DAC also improves bed homogeneity and efficiency while at the same time being very fast, with only one packing step. It can be used with both compressible and incompressible media making it more of a universal packing method for any type of chromatography resin. DAC scales easily and works well in automation, which ensures reproducibility. The main challenge with this method is the need to know the precise slurry percentage and bed height, but there are well-documented methods for calculating these variables. Choosing the right method for your situation will require some homework. The important aspect is getting a homogeneous packed column that will ensure reproducibility.
Tip: Once the manufacturing scale is defined, be sure that you have a small-scale process that mirrors the larger scale. This comes in handy when the need for validation, discrepancy investigations, trouble-shooting and continuous improvement is needed.
Media choice
There are many choices when it comes to media and it’s worthwhile to explore what is available. Resins today are developed with large-scale purifications in mind. Some provide unique selectivity, high recovery, and versatility as well as low backpressure at the higher flow rates. Nuvia™ cPrime™ offered by Bio-Rad is one of these versatile resins. In addition, some companies offer packing and column solutions at process-scale. Exploring the different options available upfront will save you time down the road.
Tip: Media fines, or fragments, can cause a column to clog and increase overall backpressure. One of the best ways to remove media fines, or fragments, is to not create them in the first place. Avoid stirring media excessively because stirring can shear the beads and generate fines. Fines which specifically occlude bottom column screens can cause altered peak profiles and loss of resolution.
Ask the experts
The good news is that several vendors of process-scale media not only sell their media but also offer custom solutions for scaling-up purification workflows. Companies such as Bio-Rad specialize in working with customers to optimize their pilot- or small-scale purification processes by ensuring they have the knowledge necessary to successfully pack and use their chromatography media. Although the process of scaling up of workflow can seem daunting, working with experts can help make your manufacturing journey an easy and successful one.
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