(35f) Integration of Two-Column, Open-Loop, Size-Exclusion, Simulated Countercurrent Chromatography into the Downstream Processing Train of a Viral Vector

The purification of complex biopharmaceuticals, such as recombinant proteins, antibodies or vaccine, is gaining increasing importance to fulfill the requirements of a reliable downstream purification process with high purity and yield, and, most importantly, cost efficiency. Viruses, in particular, play an important role in the vaccine and gene therapy fields.

The downstream biopurification train of viral vectors has been extensively developed in the past years by combining different chromatography steps, namely ion-exchange (IEX)) and size-exclusion chromatography (SEC), and, less frequently, affinity chromatography, intermingled with concentration and ultra/diafiltration steps.

To be more specific, the classical approach for adenovirus purification consists of three major steps—clarification, concentration/purification, and polishing—applied sequentially: (i) clarification of the harvested bioreaction bulk to remove cell and cell debris; (ii) ultra/diafiltration; (iii) anion exchange; (iv) ultra/diafiltration to concentrate the product; and (v) SEC as a final polishing step. The SEC step is usually carried out last, mainly because of its low productivity and low product titer. Usually in this final step the amount processed is reduced by 50–100 fold.

The current paradigm in the downstream processing of viral vectors is to operate each chromatographic step using classical single-column batch chromatography. However, the cost of goods and overall processing time can be reduced by switching to multicolumn, (semi-)continuous chromatography, which gives higher throughput, has lower buffer consumption, provides for higher capacity of utilization of the stationary phase (or smaller column volume), and hence gives increased productivity. Unfortunately, unlike other industrial fields, biotechnology is late to embrace continuous processing or multicolumn preparative chromatography.

The present work reports on the design and experimental validation of a simple quasi-continuous, open-loop, two-column countercurrent chromatographic process for size-exclusion purification of adenovirus serotype 5 (Ad5). As mentioned above, SEC has been used in the past for both polishing and intermediate purification of adenoviruses. However, it is often claimed that the main drawbacks of SEC, namely low productivity and high product dilution, make it a costly purification step. Here, it is shown that these drawbacks can be eliminated to a large extent by switching from single-column batch operation to two-column SMB-type operation.

Because of the anticipated boost in performance obtained by switching to multi-column (quasi-)continuous operation, the Ad5 purification train employed for assessing the effectiveness of the enhanced SEC step was streamlined: the SMB-SEC step was applied after the first ultra/diafiltration step and replaced the last three steps of the standard purification train. The main reason for working with the streamlined purification train was to challenge the SMB-SEC step with a less purified bulk in order to prove that, once the classical limitations of SEC are alleviated, the standard purification train can be changed to meet specific needs in terms of cost reduction or purity requirements. Moreover, running the process with a less clean bulk gave more confidence on the performance assessment of the SMB-SEC step.

In a two-column SEC process the cycle is divided into two equal-length time intervals, which we shall henceforth refer to as switching intervals. To design the cycle it suffices to define the sequence of flow-line configurations for one of the switching intervals; the sequence for the other twin switching interval is similar, but the positions of the two columns are exchanged. In practice, it is easier to keep the columns fixed in space and to move the positions of the inlet and outlet lines by means of an automated arrangement of two- or three-way valves. Over a full cycle, each column undergoes the same sequence of steps but phased out in time by one switching interval.

In the developed process, each half-cycle is divided into the following sequence of steps:

(1) Elution of the upstream column while the effluent of the downstream column is diverted to the waste line.

(2) Elution of the upstream column but its effluent is diverted to the waste line instead of being directed to the downstream column; the latter column is fed with the clarified bioreaction bulk while is effluent is collected as purified product;

(3) Operation of the system as in step 1, but the effluent of the downstream column is collected as product.

(4) Elution of the upstream column but its effluent is again diverted to the waste line instead of being directed to the downstream column; the latter column is kept frozen, that is, the flow through it is temporarily halted.

At the end of every half-cycle the positions of the two columns are exchanged to implement the simulated moving-bed contact between the solid and the fluid.

Clearance of impurities, namely DNA and host cell protein (HCP), were experimentally assessed. The obtained performance compares very favorably against single-column batch chromatography for the same volume of size-exclusion resin. In particular, our two-column SEC process achieves about the same DNA and HCP clearances as the standard batch process. However, and most importantly, the virus yield is increased from 57% for the batch process to 86% for our two-column because of internal recycling of the mixed fractions of contaminated Ad5 even though the process is operated strictly in an open-loop configuration. And last, but not least, the productivity is boosted by 6-fold with the two-column process. This is a clear demonstration that with this increased productivity, size exclusion can do much more than just final polishing and buffer exchange in the downstream train of a biopurification.

In conclusion, the main drawbacks of size-exclusion chromatography, namely low productivity and low product titer, were easily overcome by an innovative two-column configuration that keeps the mixed fractions inside the system at all times.