The Downstream View: Using Technological Innovations to Put Product Knowledge

After working in the field for more than 25 years, Margit Holzer is a well-known expert in the downstream processing of biopharmaceutical products. She has held leadership positions in technology development, quality, and manufacturing at Novasep and was director of downstream processing (DSP) and manufacturing at Boehringer Ingelheim. Holzer has a PhD in biotechnology from the University of Natural Resources and Applied Life Sciences in Austria. BPI readers know him as an author and as a meeting participant (1, 2). In May, she gave her first course at the BPI Academy. It was about bioprocess qualification, characterization, and validation. In this article, we talk about monoclonal antibodies (MAbs), high-throughput screening, modeling and analytics, and membrane adsorbers as cutting-edge goods for making tools and technologies better.

Systems for one use

How can tools that are only used once, like membrane adsorbers, help with a speed-to-IND plan? Single-use materials have been used for a long time in study labs for early stage development. New inventions include membrane adsorbers and chromatography columns that can only be used once. Single-use systems have made sense for companies that need to meet tight recovery and confirmation times and don't have a lot of the goods they need. Single-use filters, membrane adsorbers, and chromatography columns have been easy for research labs to accept because they are used to using disposable items like pipettes and tubes. Some new technologies, like spin-filters or diafiltration for concentrating products, also make activities further down the line easier.

Single-use features that help to speed up product recovery for original target design and confirmation, tests on animals, and in vitro studies include being easy to use (most come as ready-to-use materials), not having to worry about carryover, and keeping microbes from getting into the product. Materials that are only used once and their packaging are made to be used in ISO-standard labs. Keep in mind that scalability is rarely a problem in the early stages of development. On the other hand, it does become important when making plans for toxicological material.Figure 1 shows examples of multistep DSP; (a) Three-column process with reaction and process-stream conditioning built in; (b) Four membrane adsorbers with reaction and process-stream conditioning built in; (c) Three-step process with two columns, one membrane adsorber, reaction, and process-stream conditioning built in;

Automating Things

I've seen how automatic clone picking and microscale bioreactors have cut upstream timelines by a huge amount. What changes are these tools making in DSP? A lot of success has been made in DSP as well thanks to automation. One example of this kind of progress is systems for multistep processing (Figure 1). For example, they let you load harvested broth onto one column and get back almost pure material at the multistep DSP's output. A platform process should be able to clean up target goods as long as the harvested culture broth doesn't change too much in terms of its make-up. For multistep processing, equipment needs extra features like automated controls and triggers that let the product be collected and set up for the next steps in DSP. These steps include product conditioning (for example, changing the pH or diluting the product), in-line filtration, and holding the intermediate product for a set amount of time under certain conditions.

Is automation a must for continuous chromatography to work? For constant chromatography, yes, automated equipment is a must. It would not be able to run continuously without "orchestrating" valves and instruments from different columns (see above). This is also true for the multistep DSP systems we just talked about. Not only for column chromatography, but also for membrane-based processes, there are now systems that can automatically screen media and buffer solutions. Several columns, membranes, or filter units can be checked at the same time, which is another step forward in this field. The screen conditions that are chosen depend on what is known about the material, impurities, and result.

Screening with a high throughput

In the same way, we've seen high-throughput methods for screening media mixtures earlier. What can HTS do for growth further down the line, like choosing chromatographic media? Different types of HTS devices have been made to work with DSP, and they can screen chromatographic media, membranes, and filters in different ways. 96-well filter plates, pipette tips, and miniature columns are some examples of these types of forms. Usually, the filter plates and pipette tips only need 50–100 µL of media and the same amount of sample volume. This means that sample sizes can be lowered for each test condition. Also, membrane adsorbers work the same way. One type has a surface area of 3 × 0.7 cm2 of membrane per well for sorting.

A sample with the target protein is mixed or aspirated with a resin in 96-well filter plates. The resin then binds the sample, and different pH levels or salt amounts are used to separate the sample from the resin. You can use pressure or centrifugation to collect the flow-through material and eluates while leaving the resin on the filter plate. It is possible for different loads and buffers to go through a pipette tip, and fractions are collected at the tip exit. For robotic HTS, columns have inner diameters of 5 mm and heights of up to 30 mm. Typical media amounts are 100–600 µL.

You need to know what the goal of a screening, development, or characterization study is before you can choose the right style and device. One of the goals could be to get a basic idea of how adsorption and desorption work or how process ranges relate to quality traits or step yields. A developer looked at several HTP platforms using a protein A chromatography monoclonal antibody (MAb) capture process and came to the conclusion that all of them could help with high-level scientific understanding of product yield and monomer purity (4). When robotic liquid-handling platforms like Tecan and PerkinElmer liquid handlers are combined with extra support tools that let the above-mentioned screening devices be used, the study rate is very high. Keep in mind that a robotic system for handling liquids needs to be designed with certain chromatography or membrane process methods. To keep things from getting contaminated, it's important to keep liquid streams, buffers, samples, and parts separate. Even more work could be done if 96-well plates were moved between devices by mobile robots (5).

Process Analytical Technology (PAT): Can testing be done on the floor, in the process, or online to speed up DSP? Does it really make a difference in time? And are there ways that these tools could be made better? Important parts of screening systems are standard process control instruments that measure things like pH, conductivity, temperature, and pressure. However, impurities related to the process, like host-cell proteins (HCPs) and DNA, and substance concentrations related to the product cannot be measured easily just yet. A lot of very small samples are used in HTP platforms, so most quality traits need to be checked using off-line methods that only need small amounts of samples and can do multiple analyses at the same time.