In a previous post, I outlined the basic departments and specialties, along with some of the easily accessible transition points between them, and am now running a deeper dive into each of those departments. The second in this series covers the Downstream department, which takes the raw cell culture harvest and turns it into the purified drug substance that is suitable for treating patients.
Chromatography – The Backbone of Purification
Name any biotech product currently in production, and it will contain at least one chromatography step – that’s how powerful and varied this technique is. From affinity resins used for product capture to multi-modal resins that can separate product-related impurities with ease, there are multiple varieties of chromatography to solve nearly any purification problem. Each of these types of chromatography may be available as resins or as membrane adsorbers, depending on the type and vendor, resulting in a wide variety of options to fit many different process requirements.
- Affinity – These resins use targeted ligands, typically some form of Protein A but can also include other options, that specifically bind the product of interest while letting other impurities flow through to be discarded. These are often used as the initial capture steps, thereby allowing the cell culture fluid and its impurities to be removed with ease. The product of interest is typically eluted from these resins with a low pH solution, which can be challenging if the product has sensitivity to acidic pH or a tendency to aggregate. Finally, these resins are often more difficult to clean and may have shorter lifetimes, as many of them will lose their ligands with harsh cleaning methods.
- Ion Exchange – These are broken up further into anion exchange (AEX) and cation exchange (CEX) options, depending on whether the resin is positively or negatively charged. Both types of resins can be operated in either bind and elute or flowthrough mode, depending on the exact conditions used, but typically CEX is run in bind and elute and AEX is run in flowthrough mode. Ion exchange resins are usually great at removing process-related impurities, such as DNA or HCP, and often provide viral clearance as well. Product-related impurities are also often removed using these steps, although significant development work may be required depending on the impurities that need to be removed.
- Hydrophobic Interaction (HIC) – This type of ligand relies on hydrophobic interactions to provide the binding activity, typically to the protein of interest, and can be very powerful at splitting apart closely related product variants. HIC often requires significant development, including screening of different strengths of ligand as well as different pH and salt options, but if there is a tricky separation required it is often the best option. Many HIC behaviors are impacted by temperature, so that should be kept in mind during both development and scale-up to ensure consistent results from bench to manufacturing and throughout the year.
- Mixed-Mode (MMC) – These newer types of resins are exactly what they say in the name, as their resin and ligands are developed to combine the effects of both ion-exchange and hydrophobic interaction chromatography. The results from these resins can be fantastic, but can also vary significantly with surprisingly small pH or salt variations, so careful development and operating range setting is important.
- Other Resin Types – Many early researchers use more analytical type of resins, such as size-exclusion (SEC) or immobilized metal affinity (IMAC), which are great for initial screening but typically are not suitable for actual production. There are also specialty resins, such as hydroxyapatite, which can be perfect for tricky purification problems but due to more difficulties using them at manufacturing scale are often not evaluated until after other options have failed.
Other considerations when developing chromatography steps include sizing the columns, choosing the right resin bead type, understanding how the resin behaves during column packing, and the cleaning and storage conditions required for the resins between purification campaigns. The experienced purification developer will also place the steps in an order that will minimize the need for large dilutions or buffer-exchange steps, while still meeting the product quality requirements and handling any impurity removal challenges along the way.
Viral Safety – No Bugs Here!
Nearly every successful downstream process will have two orthogonal steps dedicated to viral safety, specifically a viral inactivation step and a viral filter (removal) step. The inactivation step has two options, using either low pH or detergent to disrupt the lipid envelope of enveloped viruses and thus causing the virus to lose its ability to replicate and infect. Low pH viral inactivation is the most common option used in biotech processes, because it is simple and fits in nicely after a low pH affinity chromatography elution. Detergent inactivation is typically used for products which cannot handle even short exposures to the low pH values needed for viral inactivation, but the detergent used must be removed during the remainder of the process.
Virus filtration is used as a physical removal step, using a nanofilter sized to allow product to pass through while retaining any viruses that might be in solution. This step is typically the last one prior to the final TFF into the drug substance conditions, and often is when the process passes from a production suite with a higher ISO rating to one with a lower (more stringent) ISO rating. Multiple types of virus filters are available, including hollow fiber and flat sheet options, and some also have different pre-filters available to further improve throughput.
UFDF vs TFF – Buffer Exchange by Any Other Name
Buffer exchange steps are also referred to as Ultrafiltration / Diafiltration (UFDF), which describes the actions that take place during the step, or as Tangential Flow Filtration (TFF), which describes how the filter itself operates. Either way, the step operates the same and is intended to concentrate the product of interest and exchange the buffer solution for a new one. Nearly every process will have at least one UFDF step, in order to put the product in the final drug substance formulation conditions, and it’s not unusual for there to be another UFDF during the process especially if a high-salt or high-dilution step is required. These steps tend to be fairly robust and as such many developers don’t spend much time working on them, but the details of what operating conditions are used can significantly impact the final product, which may end up requiring additional development time prior to commercialization. Phenomena such as the Donnan Effect may also impact the results of the process, and result in differences between the formulation buffer and the final drug substance targets.
Depth & Sterile Filtration – But Wait, There’s More!
In addition to the tangential flow filtration and viral filtration steps discussed above, there are depth filters and sterile filters that are also involved in the process. Depth filters are typically charge-based filters used to remove precipitation and some process-related impurities, historically made with diatomaceous earth but synthetic options are now available and often more consistent. Sterile filters are rarely tested or sized during development, due to the sizing being inconsistent at smaller scale when compared with manufacturing scale, but the material of the filter used (typically polymers like PES and PVDF) should be evaluated to ensure no significant product or buffer component / excipient loss during filtration. Finally, there are some specialty filters such as glass fiber filters that can bind contaminants such as endotoxin, but these are typically only used during development as GMP conditions should ensure no endotoxin is generated during the process.
Disparate Steps, but a Cohesive Process
The Downstream development team must use their knowledge of a wide variety of purification techniques to produce a drug substance suitable for patients, regardless of the starting material. Platforms can often significantly cut development time for some molecules that share similarities with previous products, but new modalities and even some typical mAbs may still provide purification challenges. The goal is to create a purification process that will pass regulatory standards and produce safe product for the lifetime of the project, and the Downstream team is the group to get you there.