The world faces a clear need for innovative biological products to treat and prevent diseases that cause significant health burdens. What might be less obvious is a need for innovation in biomanufacturing processes. If these products are to be made more efficiently and cost-effectively through less wasteful and safer means, then improvements are definitely needed.

“We need innovations in downstream bioprocessing, whether the products are vaccines, recombinant proteins, or other bioproducts,” said Uwe Gottschalk, vice president of purification technologies at Sartorius Stedim Biotech (SSB), at the recent Downstream Processing Day cohosted by SAFC and Sartorius Stedim Biotech in Liverpool, UK, 26 January 2011. “It is also important to follow critical raw materials all the way through biomanufacturing because this is now being watched very closely now by the regulators.”

According to bioprocess consultant Vaughan Thomas of Tillingbourne Consulting, there is a real need for innovation in bioprocessing. “Some processes we use in the plant are expensive and difficult to run,” he said. “There is increasing interest in single-use manufacturing facilities. In the past five years, hardly a week has gone by without new single-use products coming onto the market, from bioreactors through filtration and even formulation.”

Despite its continuing need for innovation, the bioprocessing field has not remained static; numerous advances have been made in recent years. Far fewer serum-containing media are used than ever before, for example. Cell culture concentrations and expression titers are going up, while longer perfusion and cycle times yield increasing cell debris in process streams.

On the engineering front, Thomas described tangential-flow filtration as one of the most interesting advances. “Innovations in chromatography include inline dilution. Sequential multicolumn chromatography from Novasep promises significant savings in resin costs,” he said. “There are also alternatives to columns, such as countercurrent chromatography from Brunel Dynamic Extractions, prepacked columns from companies such as GE Healthcare, and membrane chromatography. Initially suited to flow-through applications, these are now more widespread.”

Viral Safety

Viral contamination has appeared in news headlines over the past couple of years, with high-profile recalls and withdrawals resulting from unexpected viruses appearing in marketed products and those in clinical testing. “The blood industry is a leader [in viral safety], having had to deal with both viruses and prions,” Thomas said. “Recent cases have shown the importance of control and highlighted the need to be careful when introducing new technology so it does not cause unexpected problems.”

As SAFC strategic relationship manager Hikmat Bushnaq-Josting explained, viral contamination is a low-frequency, high-impact event. “Estimates range from 1 in 360 to 1 in 500 bioreactions suffering from viral contamination at a production scale. It can be very difficult to identify the root cause, and few investigations into the source of a virus are successful,” Bushnaq-Josting said, “so broad preventative methods are necessary.”

Sources of viral contamination include process gases and cell banks. Filters are effective for the former; viral screening is usually effective for the latter, at least in regards to specific viruses and virus types for which cell banks are tested. The biggest risk is liquid culture media, feeds, and additives, with their dozens of components from a range of sources. “Sterile does not mean virus-free!” Bushnaq-Josting emphasized.

Historically, serum was a problem ingredient. It is still used in manufacturing some products (especially vaccines), where it presents significant potential risk. Many components and feeds contain plant hydrolysates, and glucose can also be high risk. “One of the first lines of defense,” said Bushnaq-Josting, “is to replace animal-origin raw materials, such as using synthetic cholesterol or fatty acids from non-fish sources.”

However, it is still necessary to carry out a viral inactivation operation for all biologic products. The best time to do so is just before their use, which reduces chances of reinfection. Treatment options include high-temperature short-term treatment (HTST), which is already widely used in the food industry; C-band ultraviolet (UVC) inactivation; nanofiltration; and gamma irradiation.

“I think complete viral eradication will be impossible,” Bushnaq-Josting said. “Whole ease-of-use must be considered, such as plug-and-play to avoid decanting. For example, HTST technology could be introduced between media preparation and hold tanks. It takes up about the space of two pallets.” Two types of HTST technology are available: an established shell-and-tube system from Cotter Brothers/GEA Process Engineering and an electric Actijoule heating process from ABC Actini, which saves energy by heat recovery but is less widely adopted. Validation must be performed on sample metrics because viruses cannot be introduced to actual manufacturing processes.

“We think what is required is to start off with ‘annoyances’ to industry,” Bushnaq-Josting concluded, “such as 50% glucose and hydrolysates. More complex products such as serum will be more difficult because of the number of components involved.”

Host-Cell Proteins

François Champluvier (a scientist working in downstream industrialization at GlaxoSmithKline Biologicals in Belgium) described how his company implemented membrane chromatography as a back-up step for host-cell protein (HCP) removal from a glycoprotein antigen made using Chinese hamster ovary (CHO) cell culture. The antigen is purified using three sequential chromatography steps — anion exchange (AEX), cation exchange (CEX), and hydroxyapatite (HA) — at increasing pH levels. However, the manufacturing process is largely fixed by the time a product reaches phase 3 trials, after which major changes involve repeating phase 1–2 studies.

“The two first chromatography steps discard most of the HCP, ” Champluvier said, “but the HA step is necessary to remove the final amounts. It’s an indispensable fine-tuning step. We need to ensure that the HCP level is below 5% — and in some cases it can be higher than that. The challenge was to maximize HCP removal without significant loss of antigen. Three different SSB filters were tried. About half of the antigen was lost with the Sartobind Q strong anion filter. The Sartobind HIC phenyl ligand filter provided the best HCP removal, but similar amounts of antigen were also lost. The best results came with a Sartobind S strong cation filter, which eliminated 30% of the HCP without antigen loss.”

The resulting process scaled up well and added only an hour to the classical purification process, with no buffer changes needed. “It’s easy to handle, store, and use,” said Champluvier, “comparable to using a simple filter, and is low cost compared to a chromatography filter. Th
e complete set-up and scale-up was done in nine production weeks, and it was a very good solution for us late in the development process.”

Disposables

Isabelle Uetwiller, senior validation manager at SSB’s Confidence validation service, reviewed regulatory requirements for material safety in different regions. “All have similar requirements,” she said, “and the interaction between things used in processes must be considered. These include packaging materials used in direct contact with actives.”

The European Medicines Agency (EMA), for example, requires general information about a plastic container’s composition and specifications, including performance data. For plastics, extraction studies must be done under stress conditions to increase the rate of extraction with an appropriate solvent. Interaction studies must be run under process conditions with an active substrate (using the medicinal product in at least one batch to determine container compatibility) in addition to migration and sorption studies. The overall objective of such testing is toxicological assessment to determine material safety under process conditions.

When it comes to single-use, disposable plastics, validation is a shared responsibility between suppliers and users, Uetwiller said. “A vendor can supply documentary evidence regarding performance of products with model solutions and quality control (QC) for a selection of raw materials, which are verified by user audit.”

Platform Technologies

ProMetic Biosciences makes chromatography materials and technology for biologics purification. Chief executive Steve Burton explained that the company’s products include mimetic ligands that are cheaper and safer than biologically sourced alternatives. An affinity capture step in downstream processing uses a product-specific ligand that is particularly useful in manufacturing product variants, for which a conserved region of protein is available for binding. It has high capacity and provides good product recovery, as well as efficient removal of HCP aggregates or truncated forms. The ligand can also be used in secondary purification and polishing steps.

Protein A is used the same way to purify monoclonal antibodies (MAbs), which represent only about a third of biological products in development. To apply platform purification for other products, Burton explained, “We developed Mimetic Blue adsorbents for albumin and albumin-related products such as transgenic albumin from clarified whey and albumin fusion protein. It is robust and can be sanitized with NaOH [sodium hydroxide], so it is usable for multiple cycles.”

If no known adsorbent binds to a given target, then new affinity ligands will be needed. “These can be found by modeling or optimizing existing ligands,” said Burton, “or using active-site knowledge to give a model ligand.” The alternative is high-throughput screening of library compounds using an assay that detects a ligand, followed by focused sublibrary design. “In two or three iterations,” he reported, “we will get a very specific ligand for the protein.”

Vaccines and Outsourcing

Hikmat Bushnaq-Josting, a technical manager for SAFC, gave some pointers about when it’s best to outsource critical processes in vaccine manufacture. “The development time for a vaccine can be longer than for a protein therapeutic,” he said. “Managing spikes in demand for vaccine manufacture is a nightmare! It is easier to plan the raw materials for protein therapeutics because forecasting demands works pretty well. But vaccines are much more difficult: We don’t know when pandemics will happen.”

Several factors should be considered for vaccines, some of which are often overlooked. “You need a holistic view of the process and what it will cost to do things internally,” he said, “including people, investments, and energy costs. It’s not a simple decision; it’s like a maze!” There is also a capacity issue. If your maximum buffer capacity is 2,000 L and a pandemic breaks out, you will be unable to ramp up vaccine production, but the alternative is to have large amounts of capacity unused the rest of the year. This is much less of a problem for biologic medicines, for which the demand is more steady.” As a result, Bushnaq-Josting explained, vaccine manufacturers are now increasingly moving toward outside preparation of their liquid products.

It is especially effective to outsource for small volumes, he said. “The higher the volumes are, the greater the economies of scale, and it becomes cheaper to do it yourself because of transport costs. To find the right switching point, it’s important to understand your process fully and include everything: energy, people, equipment. Also ask a contract manufacturer what safety and quality it can add that you don’t have — and what costs can be saved through simplification. You should also ask yourself what more flexibility, ease of scale-up, and back-up manufacture are worth. What would the losses be if you don’t have enough process liquids when the market demands your products? How much is back-up manufacturing worth if you have a problem such as viral contamination in your plant?”

Facing Greater Demands

As the biologics industry progresses, Vaughan Thomas of Tillingbourne Consulting believes that manufacturing facilities face an exciting future. “There is a drive to reduce facility costs with rapid deployment, and more single-use equipment is coming in,” he said. “Currently, five of the top 10 drugs by sales are biopharmaceutical products, and this is predicted to rise to eight out of 10 by 2014. I believe innovation and invention will continue.”

Author Details
Sarah Houlton, PhD, is a freelance science journalist based in Cambridge, MA, who specializes in pharmaceuticals, chemicals, and chemistry; sarah@owlmedia.co.uk.