Formulation and Delivery

What’s on the minds of biopharmaceutical formulators in 2014? They want to apply innovative technologies and phase-appropriate strategies for preformulation and formulation development of monoclonal antibodies (MAbs), next-generation biologics, non-MAb protein therapeutics, vaccines, biologics, and other product modalities. They need to be aware of the latest drug delivery options. High-throughput analytical methods are helping in preformulation and liability assessments. Stability testing and physiochemical characterization remain key.

Product Characterization: As is often the case, analytical and formulation laboratories have a lot in common. Especially at early stages of product development, the data they gather are complementary and the methods they use are often very similar if not identical. So this year, you’ll see quite a bit of overlap between the quality and analytical and the formulation and delivery programs.

“Analytical testing is a key element of control strategy for manufacturing and release of protein therapeutics,” says Alla Polozova (principal scientist in analytical sciences and process development at Amgen). “In a holistic approach, in-process, release and stability tests for drug substance and drug product can be viewed as a testing continuum. Such an integrated approach, combined with QbD-based risk assessments, supports elimination of non-value added redundancies and optimization of the entire testing strategy without compromising product quality.”

Focus on Stability: A major consideration in biologics formulation development is making the product shelf-stable for as long as possible. This can be a problem especially for highly concentrated products, such as those for delivery of large antibody doses. And sometimes the obvious answer isn’t necesesarily the right one, cautions Rachel Varney (scientist II in formulation sciences at MedImmune). Early development work on a bispecific MAb at her company showed that it had a complex chemical and physical stability profile. Measuring an unusually high loss of bioactivity in the product stored at 40 °C or exposed to UV light, the team attributed that during photostability studies to oxidation of residues in the complementarity-determining regions (CDRs). But they also identified an alternative chemical degradation pathway during thermal stress studies. Adding an antioxidant reduced soluble aggregate formation during thermal stress.

Preformulation and Case Studies: Many presenters advocate a holistic approach this year. For example, Mark Krebs (senior scientist in protein pharmaceutical development at Biogen Idec) says that early preformulation and developability studies can inform and speed up later work. “Combined with more extensive subsequent studies,” he explains in the abstract of his Wednesday afternoon talk, “this broad understanding of a molecule enables us to quickly hone in on suitable conditions for formulation.”

That same afternoon Angela Blake-Haskins (manager of biopharma drug product sciences at GlaxoSmithKline) will emphasize phase-appropriate formulation development for late-stage monoclonal antibodies. She says that such a strategy “can reduce cycle times and resource requirements and increase the chance of success throughout a product’s life-cycle. A robust formulation development strategy takes into consideration several key aspects including clinical development phase, molecule type, product profile definition, and global regulatory requirements to name a few.”

BPI’s marketing and digital content strategist, Leah Rosin, conducted the following interviews as the conference program came together this summer. Participants focsed primarily on vaccine formulations. Here, in Q&A format, is what they had to say.

Pat Ahl (Merck Research Labs)

Patrick Ahl (assistant principal scientist in vaccine drug product development at Merck Research Labs) will be joining us for the “Vaccine Formulation Development” session on Thursday morning, 23 October 2014. His presentation is titled, “Innovative Characterization Methods to Identify an Optimal Vaccine Drug Product Formulation.”

Abstract: Product formulation is typically on the critical path for both vaccine and biologic development programs alike. High-throughput screening (HTS) methods can be easily applied to biologics formulations. However, they can be difficult with vaccines because of the wide range of different antigens and adjuvants needed to develop a safe, effective, and stable formulation.

Vaccines may be live-inactivated viruses (LVV), virus-like particles (VLP), bacterial membranes, carbohydrates, proteins, or protein-carbohydrate conjugates. In addition, a vaccine drug product often can require an adjuvant consisting of an aluminum-based particle, a Toll-like receptor (TLR) agonist, an emulsion, or a lipid-based particle. There is no “short list” of formulation screening assays to accommodate that entire diverse range of potential vaccine candidate types.

Although establishing vaccine HTS methods can be a challenge, we have implemented innovative HT characterization approaches. These include Sypro-orange differential scanning fluorescence (DSF), 384- well plate dynamic light scattering (DLS), 96-well freeze–thaw plate fluorescence, and “simple” Western gel technology to increase our efficiencies. And they have increased our efficiencies in screening vaccine drug-product formulations.

Can you discuss the difficulty in formulation screening for vaccines and what pushed you towards developing a panel of tests? In my opinion, there are at least two very big difficulties in vaccine formulation development: One is antigen diversity, and the other is the low antigen concentration typically found in formulated vaccines.

Those concentrations are often in the mg/mL range rather than mg/mL (frequently found with biologics such as monoclonal antibodies). Of course, that makes the biophysical characterization of antigens in vaccines much more difficult than biologic product characterization. To overcome these vaccine formulation screening difficulties at Merck, we are introducing an automated Western gel electrophoresis assay to our vaccine formulation development program. It can quantitatively characterize protein antigens below the microgram-per-milliliter (mg/mL) concentration range.

Another difficulty often encountered during vaccine formulation screening is a severely limited quantity of available antigens during the initial stages of formulation development for preclinical animal studies. During this part of development, we need to screen all potential antigens for conformational changes and aggregation in as many formulations as possible — using as little antigen as possible. We have implemented two microwell plate-based assays that each screen up to 96 formulations at a time using <600 mg/mL of antigen.

Vaccine formulations are screened for protein antigen unfolding using a plate-based DSF instrument and Sypro orange dye, and they are screened for antigen aggregation using a highly sensitive plate-based DLS instrument.

Finally, the ultimate vaccine formulation screening difficulty is found in predicting a clinical human immune response based on the biochemical and biophysical properties of a vaccine. I understand that some people are trying to solve this huge challenge, but in my opinion this difficulty remains unsolved.

Can you discuss the accuracy of those screening methods and compare them with what you have used before? I don’t want to get specific here, particularly with respect to the screening assays mentioned above. But one method we are currently developing is an automated assay to quickly measure the amount of antigen adsorbed to an aluminum adjuvant at different antigen:adjuvant formulation weight ratios. We use this assay to rapidly measure the antigen adsorption capacity of vaccine formulations with different types of aluminum adjuvant. The automated assay is faster and requires significantly less human involvement than a manual method. At least 4–8× more tests can be run in the time it takes to run one manually. Thus, more replicate experiments are averaged together in an automated assay, which significantly improves its accuracy.

What are the challenges, if any, of using such screening methods? The decision to improve vaccine formulation screening must involve a cost/benefit question. Reducing the amount of antigen needed, increasing the number of formulations that can be screened, improving measurement accuracy, and shortening the screening time will always benefit vaccine formulation development.

At Merck, the most significant cost of improving an automated vaccine formulation screening method come from the resources and time it often takes to develop, test, and validate the new methods. Everybody has plenty of work to do supporting direct product-pipeline products. Finding the necessary people and time to make significant screening innovations can be difficult. However, we have found that improving vaccine formulation screening methods almost always pays off in the long run.

So, the methods you present are for physical screening. Can you discuss the information technology (IT) side of the process? I’m glad you asked that. Managing information generated from a successful vaccine formulation workflow is challenging and an often overlooked issue. In my opinion, appropriate information-handling software is an essential part of every formulation screening program. At Merck vaccine formulation development, we have successfully used the Spotfire software package to store and then visualize screening results.

Design of experiment (DoE) software also can be a very important tool to help identify important trends and interactions in screening studies. But unlike simple large screening studies, these DoE studies require significant preassay planning to identify potential formulation factors. The Design Expert and JMP software packages have been used at Merck to successfully conduct DoE-based vaccine formulation screenings.

Besides speaking, why are you attending the BPI Conference this year? I’m really excited about attending this large conference with a lot of attendees and a wide range of topics. I should be able to learn a lot that I can bring back to Merck. I also did postdoctoral work in Boston, and Boston is simply one of my favorite cities in the world.

Luis Brito (Novartis Vaccines)

Luis Brito (head of formulation science in the United States at Novartis Vaccines) will be joining us for the “Vaccine Formulation Development” session on Thursday morning, 23 October 2014. His presentation is titled, “Reinventing the Gene Vaccine: Stabilization and Delivery of Self-Amplifying mRNA.”

Abstract: Nucleic-acid–based vaccines such as viral vectors, plasmid DNA, and messenger RNA (mRNA) are being developed as a means to address unmet medical needs that current vaccine technologies have been unable to address. We describe a cationic nanoemulsion delivery system developed to deliver a self-amplifying mRNA vaccine. This nonviral delivery system is based on a proprietary adjuvant (MF59), which has an established clinical safety profile and is well tolerated in children, adults, and the elderly. Nonviral delivery of a 9-kb self-amplifying mRNA elicits potent immune responses in mice, rats, rabbits, and nonhuman primates (comparable to a viral delivery technology), and relatively low doses (75 μg) induce antibody and T-cell responses in primates. Our cationic-nanoemulsion– delivered self-amplifying mRNA enhances the local immune environment through recruitment of immune cells similar to an MF59- adjuvanted subunit vaccine. And both the site (within muscle) and magnitude of protein expression are similar to those found with a viral vector.

Can you describe the technology you will be presenting? How is it different from viral vector technology? The mRNA sequence basically amplifies itself. It includes a sequence that launches proteins to allow the RNA self amplify. The advantages over conventional mRNA is that if a single copy of this self-amplifying mRNA gets into the cells, then it basically turns into thousands of copies. So the amount of protein or antigen expressed from this vector is much higher than what you would get with a conventional mRNA or plasmid DNA.

When you compare it with viral-vector vaccines, those vectors are made in cell culture and are typically a little difficult to grow. They require extensive purification to remove media and exogenous materials. Additionally, viral vectors (particularly envelope viruses) are notorious for being unstable. So we are basically removing that hurdle from our vector.

We are using a cationic nanoemulsion based on the MF59 adjuvant from Novartis. It’s a squalene oil and water emulsion: Squalene oil droplets are suspended in a buffer that has a cationic lipid tethered inside the droplets. That allows for absorption of negatively charged RNA onto the surface, with a positive charge that facilitates entry into cells. There, it acts similarly to a viral vector but without the complications of producing a viral vector.

Why not use a viral-vector delivery method in this case? There is something called vector immunity. When you are immunized using a viral vector — an enveloped virus, for example — your body recognizes that as a virus and generates antibodies against it. That prevents you from immunizing again with the same vector. Schemes have been drawn up using one type of vector and then a second type of vector. But you are basically changing the vector; it’s not really a platform technology. Because we don’t have any proteins on the surface of our delivery system, it is ignored by the immune system and does not create vector immunity.

Additionally, we know how to make nanoemulsions at large scale, and we know how to make liposomes at large scale. So we have the capability to make these in a good manufacturing practice (GMP) fashion more readily than we could a viral vector.

Are there any drawbacks? Messenger RNA is known to degrade pretty readily. It is designed to degrade within the body. Through stabilization techniques, we feel that we can stabilize it in a form that would be amenable to mass production similar to other types of vaccines.

When it comes to nonviral delivery, I think tolerability and safety are always a key question. We believe that because we are using local, intramuscular delivery — and the amount of nucleic acid and cationic lipid that we have to deliver is relatively low compared with systemic applications (e.g., for siRNA) — then we will be able to minimize those complications. As with any technology, there will be some drawbacks. But we are hopeful that we won’t have many issues as we go forward.

What still needs to be done to make a technology like this work? We need proof-of-concept in man. We’ve generated data all the way up to nonhuman primates, and we are incredibly optimistic about the results we’re getting. We’ve been able to match a clinical concentration of viral vector in nonhuman primates. So we’re definitely optimistic that this is something that will work out. But until we have that proof-in-concept in man, we just don’t know.

Other than speaking, why are you attending the BPI Conference? I’m really looking for new technologies — for delivery of biologics — and trying to get a sense of what’s cutting-edge on the industry side. At academic types of conferences, you get certain angles as to what’s “the next big thing” bubbling up through academia. It really looks like key opinion leaders from industry are going to be represented here. I’m looking forward to seeing what has bubbled up through industry, what they can share, and how the biologics pipeline looks at other companies.

Listen Online! These interviews have been edited from transcripts for space and style. You can access the original conversations at www.bpiroundtables.com/bpi14.