Biopharmaceutical formulation and delivery are more than science; they also must take into account patient preferences and behavior, the biology of diseases being treated, and even the concerns of legal, sales, and marketing groups. But science is the foundation. Formulation work has become more methodical and quantifiable thanks to advancing analytical technologies — which brings quality by design (QbD) into formulation laboratories.

The vast majority of biotherapeutics and vaccines are parenteral drugs — many of them lyophilized and reconstituted, some shipped and stored as liquid formulations. High-concentration formulations are the industry’s way of delivering large quantities of many protein therapeutics to patients in the least painful and most effective way. But that introduces viscosity issues that cause normal delivery methods to be impractical. Using large-diameter needles to deliver drugs isn’t popular with patients, so formulation scientists need to develop other solutions. And new product classes such as cell and gene therapies present their own challenges.

AUDIENCE: FORMULATORS, ANALYTICAL AND PRODUCT DEVELOPMENT PERSONNEL
KEYWORDS: PARENTERALS, QBD, DRUG DELIVERY, PRODUCT CHARACTERIZATION, BIOSIMILARS, COMBINATION PRODUCTS, HIGH-CONCENTRATION FORMULATIONS
TAKE-AWAY: LEARN HOW TO EFFECTIVELY CODEVELOP DRUG PRODUCT FORMULATIONS AND RELATED DELIVERY DEVICES.

Product Characterization and Prediction

Biophysical and other analytical technologies have advanced to present formulators with new ways to characterize drug molecules and solutions. Modeling software and laboratory robotics allow scientists to test many variations on the theme of protecting fragile proteins and patients alike.

Stability is key to a successful formulation. Oxidation is one of its major enemies. Two speakers on Wednesday, 10 October 2012 at IBC’s 12th annual Formulation Strategies for Protein Therapeutics (colocated with the BioProcess International Conference and Exhibition in Providence, RI) will focus on this challenge.

Caroline Loew of Hoffmann-La Roche will discuss oxidation detection. “Oxidation can have a dramatic influence on an antibody’s activity and stability,” she says. “This chemical degradation can be efficiently counteracted by an appropriate formulation; however, the detection of oxidized species remains a time-consuming step.” Loew will present a fast and quantitative high-performance liquid chromatographic (HPLC) method based on protein A binding for high-throughput screening of Fc methionine (Met) oxidation.

“Two-electron oxidation of Met due to peroxides is one of the major pathways for protein oxidation,” explains Vikram Sadineni of Bristol-Myers Squibb, “resulting in loss of stability and activity. Attempts have been made to predict the long-term two-electron oxidation kinetics of Met during early phase pharmaceutical development, which could assist with mitigation steps such as amino acid mutation or formulation optimization.” He will present a strategy for quickly and methodically assigning a risk category for methionine at recommended storage conditions based on a limited number of accelerated degradation studies.

On Wednesday afternoon, Mark Brader of Biogen Idec considers the utility of rapid screening for thermostability. “The goal of rapid clinical evaluation often dictates that formulations and candidate molecules are selected based on short-term accelerated stability data,” he explains. “High-throughput–type instrumental methods capable of generating large arrays of protein thermostability data can represent attractive platformable approaches to efficient screening and rapid-decision making.” Brader will examine the putative relationship between molecular thermostability and protein pharmaceutical stability.

Afterward, Michael Doyle of Bristol-Myers Squibb expands the thermal stability discussion to examine native-state solubility. “Solubility is a key attribute of protein therapeutics that influences the ease with which they can be purified and formulated,” he says. Thermal stability and native-state solubility profiling can help formulators identify candidates during discovery with good biophysical behavior. Doyle will discuss biophysical characterization during the discovery of a bispecific Adnectin protein therapeutic candidate targeting EGFR and IGF1R.

Aggregation-prone molecules can be identified early on as well, according to Murali Bilikallahalli of MedImmune and Tim Kelly of KBI Biopharma. They say that a range of common variants — both conformational (e.g., misfolded and partially unfolded species) and chemical (e.g., oxidized species and disulfide variants) — can act as precursors to aggregation. Murali’s presentation will focus on novel method of stopped-flow kinetics and thermodynamics to identify aggregation prone precursors which are otherwise difficult to identify and monitor by regular biophysical/biochemical methods. Kelly’s presentation will focus on processing conditions that can cause such precursors to form, their impacts on long-term product stability and quality.

Biophysical analysis can help in both downstream process and formulation development. “Optimization of stability and solubility of a protein at low pH may be required for Protein-A chromatography and low pH viral inactivation,” says Haripada Maity of ImClone Systems (a subsidiary of Eli Lilly). “This optimization can be very challenging using conventional methods.” Maity will present a MAb case study to demonstrate exceptional utility of monitoring unfolding/refolding kinetics over other measurements. He will also discuss conformational stability, structural characterization, physical size, unfolding/refolding kinetics, pH, and concentration-dependent protein–protein interaction, as well as application of biophysical analysis in resolving contamination issues during process development.

“Charge heterogeneity can impact the physical and biochemical attributes of a MAb,” elaborates Genentech’s Zephania Kwong Glover. “This may affect its therapeutic potential.” Glover’s team isolated the major charge variants (acidic, main, and basic) of an IgG antibody using cation-exchange displacement chromatography at gram scale, then characterized them using a number of analytical, biophysical, and biological methods. “Results suggested that the charge variants do not affect the in vitro potency, FcRn binding affinity, or the pharmacokinetic properties in rats,” she said.

Clearly product characterization methods can help predict a molecule’s behavior in formulation. But they don’t preclude real examination of the solution itself. A panel discussion sponsored by Protein Simple on Tuesday afternoon, 9 October 2012, will address a number of questions involved in subvisible particle characterization, monitoring, and control. Chairperson Valentyn Antochsuk of Merck Bioprocess Development will lead panelists from Genentech, Genzyme, Merck, and Human Genome Sciences in exploring the best analytical techniques for measuring particles in the 2–10 µm range and best practices for part
icle monitoring in clinical development and commercialization. Does subvisible particle formation influence product stability, and can those particles become visible or change their character based on the properties of their container’s size? The panel will look for a correlation between subvisible particles and immunogenicity and consider how USP acceptance criteria can be adapted to current understandings. They will examine the exceptions seen in particle characterization work and discuss what additional analytical capabilities would be useful — especially in managing inconsistent results from different methods.

For example, light-scattering techniques have already shown to be powerful tools for analyzing high- and low-concentration biotherapeutics, as Michael Marlow of Regeneron Pharmaceuticals will explain Wednesday afternoon.. “The impact of molecular crowding and biomolecular self-interaction is markedly concentration dependent,” he says. “Formulated biotherapeutics typically exceed the high-concentration threshold, and the resulting nonideality complicates reliable estimation of critical attributes from measurements under dilute conditions.” Marlow will describe the utility of these techniques for bridging dilute and high-concentration approaches and provide insight regarding both the nature of the molecular interactions and the effects of formulation components.

Fill–Finish Symposium: Monday, 8 October 2012

Symposium #6
Risk Management in Fill/Finish Operations cochaired by Mark Yang, director of fill finish development at Genzyme (a Sanofi company) and Brian Roberts, process development and GMP project manager at SAIC

Fill–finish operations are critical steps in biopharmaceutical manufacturing. Problems occurring during these processes can have serious consequences, from delaying product release to losing a batch to costly product recalls. This symposium overviews and updates the state of the art, with associated risks and mitigation strategies for different stages.

Roberts will provide an overview of risk management for fill–finish and process transfers to CMOs. Yang will elaborate on risk mitigation for container–closure preparations and lyophilization process transfer. Other speakers will focus on media fill and process validation, in-process inspection and logistics, and failure investigations.

Formulation and Quality By Design

Well on the way to becoming an integral part of manufacturing process development, QbD has found its way into formulation laboratories too. A Wednesday morning session is devoted to sharing case studies that illustrate how. First, Mary Cromwell of Genentech shows how her group used a QbD approach to drug-product development for a MAb that was recently submitted to global health authorities for licensure. She will describe how that affected study design, the formulation/fill–finish process, and its related control systems — comparing the results with those of a traditional approach.

Formulation Strategies for Protein Therapeuticics: Sessions

Monday, 8 October 2012 1:00–5:00 PM Symposium #6: Risk Management in Fill–Finish Operations Tuesday, 9 October 2012 8:30 am – 12:15 PM Formulation Strategies for Biosimilars and Next-Generation Biologics 1:40–3:15 PM Panel Discussion 3:45–5:15 PM Rational Selection and Design for Improved Pharmacokinetics Wednesday, 9 October 2012 7:55–9:45 AM Quality by Design for Drug Product Development 10:30 am – 2:45 pm Product Characterization for Formulation Development 2:45–6:00 PM Predictive Methods in Formulation Development

Next, Karin Shoenhammer of Novartis shows how QbD principles supported the design of a highly concentrated protein therapeutic formulated and filled as a liquid in prefilled syringes. Her group used risk assessments to capture prior knowledge and expertise from other development activities to identify potential critical material attributes (pCMAs) of the formulation. After ranking those, the team used a design of experiments (DoE) approach to test stability both in real time and at accelerated conditions.

Amgen also used DoE in developing a highly concentrated MAb solution, as described by Holly Huang in the final presentation of the session. “Multiple challenges are often encountered during formulation development,” she says. “The solution for one problem is not always straightforward and may not apply to the others.” In her case study, a DoE based on a customized mixture-design model helped mitigate issues of high viscosity and protein instability in both liquid and frozen states. Testing the predicted results demonstrated a mixture of excipients to solve complex issues that single-excipient formulations could not.

A rational approach can even improve pharmacokinetics, as the presenters will show in a Tuesday afternoon session. “Properties that govern the half-life and clearance of antibodies and similar molecules are not well understood,” explains Abbott’s Vineet Kumar. “The utility of preformulation work during the early candidate screening of molecules thus becomes important because the selection of the candidate with the best drug-like properties and physicochemical stability is certainly expected to be the optimal candidate for any PK and formulation studies.” Kumar’s group learned from other projects in adapting an appropriate screening funnel in a rational approach — requiring early interdepartmental collaboration among pharmacokinetics, formulations, analytics, and other groups.

PEGylation — adding polyethylene glycol to a protein molecule rather than simply including it in solution — is one method of improving the drug’s pharmacokinetics. “Improvement of pharmacokinetics through PEGylation could offer less frequent and more convenient dosing regimens in the clinic,” says Anna Park of Genzyme. She will demonstrate successful pharmacokinetic/pharmacodynamic extension of a heterodimeric, cysteine-knot protein through systematic exploration of several PEGylation strategies. Her group developed novel conjugation chemistry of carbohydrates, “meeting our target product profile in animal studies.” Park’s presentation will show how the protein’s known structure–function relationship was confirmed by PEGylation.

Zhuchun Wu of Human Genome Sciences sums it all up this way: “Protein formulations are developed to impede protein degradation and to preserve functional activities. Although negligible degradation is always desirable, the tolerabl
e level of degradation could be assessed in the context of in vivo structures and functions.” Wu will present a case study evaluating protein modifications in vivo and how such information can be used to support drug formulation and stability.

Biosimilars and Next-Generation Biologics

Issues surrounding next-generation products open the formulation program on Tuesday morning, 9 October 2012, and close the delivery program on Friday afternoon, 12 October 2012.

Tuesday’s keynote presentation by Pfizer’s Kevin King will provide an overview. “The diversity of biotherapeutic modalities in development and in the marketplace is increasing,” King says. “Successful formulation and process studies and subsequent development of a commercial dosage form are key to the successful development of these new medicines through clinical trials and eventually launch.” He will highlight the scientific challenges and progress being made in designing stable, safe, and efficacious dosage forms.

Hospira’s Krishnan Sampath will focus on biosimilars shortly thereafter. “Drug product formulation and process development need to be risk-based and occur earlier during product development cycle of biosimilars,” he explains, “than for originator products to ensure commercialization success.” Sampath will offer development strategies and approaches that align with scientific considerations for demonstrating biosimilarity and QbD principles.

Asian markets present other challenges, with a number of different regulatory paths and intellectual property (IP) protections. “Development of biosimilar therapeutic proteins imposes additional requirements on the formulation that are uniquely connected to the reference product,” points out Ramesh Kumar of India’s Biocon Ltd. That can be complicated if an innovator product formulation is IP-protected. “Development of a noninfringing formulation requires a blend of knowledge of the product, types of formulation-specific IP available, regulatory understanding, and scientific expertise,” he says. He will describe the sequential approach his company followed to develop a noninfringing, lyophilized formulation for a PEGylated biosimilar product.

Afterward, Valentyn Antochshuk of Merck Bioprocess Development will delve further into biosimilar product development, what he calls “a widely debated topic. Realization of the concept requires ultimate understanding of two products — the originator and the intended biosimilar — and an extensive development in the preclinical phase.” As regulatory expectations are developed, biosimilar pipelines continue to expand. He will evaluate process and product development challenges associated with such products.

In some cases, the answer might lie in an entirely different delivery method or route of administration. Some options will be discussed at the end of the week: from nanoparticles to oral and inhaled formulations. “Developing bioadhesive delivery systems (particularly nanoparticles) has been the focus of many research groups,” says Edith Mathiowitz of Brown University, “due to their potential as carriers for oral drug delivery of proteins.” Despite years of research, however, such a system has not yet been realized. She will present steps involved in developing engineered bioadhesive nanoparticles with their mechanism of delivery as well as the total uptake taking place when they are delivered to specific region of the gastrointestinal tract.

Entrega’s Jonathan Behr will follow her with a case study describing a proprietary, spatially directed oral formulation technology for peptides, proteins, and poorly bioavailable small molecules. “Drug is delivered through the intestine using adherent wafers,” he explains, “that provide a controlled microenvironment for drug delivery. The potential of the technology has been demonstrated with a variety of molecules using in vitro and in vivo models.”

Delivery Strategies for Biologics: Sessions

Monday, 8 October 2012 1:00–5:00 PM Symposium #6: Risk Management in Fill–Finish Operations Thursday, 11 October 2012 8:15–9:00 AM Keynote Presentation: The Next Generation of Biotherapeutic Dosage Forms 9:00 AM – 12:00 PM Delivery Strategies for High-Viscosity and High-Concentration Drug Products 1:25 AM – 5:00 PM Codevelopment Strategies for Biologic–Device Product Combinations Friday, 12 October 2012 8:30 am – 1:30 pm Localized and Targeted Delivery of Biologics 1:30–3:00 pm Next-Generation Routes of Administration for Biologics

Andrea Leone-Bay of MannKind Corporation will focus on inhalation without nebulizers. “Advances in dry-powder formulation, particle engineering, and inhalation device technologies have been integrated to simplify drug delivery by oral inhalation,” she says. “These advances have made possible the development of dry-powder formulations of biologics, including peptides and proteins, and their administration by oral inhalation using breath-powered, patient-friendly inhalers.” Leone-Bay will describe product development using such technologies for diabetes, osteoporosis, and pain.

In a Thursday morning keynote, MedImmune’s William Lambert will address drug delivery devices in the context of the next-generation biologics. “Biologics continue to play a greater role in modern medicine,” he says. “Drug delivery and device technologies will play a key role in providing successful drug products for second-generation biologics and biosimilars.” The value of those technologies will be tied to their ability to meet both clinical and marketing needs. Lambert will review their use in marketed products and provide insight for future development approaches.

Combination Products

Many of the most innovative biologics in development are paired with specialized delivery devices. Codevelopment requires specialized strategies. For example, as Merck’s Soumendu Bhattacharya explains in a Thursday afternoon session on the topic, regulatory agencies require protein-based formulations to be tested for compatibility with process materials (e.g., elastomers, stainless steel) and dosing devices. He will describe compatibility tests his company runs to consider different manufacturing attributes such as duration and contact area as well as compatibility with dosing devices (e.g., prefilled syringes). Bhattacharya will suggest manufacturing process materials and devices backed up by protein quality data.

Clinical in-use stability evaluations represent another important kind of testing. The results can guide dosage handling and instructions for intravenous administration of antibody products
. “During preparation of dose solutions,” says Camellia Zamiri of Abbott Biotherapeutics, “the dilution of the product in the infusion diluent alters the formulation composition, which may result in stability issues.” She will present a case study illustrating the importance of evaluating a diluted dose solution stability before locking down a formulation.

Gerhard Mayer of Sensile Medical will describe a specific delivery option: patch pumps (“micropumps”) for large-volume subcutaneous products.

He says they enable patients to subcutaneously self-administer large-volume (2–10 mL) of sometimes highly viscous formulations over a relatively short time span (e.g., 2–20 minutes). “This is important for new biotech drugs,” he says, “allowing formulators to circumvent the volume constraint of 1 mL that is typical of prefilled syringes and auto-injectors.”

“Delivering large volumes (>2 mL) of biologics requires unique delivery devices that can handle higher viscosity and programmability,” adds Anand Subramony of the Novartis Institutes for BioMedical Research. “One strategy for successful product development is to start early on the choice and functionality of devices and understand the various synergistic aspects they can bring.” He will present key steps in identifying a device choice and subsequent product development, along with an overview of the device landscape for the parenteral delivery of concentrated biologics.

Delivering Highly Concentrated Products

Highly concentrated and viscous formulations are particularly challenging to modern formulation and delivery experts. “There are currently a large number of high-dosage MAbs under development,” says Robin Hwang of ICP Consulting. “Due to viscosity and stability issues, concentrating the drug volume down to 1 mL is not always possible.” But rapid subcutaneous injection of 10 mL may be possible with the help of hyaluronidase. Hwang points out that “slow injection of 10 mL without hyaluronidase is also possible” with a patch pump as described above. To ease patient concerns and lessen pain, this approach causes none of the tissue extortion that would otherwise occur. In a Monday morning session, Hwang will present some options for large-dose MAb injections. Afterward, Jeff Abel of Amgen will suggest some “novel yet practical solutions” to filtration and processing challenges that come with high protein concentrations and “excipient-based strategies” for such products.

Protein crystallization is one technology that could reduce the viscosity of those product formulations. “There are limited options for delivering the large quantity of therapeutic MAbs needed for clinical efficacy,” says Tod Lauerman of Althea Technologies. Solubility, viscosity, and potency limitations often require intravenous infusions that can be time consuming and costly, requiring administration by healthcare professionals. Lauerman will describe his company’s Crystalomics platform for producing highly concentrated (≤350 mg/mL), low-viscosity protein formulations suitable for subcutaneous administration. Some products have completed phase 2 human trials with this technology.

Targeted Biologic Delivery

In some cases, once a biotherapeutic molecule is in circulation, getting it to its site of action is a challenge in and of itself. The most obvious difficulty is that presented by the blood–brain barrier (BBB). In a targeted-delivery session on Friday morning, Reinhard Gabathuler of biOasis Technologies will explore physiological approaches for drug delivery to the brain, offering critical opinions and pointing out the advantages and disadvantages of each. For example, antibodies and ligands for specific receptors expressed at the BBB (e.g., antibodies to the transferrin receptor and ligands to the LDL receptor–related protein) may be able to help deliver biologic compounds to treat brain diseases.

Options for other types of targeted delivery include microencapsulation and nanoparticles. “Controlled-release depot development for biomacromolecules, particularly proteins and other process-sensitive drugs, has been problematic,” explains Steven Schwendeman at the University of Michigan, “owing to protein instability, elevated manufacturing costs, difficulties associated with organic solvent use, insufficient control of release kinetics, and other factors.” Despite advances in stabilizing biomacromolecules encapsulated in polylactic-coglycolic acid (PLGA), Schwendeman says that microencapsulation technologies need to minimize protein damage, organic-solvent use, and costly aseptic manufacturing. He will describe two aqueous approaches using PLGAs under mild conditions without organic solvents that have succeeded both in vitro and in vivo.

Christopher Cheng of Yale University will focus on nanoparticles and ligand-conjugates for tumor-targeted delivery of RNA-based gene therapies. “Often characterized by aberrant microRNA expression, cancers can become dependent on oncogenic microRNAs (oncomiRs),” he explains. “Inspired by personalized therapies that exploit addictive oncogenes such as HER2 and ABL, addictive oncomiRs are promising lynchpin targets for cancer therapy.” Cheng will describe advances in engineering peptide- and polymer-based therapeutic systems to elicit oncomiR inhibition in solid tumors. Research results detail their therapeutic efficacy in microRNA-targeted therapy.

Gene therapies aren’t the only new products that present unique challenges in formulation and delivery. David Kaplan of Tufts University will discuss delivery matrices for cell therapy and tissue engineering applications. “Fibrous proteins are intriguing polymers for biomaterials-related needs,” he explains, “due to their functional properties and their similarities to synthetic polymers.” Such features can facilitate design of protein–polymer systems for delivering cell therapies, tissue engineered products, and other regenerative medicines. Kaplan’s team uses silks to bioengineer new protein polymers. Their goals are to better understand the structure–function relationships involved and fine-tune features for specific applications.

About the Author

Author Details
Cheryl Scott is senior technical editor of BioProcess International. Quotes not otherwise attributed are from presentation abstracts.