Preformulation Means Characterization

A biopharmaceutical drug can go into development before anyone knows much about how it works. The protein may be identified through genomics or proteomics activities or through more traditional medical research. It may initially be associated with a particular disease process or a certain metabolic event. In any case, its mechanism of action — as well as many of its structural characteristics and biochemical properties — may be unknown. One of the more challenging aspects of developing protein pharmaceuticals is dealing with and overcoming the inherent physical and chemical instabilities of proteins. This inherent instability has the potential to alter the state of the protein from the desired (native) form to an undesirable form (upon storage), compromising patient safety and drug efficacy. The set of activities related to overcoming the inherent instability of the drug is referred to as formulation development.

A successful formulation process has four stages: preformulation, stabilization of the active substance in bulk form, formulation in the designated dosage forms (drug delivery), and fill and finish of aseptic manufacturing activities.

Preformulation is an exploratory activity that begins early in biopharmaceutical development. Preformulation studies are designed to determine the compatibility of initial excipients with the active substance for a biopharmaceutical, physicochemical, and analytical investigation in support of promising experimental formulations. Data from preformulation studies provide the necessary groundwork for formulation attempts. Successful formulations take into account a drug's interactions with the physicochemical properties of other ingredients (and their interactions with each other) to produce a safe, stable, beneficial, and marketable product.

Corporate concerns. Some companies have a preformulation/formulation team specifically devoted to these tasks. In other (usually smaller) companies, formulation development may be the responsibility of the fermentation, process chemistry, or quality control group. Industry experts claim that preformulation teams are often understaffed, underresourced, and not well understood by company management. Formulation is sometimes considered a sort of "black art" in drug development. This is because in the history of protein formulation, development began as an empirical science due to the unique nature of each protein. However, a better understanding of protein behavior and an increased awareness of the adverse effects of an improperly formulated protein product on clinical trials and on the marketed product have highlighted the need for a more rational approach to formulation development. Such an approach has been developing over the past decade. A dedicated formulation development group that is adequately staffed and resourced has thus become necessary in order to successfully incorporate protein formulation development in the overall drug development strategy. Bio-pharmaceutical companies should consider their formulation teams to be just as essential to their survival as process development and finance. Doing things right the first time is always preferable to going back to the drawing board and starting over — and it's also the faster way to market.

Formulation and delivery issues must be considered early in development. An initial formulation is needed for preclinical studies, and a better formulation will be needed for clinical trials. The formulation may be critical to the safety and efficacy of a therapeutic protein, and, as development progresses, it will become harder and harder to make changes.

Marketing concerns come up earlier than you might think. Route of administration is determined by the target product profile: Will our product treat a chronic or acute disorder? Will it need specific targeting — a broad or narrow therapeutic window? Will it be administered at home or in the clinic or hospital? What is the competitive landscape; are other drugs already treating the same indication? What will give our project the advantage over existing treatments and those that may emerge?

For example, marketing considerations arise early in product development for monoclonal antibodies (MAbs). Typically, MAbs are needed at high doses (hundreds of milligrams per dose) and are normally delivered intravenously. The drive to reduce healthcare costs has created a need to administer MAb therapeutics more conveniently, at home, subcutaneously. Thus, MAbs must be available at high concentrations (~200 mg/mL) in the vial. At these high concentrations, MAb-containing solutions are viscous, making them difficult to administer conveniently. Hence, a preformulation activity that needs to be considered is a concentration study investigating solubility behavior, effect of concentration on viscosity, and increased potential for aggregation. These studies have the potential to strongly influence the target product profile as well as the design of the clinical trial.

All of these questions can affect the optimal formulation of a drug. For example, an early formulation question is whether the product will be lyophilized (freeze-dried) or sold as a liquid. The advantages of a liquid include time savings, lower cost, and ease-of-use for patients and clinicians, all of which are good sales points. But stability questions often make freeze-drying necessary for protein and peptide pharmaceuticals. Freeze-dried drugs have a longer shelf life and better stability for shipping and storage, even if they cost more and take longer to make.

For these reasons, the formulation team must communicate with other teams within the company right from the start: protein biochemists, purification scientists, quality control and regulatory affairs personnel, clinical investigators, manufacturing technicians, marketing specialists, and managers. There is a tendency on the part of upper management to rush a formulation along into initial clinical studies with little or no stability data. This can be a costly move; FDA will stop any clinical study where the formulation has lost or is losing its potency.

Development Steps

The seemingly endless variation of polypeptides makes them interesting as potential therapeutics, but it also makes them a challenge to develop into products. Each protein is unique, and just as variation from protein to protein affects biologic production and purification, so it is central to the formulation development process. Methods developed for one biopharmaceutical are not always directly applicable to others. Similarly, it is quite likely that a formulation developed for one biopharmaceutical may not provide the same level of stability for a different biopharmaceutical.

While there are numerous ways for a protein to lose its stability, the three most commonly encountered modes of denaturation and degradation are aggregation, oxidation, and deamidation. The commonly accepted strategy for rational formulation development relies on identifying mechanisms of denaturation and degradation in order to develop effective countermeasures. Once the specifics of any particular degradation pathway are understood, a more informed choice regarding excipients and formulation can be made, accelerating product development.

Using the scanning electron microscope

Characterization. Information gathering begins as soon as a molecule is chosen for development. Internal data sources include the research, discovery, and analytical groups, along with the company's history with any similar molecules in development or on the market. Formulators need to know the composition (amino acid sequence and molecular weight) and other characteristics of the protein with which they'll be working, so they want to start with as much information as possible. External data sources include published literature and other products already on the market. Talking to other formulators at industry meetings and conferences can be a great help. One of the characteristics the formulator is trying to generate is the stability profile.

No matter where the process ends, preformulation begins with a study of the drug's pharmacokinetics and physicochemistry. If the active molecule is not stable on its own, formulators can usually make it so by adding buffers or other excipients. It's wise to choose a buffer that is familiar to regulatory authorities, easy to get from vendors and suppliers, and compatible with relevant characteristics of the active molecule (such as pH range). Formulators may also choose other excipients to protect the protein and enhance its stability — and these excipients also must be compatible with the necessary conditions, the buffer, and each other.

Product concerns. As early as possible, formulators want to know the drug's indication, which is based on the drug's pharmacokinetic profile. Early research should indicate where the drug travels in the body, what it will treat, and its site and method of delivery. Formulators also need to know early on whether the product will be sold as a single dose or in multidose vials. Single-dose formulations are easier to protect against contamination. Multidose vials, which are entered several times over several weeks, can become contaminated, so preservatives must be chosen and tested for stability and compatibility with the protein.

International disagreement over preservatives in food and drugs may present a problem at this stage. US, EU, and Japanese compendial standards differ regarding the timing of antimicrobial tests and which preservatives and excipients are allowed. Japan, for example, does not accept phenol, a preservative used commonly in the US. So at this early stage, companies must decide if and where their products will be distributed outside the US. The EU is known to have the toughest acceptance criteria for preservatives. The International Conference on Harmonization is working toward a common standard, but many formulators have criticized its slow progress.

As discussed, an early preformulation decision is whether the final formulation will be liquid or lyophilized, a choice that determines which excipients will be needed. (Liquid and lyophilized products require different excipients.) Some industry experts advocate developing both types of formulation in parallel. In concert with all these steps, a placebo formulation containing all but the active ingredient also must be developed for use in blinded clinical trials.

The package. Four important preformulation stress tests are the shake test (agitation), surfactant test, freeze-thaw test, and heating experiments. Each formulation configuration is shaken in a vial to determine whether it forms aggregates. Then a surfactant (usually a polysorbate detergent such as Tween) may be selected to prevent formation of precipitants by making it harder for proteins to aggregate. Formulations are checked through multiple freeze-thaw cycles (which can take about a week) to check for the effects of temperature and freezing-process stresses. Most proteins are stable around 2-8°C, but few are stable at room temperature. Heating experiments help scientists examine degradation at temperature extremes by heating them to 30°C (about 86°F), and maybe even 45°C (about 113°F). At high temperatures, different mechanisms of protein denaturation may arise. (For more information on what can go wrong, see the next chapter.)

Formulators cannot ignore any potential interactions, which means paying attention to container materials. Is the formulation compatible with the vessel and closure? Some proteins may stick (adsorb) to the walls of a vial or other container, particularly in low-concentration formulations. Glass is reactive; it can be delaminated, for example, through contact with a formulation. Certain kinds of plastic may be less reactive than glass.

Will the formulation react with the stopper or elastomer? Such contact could destroy the protein. Stopper materials are selected based on their reactivity with the formulation. To test for that, vials are inverted — to give their rubber stoppers total contact with the formulation — and then stored horizontally to create more surface area — and more chances for the protein to degrade. More oxidation of the protein can occur as the surface area grows larger.

The filling process (discussed in the final chapter of this guide) should remove as much oxygen as possible from a vial's headspace because oxygen can cause a certain kind of degradation. Potential product extractables and packaging leachates must be listed in regulatory submissions. Preformulation development may take three to six months or more, depending on the instability and reactivity of the active bulk substance with its excipients. Interrelated preformulation activities may determine whether a molecule can be developed into a marketable drug.

Know Your Molecule

Development of any protein formulation should be done empirically, considering the final dosage configuration, because of the enormous potential for interaction between the ingredients, the packaging or delivery device, and even the patient's body itself (for example, at the site of injection or delivery). Protein molecules interact with other molecules following their own rules and tendencies. They have had billions of years of evolution to reach this state of complexity, and we have had only a couple of centuries to learn about them.

"Characterization is . . . about knowledge and intimate understanding of your biopharmaceutical and any product- or process-related impurities," notes biopharmaceutical analytical scientist Jim Faulkner. "Ultimately, characterization is about deciding what features of your molecule need to be under control and what features are noncritical to its overall quality. Ideally, that information would be in hand at the beginning of the product development program. Clearly, that is unrealistic. Therefore, product characterization is an evolving process that involves contributions at different stages of the product life cycle. Assuming that characterization is purely a development exercise is wrong; the moment a biotherapeutic is identified for development, the process of characterization has begun."

The new biologics regulatory paradigm in place since the late 1990s refers to biopharmaceuticals as "well-characterized biologics." But to take advantage of the lessened restrictions for certain products overseen by FDA's Center for Biologics Evaluation and Research (CBER), companies must prove that their products are indeed well characterized.

Meanwhile . . . Documenting the Work

As a product moves through production, purification, formulation, and fill and finish, the quality control (QC) department tracks every process component from the point of purchase or in-house development. QC ensures the safety of development scientists and patients. As soon as a company moves its formulation from research and development (R&D) into development and manufacturing, QC starts designing systems for documenting the company's compliance with good manufacturing practices (GMPs) for FDA.

Documentation comprises a complex and interconnected network of records and forms — merely introducing the subject in BioPharm International required an 11-part column series (see References). GMP articles and columns continue to explain and define critical documentation elements.

Part numbers are assigned as identification codes for tracking purchases, material flow, and inventory. They are especially important for "critical" items, those that form part of a product or come into contact with it (vials, stoppers, and seals, for example). Specifications describe in detail every numbered item (purchasing information, chemical formulas, handling precautions, storage conditions, expiration dates, testing requirements, and so on). Incoming materials are monitored through receiving codes and assigned lot numbers — as are solutions prepared in-house, cell-lines, processing events, and production batches. Standard operating procedures (SOPs) provide step-by-step instruction for technicians in QC, production, maintenance, and material handling. A Master Production Batch Record (MPBR) details the production process step by step, ensuring that all vials are exposed to the same processing conditions as the product moves through formulating, sterilizing, filling, sealing, inspecting, and labeling. The MPBR begins with cell-culture inoculation and proceeds through harvest and even initial purification steps.

Meanwhile, equipment installation qualification evaluates the assembly and installation of critical processing equipment — including its own set of SOPs and materials specifications. Log books and work orders track equipment monitoring, repairing, cleaning, and preventive maintenance.

And what of the big picture? As documention is developed throughout a facility, protocols are written as "umbrella documents" to tie individual SOPs together and to direct the work. Protocols even describe where in the company to find specific files and documents. They tell who directs which activities, who approves what, and who is allowed to sign off on materials and products. Performance qualification sections, for example, describe preliminary operations and validation acceptance criteria. Master method validation protocols help scientists develop, monitor, evaluate, and adapt analytical protocols. And process validation protocols help them determine whether and how to validate various critical aseptic manufacturing processes.

Documentation (which includes much more than is mentioned here) supports the work in development laboratories and throughout scale-up, production, manufacturing, and even postmarket surveillance. It helps ensure product consistency from batch to batch — and helps instill physician and patient confidence in the safety of the final formulation.