Although no biopharmaceutical pills are yet on the horizon, formulation and delivery have advanced over the past 10 years. Formulators have new biophysical technologies and new product types (such as protein–drug conjugates) to work with. The most important issues haven’t changed much, though — from aggregation to stability, freezing to freeze-drying — although the FDA’s quality by design (QbD) initiative changes the strategies used to address them.

Fragile proteins and other biologically sourced macromolecules need protection to achieve product stability in final formulations. The larger a molecule is, the more difficult it will be to make, ship/store, and administer to patients. Biotech drug formulators have many concerns to juggle in their work, beginning with the physicochemical characteristics of an active molecule and including the reliability, cost, and availability of analytical methods used in formulation work; the array of excipients and adjuvants on the market (and their chemistries); evolving delivery methods and devices; patient preferences and behavior, as well as the biology of diseases being treated; and even the concerns of legal, sales, and marketing groups.

Formulation is more than science, as a result, but science is its foundation. For years, it has been considered by many as an arena where luck and intuition play a role as well (1). But formulation work has become more methodical and quantifiable over the past decade as QbD and new analytical technologies (2, 3) have crept into formulation laboratories.

This stock photo of formulation work appeared on the cover of our very first issue. ()

The vast majority of biotherapeutics 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 (4). But such solutions introduce 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 options. This is the most challenging issue for them to face in the 21st century so far. Formulation Strategy

Outsourcing is a big part of the product formulation strategy for many biopharmaceutical companies (5, 6). No matter who’s doing the work, though, the smartest approach is for preformulation (characterization) work to begin as early as possible in product development — and here, formulators can work hand-in-hand with other laboratory personnel who are characterizing the active molecule for quality and risk assessment purposes. As a team of authors from Abgenix, Inc., wrote for an early BPI issue, “Using an integrated design based on scientific literature, rationale, and experience combined with a proven statistical design can accelerate the formulation development process and increase productivity” (7). Four years later, a Genzyme team described preformulation explorations of how variables such as pH, ionic strength, and excipients affect the solution behavior of a protein (8). Those findings can guide downstream formulation development while providing valuable information concerning protein stability, solubility, and structure. Aggregation and Stability

One important aspect of that early characterization is determining what environmental circumstances cause proteins to aggregate and fall out of solution. Aggregation problems have been implicated in adverse reactions and other safety issues since the beginning of clinical applications of protein pharmaceuticals. Antibody aggregates have long been known to cause anaphylactic reactions, so formulations must be optimized to reduce aggregation during storage, handling, and shipping (9). The past decade has seen a shift from the traditional size-exclusion chromatographic method of detecting and quantifying protein aggregation toward column-free techniques such as dynamic light scattering analytical ultracentrifugation, and field-flow fractionation (9). Photon-correlation spectroscopy — as performed by the Zetasizer Nano ZS analyzer from Malvern Instruments — can detect subvisible particles “in a broad dynamic range from a few nanometers to a few micrometers” (10); and microflow imaging — as commercialized by Brightwell Technologies — is helping scientists evaluate populations of microparticles in liquid formulations (11).

As part of BPI’s close relationship with the California Separation Science Society’s (CASSS) CMC Strategy Forum series, we published a two-part update on the analysis and immunogenic potential of aggregates and particles late in 2011 (12, 13). According to the authors, “Discussions between regulators and industry have led to development of novel techniques to detect and characterize aggregates and increased research into the role of protein aggregates of all sizes in immunogenicity. In addition, the pharmacopoeias have been revising monographs to improve subvisible particle testing of biotherapeutics and clarify terms such as practically or essentially free of particles.”

Once you know that aggregation may be a problem, you can take steps toward preventing it — such as incorporating specialized excipients intended to aid in formulation stability (14). Some companies choose freeze-or spray-drying too (15). But even as lot-release testing has diminished somewhat in importance due to risk management and QbD, stability testing is still a vital aspect of formulation development. Although analytical methods such as intrinsic tryptophan fluorescence measurement can help formulators make predictions (16), long-term studies are still the only way to determine a product’s true expiry date (17).

Talking About Opalescence with Mary Cromwell (Genentech)

Mary Cromwell, senior scientist at Genentech, spoke on opalescence in formulations at the BPI European Conference and Exhibition in April, 2008. Before the meeting, she spoke with then associate editor Leah Rosin. Cromwell explained that this phenomenon is common in formulation science. The concern about opalescence involves its correlation with aggregation and thus related concerns about product immunogenicity.

However, Cromwell explained that the connection between immunogenicity and opalescence isn’t straightforward. Her case study showed how opalescence is actually one type of a more commonly observed phenomenon that occurs when a solution reaches a critical point (e.g., pressure, temperature, or pH) and starts exhibiting different behavior. Opalescence is related to temperature.

“In this system, when the critical temperature comes into play, you start getting liquid–liquid phase separation,” said Cromwell. She compared the phenomenon to what you would see in chemistry class when cooling a mixture of cyclohexane and isopropynol. “So opalescence doesn’t necessarily cause a problem. On the other hand, it may indicate that you have a solution that when taken to the right temperature may show phase separation, and thus you may need to control your temperature a little bit differently.”

The product in her case study was a monoclonal antibody, but the problem of opalescence is a prevalent issue throughout the formulation discipline. “It’s not limited to MAbs,” said Cromwell. “All proteins can do this potentially. As long as we’ve been producing protein formulations, there have been observations of opalescence.”

References

1 Rosin L. Tackling Formulation and Delivery. BioProcess Int. 6(4) 2008: 72.

2 Woods JM. Nesta D. Formulation Effects on Opalescence of a High-Concentration MAb. BioProcess Int. 8(9) 2010: 48–59.

Q&A with an Expert: Kent Simmons

Kent Simmons has been producing IBC’s “Formulation Strategies for Protein Therapeutics” conference for the past 10 years. I asked him to comment on the changes he’d seen in its programming in that time, and here’s what he had to say:

“In 2002, we did a whole workshop on the use of supercritical fluids in biologics formulation development, but I haven’t heard about this technology in years.

“The early conference was very much dedicated to very fundamental issues of stability, aggregation, and dosage-form development. Recent conferences have addressed the newer circumstance of formulating high-concentration and high-volume drugs (in response to the need for much higher doses in a patient-convenient format), issues with subvisible particles (covered for the first time at the 2009 conference), and recent issues with prefilled syringes and packaging interactions.

“Quality by design has now become real for formulators. Rather than being an abstract concept that companies might implement someday, there is now widespread work on understanding design space and critical quality attributes (CQAs) for formulations.

“Analytical technologies have evolved too — with more use of biophysical characterization methods and high-throughput approaches to formulation screening.”

This must-atte
nd conference for protein formulators is colocated with the BPI Conference and Exhibition every year — this year at the Rhode Island Convention Center in Providence, RI, 9–10 October 2012. For more information go online to www.ibclifesciences.com/formulation.

Freezing and Thawing: Many commercial biotherapeutics are stored frozen either in bulk form or after product formulation (18). Biologics can be frozen at large scale in improvised, catalog-purchased, or purpose-designed systems (19). The choice is often dictated by the volume to be frozen, available technology, shipment needs, stability of the frozen matrix, and so on. Companies such as Sartorius Stedim Biotech (Celsius S-cube system and cryovessels with Cryofin technology) and Zeta Holdings (FreezeContainer system) have offered advanced options over the past decade that take into account the needs of freezing stability studies as well (18,19,20,21). Studying this process cannot be meaningfully accelerated — like liquid-state stability can — so freezing practices are often empirical. And companies such as BioLife Solutions Inc. offer specialized media for cryopreserving cells (22, 23).

Q&A with an Author

Edward T. Maggio, PhD, is president and chief executive officer of Aegis Therapeutics LLC, 16870 West Bernardo Drive, Suite 390, San Diego, CA 92127; [email protected], www.aegisthera.com. He wrote on excipients for BPI’s November 2008 issue (14).

BPI: What new excipients have been introduced to the biopharmaceutical arena over the past 10 years?

ETM: Intravail alkylsaccharides for enhancing transmucosal absorption enhancement of peptides, proteins, and BCS class III–IV small molecules; ProTek excipients for preventing aggregation and reducing or eliminating aggregation induced immunogenicity for monoclonal antibodies (MAbs)

BPI: What new challenges do formulation scientists face?

ETM: One of the biggest challenges is finding a replacement for the polysorbates used in essentially all monoclonal antibody therapeutics and many other biotherapeutics. Polysorbates — and presumably the other polyoxyethylene-containing surfactants (e.g., Brij-35 and poloxamers) — autooxidize to become protein-damaging peroxides. Protein damage gives rise to increased immunogenicity, which can be a serious problem. A recent FDA guidance and EMEA opinions have highlighted the importance of assessing immunogenicity of biotherapeutics, including a need for comparison of immunogenicity of biosimilars to the innovator product.

BPI: What analytical methods are most vital to a formulation laboratory?

ETM: High-pressure liquid chromatography (HPLC), affinity chromatography, a number of light-scattering techniques (e.g., forward light scattering, dynamic light scattering), gas chromatography with mass spectrometry (GC–MS) for analysis of oxidation products caused by peroxide damage, and in vivo assessment of immunogenicity

BPI: Do protein conjugates present a new frontier in formulation science?

ETM: Yes. It’s unclear how these will be treated from a regulatory perspective. Will they be treated as new chemical entities (NCEs)? PEGylation may reduce aggregation, which could be a positive step. But it needs to be shown that modification does not increase immunogenicity. Combos and Conjugates

Combination products — whether combining small molecules and biologics in one formulation or requiring a specific delivery device — and protein conjugates are presenting new challenges to formulation science (24, 25). They introduce new characteristics and requirements, new degradation pathways, and new excipients. The addition of polyethylene glycol to formulations is nothing new — but conjugating it to the therapeutic protein itself is (26,27,28). The process improves that active molecule’s life in vivo, but it may create instabilities in formulation and problems with immunogenicity that must be dealt with. Solving one problem can sometimes create new ones. But that’s what risk management is all about. Dried and Particulate Formulations

Lyophilization is a three-stage process: freezing (solidification), primary drying (ice sublimation), and secondary drying (moisture desorption). A team of authors at Praxair, Inc., wrote in 2009:

Pharmaceutical and biopharmaceutical manufacturers often must dry their products to meet shelf life targets by inhibiting chemical, microbiological, and physical degradation pathways that occur in the presence of moisture. A wide variety of drying methods have been used including spray drying, freeze-drying (lyophilization), vacuum drying, and fluidized-bed drying. Lyophilization has become the preferred method for sensitive, high-value pharmaceuticals because it enables moisture removal at relatively low temperatures under easily maintained sterile conditions. Furthermore, the technique often imparts the valuable product attribute of rapid reconstitution at the point of use. Lyophilization has a long history of regulatory acceptance and will continue to occupy a prominent role in pharmaceutical fill–finish operations for decades to come.” (29)

Dried products need reconstitution: mixing the drug product with a fluid to create an injectable solution (30). Until fairly recently, that process was up to clinicians and/or clinical pharmacists — or even patients themselves, when managing chronic conditions — and thus involve risk of accidental needle sticks, inadvertent exposure from spray-back, inaccurate dosing, and patient noncompliance. In the 21st century, companies such as West Pharmaceutical Services have developed safe, convenient, and easy-to-use systems for reconstituting and administering injectable formulations. Meanwhile, the process of lyophilization itself has improved as companies implement liquid nitrogen–based cooling systems. They provide better control over a freezing process and broaden the range of available operating parameters (31).

Particulate Formulations: In vivo stability is another concern for biological drugs. Sustained release has been a goal for many products with limited in vivo halflives. One solution that was put forth in the 1990s and further expanded upon in the first years of the 21st century is polymeric drug delivery. Lipids and polymers are used to create matrix formulations for sustained delivery — but the better approach to biologics so far appears to be in conjugation rather than formulation. For gene delivery, viruses have had millions of years to perfect their skills, so they so far show more promise than other types of encapsulation. Supercritical fluids and microfluidics have been offered as a means of improving particle size distribution for inhaled formulations (32,33,34), but so far those have succeeded only with small proteins (e.g., insulin).

Looking to the future, polymer hydrogels have been proposed as a means of controlled delivery (35). So-called “smart” polymers could release their payload drugs in response to certain chemical or environmental signals. “Associated difficulties cannot be ignored,” however: “possible toxicity or nonbiocompatibility of the material(s) used, potentially undesirable byproducts of
degradation, and the higher cost of controlled-release systems compared with traditional pharmaceutical formulations” (35).

Polymeric delivery systems for macromolecules are not likely to gain broad acceptance, wrote Larry Brown of Epic Therapeutics in our five-year anniversary issue, as experts question the value of particulate formulations (36). Brown suggested that the presumed advantages of sustained-release dosing “need to be critically compared with chronic but virtually painless daily injections using 30-gauge needles.”

About the Author

Author Details
Cheryl Scott is cofounder and has been senior technical editor of BioProcess International since the first issue; 1-646-957-8879; [email protected].

REFERENCES

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