September 15, 2013 (Vol. 33, No. 16)

Formulation of biopharmaceuticals poses numerous challenges related to the chemical and physical properties of drugs, and the fact that nearly all such products are injected.

Formulation development is a “big piece of the drug development puzzle,” says M. Byron Kneller, Ph.D., associate director for analytical and formulation development at contract manufacturer and development firm CMC Biologics. The company specializes in early- and production-stage formulation development, not only for its manufacturing clients but also through standalone contract work.

Most of CMC’s customers are early-stage developers of biotherapeutics preparing for their first clinical cGMP batches. With such “young” projects, the key hurdles are logistical.

“Our main challenges are acquiring enough material to conduct all the formulation and stability studies,” notes Dr. Kneller. Material demand is particularly high for studies on highly concentrated protein drugs.

In many instances, when customers ask CMC to redesign the process from the cell line up, there is no material at all to work with. Formulation development may even stall until CMC’s manufacturing team can prepare sufficient material and work out upstream and downstream steps.

A related issue involves the stage at which defining formulation studies occur. “We try to do as much as we can early in development, but we attempt to save our pivotal studies for the end, when we have lots of material and the process is more or less locked. The final material we use should be representative of the drug substance produced during Phase III and beyond,” explains Dr. Kneller.

Even when material is scarce, or not representative of the final process output, it is possible to perform stress-degrading studies and to obtain some idea of factors that promote stability, and those that don’t. “For some molecules it might be oxidation, for others pH or heat,” continues Dr. Kneller. Such “preformulation” studies also help identify suitable analytical methods, and even guide downstream processing.

CMC has employed high-throughput and design-of-experiment approaches to obtain the most data, in timely fashion, from the smallest quantity of material. This strategy applies to liquid formulation as well as lyophilization.

Recent articles in GEN have highlighted the power of design-of-experiment and high-throughput approaches to formulation development. CMC is capable of conducting formulation development in that manner.

But because of development time constraints, the final formulation may not have been thoroughly and systematically optimized for that particular protein. “Sometimes customers only have time for a formulation that is good enough, that is suitable for clinical testing” admits Dr. Kneller. “They want a formulation that will serve them in Phases I and II, but in many cases it’s perfectly fine beyond that as well.”

According to CMC Biologics, it employs high-throughput and DOE approaches to obtain the most data, in a timely fashion, from the smallest quantity of material.

Biosimilars: Not Quite an Open Book

Formulation of biosimilars is somewhat facilitated by the requirement that manufacturers disclose formulas for all injectable drugs. According to Sarfaraz K. Niazi, Ph.D., chairman and CEO of Therapeutic Proteins International, “We know exactly what’s in every product. What we don’t know are the in-process limits and controls, grades of materials used, and mixing and formulating processes. For protein drugs those factors are much more important than for small molecules.”

Since seemingly innocuous formulation changes can pose grave consequences, it is imperative for biosimilar producers to get things right. Dr. Niazi relates the example of erythropoietin (EPO) from the late 1990s to early 2000s. The switch from a human serum albumin stabilizer to polysorbate 80 and glycine was believed to cause the “epidemic” of pure red cell aplasia among patients taking EPO. According to some reports, the new formulation caused extraction of chemical agents from uncoated rubber stoppers on vials.

The incident is perhaps over-cited, says Dr. Niazi, and was the only known example where such drastic formulation-related effects were observed, “but it nevertheless points to the significance of formulation.”

The EPO case also alerted formulators to the interplay between drug, formulation, and the nearly ubiquitous delivery or storage device that accompanies parenteral biologics. “The device is often the most neglected factor, even though it is an integral part of the formulation,” says Dr. Niazi.

Many biotech drugs come in prefilled syringes and auto-injectors. Even the vials in which lyophilized drug is reconstituted may be considered a device. Materials and ancillary components of delivery and storage devices can impart properties to formulations that render them potentially immunogenic, or interfere with quality assays.

For example, the barrels of prefilled syringes are often lubricated with silicone oil. Unless formulators take special care, the lubricant could spill into the drug product, where particle-sizing instruments may mistake it for protein aggregates.

Automation to the Rescue

Contract manufacturing/development firm KBI Biopharma has recently put into place a high-throughput robotic liquid handling system from Freeslate. The Freeslate Core Module 3 resembles common liquid handling workstations, but incorporates functions and software suitable for formulation development in 96-well plate format.

Prathima Acharya, Ph.D, chief scientist at KBI, notes that the Core instrument has built-in capability for analyzing viscosity, osmolology, and concentration, as well as standard solution parameters. Because it relies on robotic plate handling and dispensing, the system facilitates highly parallel, design-of-experiment formulation development.

“We adopted the Freeslate technology because of the frequency with which initial formulation development faces material shortages,” says Dr. Acharya. “The Freeslate product enables us to do a lot more with very little material and provides answers quickly. We can set up an experiment before we leave for the day and have results by the next morning.”

Despite high-throughput capabilities, KBI does not frequently perform highly systematic formulation screening or attempt to cover every parameter, buffer ingredient, or stabilizer. Based on a molecule’s structure and type, formulators can achieve an a priori elimination of many variables, or at least narrow their expected ranges. “Then, when you add high-throughput methods, timelines accelerate even more. It’s all about timelines,” explains Dr. Acharya.

Bridging the gap between early-process and late-process material has always been challenging for formulators. With the “product is the process” being the prevailing view, one may ask where in the development timeline one would find the optimal point to initiate formulation development, or where one should become concerned over impurities, misfolded protein, or aggregates.

Early versus late impurity profiles can alter the endpoints of formulation screening. But Dr. Achayra believes that early-late difference matter less if one relies on experience and high-throughput methods. Dr. Achayra’s group runs early material in select high-throughput screens, then runs confirmatory experiments after the process is set. “That’s how to bridge the gap between development and manufacturing material,” says Dr. Achayra.

KBI Biopharma is using Freeslate’s Biologics Formulation System to support biologics formulation screening and accelerated stress studies. The system capabilities include formulation/sample preparation and measurement of formulation pH, viscosity, particle counting, turbidity, color, and protein concentration and aggregation.

Stability = Quality

To Ahmed Yasin, Ph.D., head of formulation and stability research at GlaxoSmithKline’s Stevenage, U.K. facility, the greatest formulation-related challenges are promoting stability, minimizing aggregation on storage, and dealing with viscosity in high-dose formulations. These issues are related.

Because some monoclonal antibodies are administered at high doses by injection, viscosity problems are more or less built into the product. Highly viscous solutions are more concentrated, so molecules have a greater opportunity to interact and therefore aggregate. That being said, Dr. Yasin noted that highly viscous monoclonal antibody therapeutics are common and, without elaborating, stated that strategies exist for dealing with them.

Aggregation sometimes affects a molecule’s activity by shielding the active region from its binding site. This effect is not always noticeable. Regulators and bioprocessors are more concerned about aggregates being immunogenic.

Selecting from among the many techniques that quantify aggregation is difficult. Many methods lack robustness and reproducibility. Those based on light scattering do not always distinguish aggregates from non-protein interferences. The presence of even small amounts of silicone oil can sometimes thwart accurate characterization of aggregates.

Aggregates are typically monitored using size-exclusion chromatography, but the gold standard analytical method is analytical ultracentrifugation (AUC). Even with AUC, results depend on who runs the method and on what instrument.

Another concern with monoclonal antibodies is deamidation. By transforming an amide to a carboxylic acid, deamidation alters a protein’s physical and biological properties. “It is essentially a chemical reaction that takes place in a highly ionic environment,” says Dr. Yasin.

The process involves isomerization followed by elimination of –NH2. Deamidation is one of several degradation mechanisms that are innate to proteins. Particularly affected are proteins containing exposed asparagine and glutamine sidechains with nearby glycine residues.

Development teams will therefore seek formulations that minimize or eliminate deamidation. “If deamidation occurs in the complementary-determining region (CDR), which contains the binding points of the monoclonal, there’s a huge chance it will affect stability,” notes Dr. Yasin.

However, if it occurs in the heavy or light chains, or far from the CDR, the effect will be lessened.

Deamidation is also a possible avenue toward immunogenicity. While this idea is entrenched in theory, finding real-life examples, even experimental evidence, is difficult.

Small Molecule Drug Formulations

One of the most common challenges in small molecule drug development is formulating low-solubility drugs as manufacturable and bioavailable products. At least 40% of new chemical entities (NCEs) in the global pipeline have poor solubility and low bioavailability.

For these compounds, low aqueous solubilities in the region of 0.1 µg/mL are common. The implication is that a 10 mg dose requires 100 L of aqueous media to dissolve, making administration practically impossible.

Significant bioavailability issues include variable absorption due to precipitation in the gut, food effects, and atypical gastrointestinal physiology in some patient populations.

“Typically, for a drug molecule to be absorbed into the systemic circulation it must be adequately soluble in the aqueous milieu of the gut,” observes Peter Pekos, CEO of Dalton Pharma Services. “The drug must also be sufficiently permeable to cross the intestinal barrier by passive diffusion, the primary process involved in drug absorption.”

Drug solubility and permeability work in opposite directions, giving rise to a seesaw effect. To deal with this effect and ensure that therapeutic goals are met requires skill and art.

“It follows that potential drug compounds that are highly soluble tend to be poorly permeable, and vice versa,” Pekos adds. Formulation scientists must balance the impact of these two competing properties to achieve the desired solubility and bioavailability goals.

Given that most molecules Dalton Pharma sees are poorly soluble, the company draws on myriad strategies to provide these compounds with the best opportunity to succeed in early-stage clinical trials. These include liposomes, nanoparticles, photo activation, and amorphous dispersions within a dissolving polymer matrix.

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