January 15, 2006 (Vol. 26, No. 2)

Desire to Improve Therapeutic Proteins at Molecular Level Drives Bioformulation Advances

While pharmaceutical formulations have always been part science, part art, the complexity of todays drugs has shifted the balance toward the former. Aided by analytic tools that detect subtle changes in proteins over time, biomanufacturers employ rigorous science to create formulations that stabilize and enhance sensitive products. Driving formulation technology are a greater appreciation for consistency, novel delivery mechanisms and dosage forms, and the desire to improve therapeutic proteins at the molecular level.

Proteins undergo numerous physical and chemical changes that affect potency and safety. Among these are aggregation, which includes dimerization, trimerization, and higher-order aggregates, plus crystallization and precipitation. During fill-and-finish operations, concentrated protein solutions squeeze through piston pumps, which imparts high-shear and mechanical stresses that cause denaturation and aggregation.

Once formulated, manufacturers rely on additives, such as amino acids (particularly glycine), salts, and surfactants, to enhance protein solubility and inhibit aggregation.

Preventing aggregation has become a major issue for formulators since the trend toward high-concentration solutions increases the likelihood of protein-protein interactions favoring aggregation. Many products are only effective when delivered by injection in relatively high concentration, notes Wayne Gombotz, Ph.D., vp of pharmaceutical operations at Omeros (www.omeros.com).

In some cases manufacturers can exploit proteins inherent tendency to aggregate by delivering them as suspensions, or depots, which are slowly dissolved by interstitial fluid between cells and delivered systemically. No products are currently approved with that type of formulation, notes Dr. Gombotz, but people are looking at this approach.

For example Altus Biologics (www. altus.com), which has long been interested in crystalline protein drugs, has published on a technique for delivering both crystalline vaccines and Mabs.

Every protein exhibits its own peculiar degradation behavior. Degradation occurs through multiple mechanisms, including simple oxidation, deamidation of asparagine residues, disulfide bond rearrangements, chemical hydrolysis of peptide bonds, and enzymatic proteolysis.

Simply formulating at the right pH can sidestep many of these problems. For example, deamidation occurs above pH 7, and proteases have optimal pH ranges that can be avoided in straightforward manner by choice of an appropriate buffer. Adding EDTA or citric acid to the final formulation can prevent many types of chemical oxidation.

Usage and delivery trends are the two emerging factors affecting bioformulation strategies. Insulin is the classic example of a self-administered, injectable biotech drug. As more replacement proteins and Mabs gain approval for chronic, nonlife-threatening diseases, manufacturers will devise formulations, unique to user-friendly devices, such as pre-filled syringes, auto-injectors, and needle-free delivery systems.

Formulation and delivery go hand in hand as part of a drugs life-cycle management. Lyophilization, which is an excellent formulation for stability and shelf-life, may not be appropriate for certain products.

Human growth hormone (HGH) was originally supplied as a freeze-dried preparation that required reformulation in the vial, dispensing an aliquot by syringe and injection. Lyophilization may be appropriate for drugs administered by professionals but is probably not, as is the case with HGH, for drugs administered at home.

Eventually, HGH was available in a pen delivery system that required reconstitution but with easier injection, and finally by needle-free injector that has helped boost sales of this product, according to Dr. Gombotz. HGH is a great example of how a new liquid formulation can help drive a new dosage form, which drives sales.

Enbrel, Amgen/Wyeths rheumatoid arthritis (RA) Mab, was also originally manufactured as a freeze-dried product, but patients with RA had difficulty reformulating and injecting the drug. Enbrel is not available as a solution.

Reducing Cold Chain Requirements

Formulation will play a significant role in the approvals of biogenerics or follow-on biologicals. Since generic proteins will almost certainly differ in subtle and not-so-subtle ways from the original, developers will rely on formulation science to iron out problems associated with aggregation, misfolding, and other common mishaps.

Formulation strategies can mitigate and, in some cases, eliminate the need to cold-chain biopharmaceuticals. This is especially true for vaccines that, while not as costly or sensitive as Mabs, are desperately needed in parts of the world that lack refrigeration.

Avant Immunotherapeutics (www.avant immune.com) VitriLife technology preserves vaccines in a glass-like physical state that indefinitely remains stable above room temperature. VitriLife uses a proprietary sugar solution followed by drying and milling to generate the vaccine as a white powder. The product is simply reconstituted with water or saline and simply ingested.

Avants vaccine platform itself may be viewed as a type of formulation. The company uses nonpathogenic Salmonella organisms to carry bacterial or viral antigens into the immunized individual through the digestive tract, a strategy that CEO Una Ryan, Ph.D., calls a Trojan horse. The bacterium itself is immunogenic, inducing a generalized immune response, similar to but more specific than that from an adjuvant.

Novel delivery systems demand equally innovative formulations. Aradigms (www.aradigm.com) AERx pulmonary delivery system targets the lungs huge surface area for pulmonary or systemic medications, while the disposable Intraject is a disposable, needle-free injector. The companys inhaled insulin, co-developed with Novo Nordisk, is in Phase III testing. Aradigm has demonstrated inhaled delivery of other proteins, among them interferon alfa.

According to David Cipolla, Ph.D., director of pharmaceutical sciences, attenuated bioavailability limits pulmonary delivery to peptides up to the 2050 kD molecular weight range. For example inhaled insulin is only 25% as bioavailable as the peptide delivered by subcutaneous injection. If you go to much larger molecules, such as monoclonals, only one to two percent is absorbed by the pulmonary route. At those levels you can deliver peptides to treat lung diseases, but systemic delivery is impractical.

Delivery Driving Formulation

Inhaled drugs are available in a number of different formulations. Dry inhaled formulations for metered dose inhalers are difficult to implement for proteins, since the drug must be dried into micron-sized particles, which promotes aggregation and degradation. Aradigms inhaled formulations are delivered as simple aerosols, consisting of protein, buffer, and surfactant.

Dr. Cippola believes that needle-free injection may be the answer for chronic, self-administration of highly concentrated Mab formulations. Delivering a viscous solution by needle is painful, slow, and may require multiple injections. An injection of the antiasthma Mab Xolair (Genentech/Novartis), for example, can take as long as five minutes, whereas a needle-free system can deliver a dose in 0.1 second.

Best of Both Worlds

Unigenes (www.unigene.com) oral and nasal peptide pharmaceuticals demand formulation expertise for both stability and delivery. To enhance stability, Unigene employs a combination of alcohols to inhibit bacterial growth in Fortical, its nasally delivered calcitonin product.

Peptides resemble proteins in their susceptibility to proteolytic degradation but being much smaller than, say, Mabs, peptides lack higher-order structure, which can be adversely affected by pH and temperature.

Unigene has developed delivery technology that essentially overcomes the proteolysis knock against peptides. Its nasal formulations contain ingredients that facilitate peptide transport across membranes, thereby increasing bioavailability.

For oral peptide delivery, Unigene has used organic acids to inactivate digestive enzymes and a detergent, or bile acid for temporarily opening up the tight junctions within the intestine to facilitate transport into the bloodstream. This is a classic example of how formulation provides a pharmacologic advantage, in this case allowing oral peptide delivery, says CEO Warren Levy, Ph.D.

Unigene has demonstrated effective oral calcitonin delivery in seven clinical trials. GlaxoSmithKline (www.gsk.com) has used Unigenes technology to deliver parathyroid hormones in a Phase I trial. The technique has been shown to work in animals with insulin, luprolide, desmopressin, and other peptides.

Chemical modification of proteins is increasingly viewed as a type of formulation. For example, polyethylene glycol (PEG) imparts greater circulating half-life and, in some cases, improved pharmacokinetics to protein drugs.

DowPharma (www.dowphar ma.com) manufactures and sells high-quality, well-characterized polyethylene glycol (PEG) and related reagents for biopharmaceutical formulation. PEG imparts greater circulating half-life and, in some cases improved pharmacokinetics, to protein drugs.

DowPharma has developed processes that produce a narrow molecular weight range (+/- 10%) for its PEG reagents, assuring that the finished product will be relatively homogeneous. The process also minimizes PEG diol, which can attach to two proteins. The key to PEGylation is starting from a high-quality PEG, says Eric Schlorff, PEGylation commercial leader.

PEGylation is becoming one of the most popular ways to add value to existing protein drugs. Molecules employing PEGylation include Neulasta (Amgen), PEGasys (Roche), PEG Intron (Schering), Macugen (OSI Pharmaceuticals/ Pfizer), and Somavert (Pfizer).

Since PEGylation is expensive, its application is limited to high-value pharmaceuticals such as proteins, peptides, Mabs, and Mab fragments. Not coincidentally, all these product classes are injectible, which presents high-quality and consistency requirements for both protein and PEG reagents. The use of high-quality PEGs facilitates the regulatory approval of PEGylated molecules, says Schlorff.

Formulation by Design

Thus far we have discussed two general rationales for formulationstability and pharmacology. The former will always be part of therapeutic protein development; formulation for pharmacology is increasingly employed as part of biopharmaceutical life-cycle management.

A third strategy involves designing desirable characteristics into molecules from the earliest development stages that could lead to therapeutic proteins that may possess an inherently higher degree of stability, effectiveness, and safety.

Molecular design will not eliminate all formulation considerations. But by enabling the manufacture of proteins that are more potent, less sensitive to heat, or that possess more desirable pharmacokinetics, design can greatly improve the effectiveness of subsequent formulation efforts.

If technologies or formulation enhancements are product enabling, well go down that path, says Christian Allan, Ph.D., associated director of formulation development at MedImmune (www.medimmune. com). However, we try not to put ourselves into a situation where we need to use technologies, such as PEGylation.

Historically, protein drug developers apply narrow criteria for choosing among candidate molecules and promoting them from research to clinic. MedImmune has expanded the wish-list for candidates to include stability and pharmacokinetic properties and is exploring the ability to engineer those properties into the protein at the research stage.

Instead of looking into controlled release, PEG, or other enhancement strategies we attempt to design those properties into the molecule from the beginning, says Dr. Allan. We believe this gives us a better chance of succeeding at the development stage.

For example, MedImmune is working toward improving Mab solubility by modifying the proteins isoelectric point (presumably by swapping out acidic and basic amino acid residues). According to Dr. Allan, his team also seeks to understand thermal stability and its relationship to shelf life and to provide this information to the antibody engineering groups at MedImmune to engineer the property into antibodies.

Medicinal chemists almost always seek to optimize activity and pharmacokinetics simultaneously, when designing new drugs, but this idea is relatively new for biotech. Biotech is relatively new and proteins are more complicated than traditional small molecules drugs, which is why this hasnt been tried much, says Dr. Allan. In 10 to 20 years this approach may be commonplace.

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