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Tutorials : Nov 15, 2009 (Vol. 29, No. 20)

Analytical Considerations in Formulation

Timing, Thoroughness, Simplicity, and Extensive Product Knowledge Are Essential Components
  • Sian Estdale, Ph.D.

Formulation is arguably the most critical—and challenging—component of biopharmaceutical drug development, second only to the molecule itself. Formulation’s far-reaching consequences help sponsors satisfy the scientific, medical, and regulatory conditions for drug approval and, subsequently, support marketplace success.

A biopharmaceutical formulation consists of active pharmaceutical ingredient and other ingredients that help maintain the drug product’s chemical, biochemical, and physical attributes, particularly those related to stability, safety, efficacy, and potency.

Dosing and mode of action are two factors that strongly affect formulation design. Monoclonal antibodies (mAbs), which are typically delivered in high doses, require concentrated formulations that remain active and potent after extended storage without aggregation or precipitation. By contrast, vaccines are administered in extremely small doses so their formulations may be dilute.

Monoclonal antibody and vaccine formulations, though quite different in composition and concentration, must both provide long-term stability, be amenable to the chosen route of administration, and ensure activity/potency while supporting regulatory requirements for stability and related analysis.

No One-Size-Fits-All

Since nearly all biopharmaceuticals are administered by injection or infusion a platform approach to formulation might seem attractive as a way to streamline biopharmaceutical development. The diversity of biopharmaceuticals, however, even within certain molecular classes, unfortunately precludes a one-size-fits-all approach to formulation and formulation analysis. Platform formulations often fail to provide stability, deliverability, potency, and amenability to analytics.

Most biotherapeutics are prepared and stored in buffers, but formulations differ significantly from basic compositions of protein, salt, and water. Even within specific molecular classes, balancing effectiveness, safety, and ease of analysis often requires the addition of excipients, modifiers, detergents, adjuvants, and/or stabilizers. Each additive introduces a new set of issues for the subsequent analysis of the drug product.

For example, the common stabilizer human serum albumin, significantly increases ultraviolet absorption at 280 nm, a standard concentration measure used in HPLC analysis. Other formulation components may interfere with analysis by electrophoresis or HPLC. Introducing excipients or changing the buffer to improve stability, while scientifically justified, will cause at the very least a reassessment, if not an overhaul, of associated analytics.

Comparability Studies

In these situations comparability studies may be required. Comparability studies compare quality attributes of product produced before the manufacturing change with that manufactured afterward, against a reference standard. Comparability studies encompass a variety of tests, sometimes including cell-based assays or potency assays in animals.

According to ICH guidelines, the purpose of a comparability study is to “assess the comparability of biotechnological/biological products before and after changes are made in the manufacturing process for the drug substance or drug product. Comparability can be deduced from quality studies (partial or comprehensive), but might sometimes need to be supported by comparability bridging studies.” Comparability studies introduce timing and scheduling issues and may cause delays in a development project.

Formulation Matrix

A formulation matrix can assist in achieving an optimal formulation by simultaneously investigating multiple parameters that may include pH, buffer composition, drug-substance concentration, salts, and excipients. The samples are formulated and stored at ideal and accelerated degradation conditions, and analyzed over time. Specific tests employed during the formulation matrix include electrophoresis, chromatography, spectroscopy, and visual examination to assess gross changes such as the presence of particles or precipitates.

A formulation matrix is ideally run as early as possible during development. A formulation matrix conducted during Phase I or earlier may uncover stability issues that may be addressed, through reformulation, before considerable resources are devoted to clinical or analytics development. However, due to financial limitations the formulation matrix may be performed as late as Phase II.

Case Studies

Interference between excipients and other components is a perennial problem in formulation analysis. In one mAb formulation encountered at Covance, Tween co-eluted with one of the product aggregate peaks. It was subsequently found that the Tween absorbance was below detection limits for this particular formulation. Had Tween been present at a higher concentration, the analytic method may have required modification, for example by monitoring protein concentration at wavelengths other than 280 nm, which would have likely led to reduced sensitivity.

One seemingly small alteration in formulation can introduce significant changes in analytic method. Recently, a trimeric cytokine fusion protein submitted by a Covance client, appeared to be unstable above -80°C, which necessitated production and storage as a lyophilized product. This change, in turn, required a switch from a standard biconchoninic acid (BCA) assay (the protein’s extinction coefficient was unknown) to a microBCA assay.

A number of proteins conjugated to polyethylene glycol (PEG) have been tested. Assays for these molecules must include assessment of unconjugated, free PEG within the formulated drug product. Since PEG lacks a UV chromophore, until recently the most common method employed was size-exclusion HPLC with refractive index (RI) detection. The size-exclusion method was necessary since it employs an isocratic gradient that provides a stable signal for the RI detector. The sensitivity of this method is also limited, which restricts its applicability as an impurity assay. In addition, this method is naturally unsuitable where separation is not achieved by SE-HPLC.

Covance is currently evaluating a gradient HPLC method for separating free-PEG and PEG-protein conjugates that incorporates a charged aerosol detection, which has a greater sensitivity and should have greater applicability for PEGylated proteins.

Discussion

Timing, thoroughness, simplicity, and knowledge are critical components of formulations, particularly with respect to the ongoing analytical work conducted during preclinical and clinical development.

While small molecule drugs rarely fail due to formulation problems, the same cannot be said for biopharmaceuticals. The cytokine fusion protein mentioned above would not have been approved had it not been reformulated in a lyophilized form. Stability failures can occur as late as Phase II, after tens of millions of dollars have been invested, and the prospect of re-formulating is a costly one.

With formulation, the more effort invested up front the better. Establishing analyses appropriate to both formulation and stability-determining assays before entering human studies is ideal since these drugs are unlikely to experience formulation-related issues. The most efficient way to do this is to perform a formulation matrix construction and develop assays to circumvent interference from excipients. 

A thorough understanding of one’s biological agent, route of administration, and pharmacokinetics is indispensible for a successful formulation program. Within that framework, the aim is to arrive at the simplest formulation that satisfies requirements of stability, activity/potency, and analytics. This is not always easy to achieve as formulations might contain excipients that are suitable for use in the laboratory but not for use in humans, that interfere with common analytical methods, or that do not support long-term stability studies.

It is no exaggeration to say that for practical purposes formulation carries approximately the same significance for the commercial success of a biopharmaceutical as the drug substance itself.