CHAPTER 3

Proteins as Drugs: Analysis, Formulation

and Delivery
c.R. MIDDAUGH and R. PEARLMAN

A. Introduction
The use of proteins as drugs is by no means new. Insulin, gamma-globulin and
protein-containing vaccines have been routinely employed for decades. How-
ever, the advent of recombinant DNA technology has resulted in a dramatic
expansion of interest in their pharmaceutical applicatiobs. It now appears that
we can make virtually any desired protein in sufficient quantities for therapeu-
tic use, although often with significant difficulty. It is considerably more prob-
lematic, however, to take the appropriate macromolecule and prepare it as a
clinically acceptable drug substance. This problem arises from several sources.
First, proteins are intrinsically less stable than their lower molecular weight
(MW) pharmaceutical counterparts. Although this has turned out to be less of
a problem than first anticipated, it remains a continuing challenge to formulate
proteins that can be handled without damage throughout their entire lives;
from their initial preparation, through distribution within the complex bio-
medical system, into their ultimate clinical use in a hospital or doctor's office
or perhaps even in the home. Second, to make optimum use of a protein as a
pharmaceutical agent, it is necessary to get it to the relevant in vivo site of
action with maximum efficiency. A major potential power of proteins as thera-
peutic agents resides in their intrinsic compatibility with living systems. As
critical components of virtually all biochemical processes, the presentation of
a natural protein or one with specifically altered functional properties offers
the opportunity to intervene in a pathological process with a high degree of
specificity and minimal perturbation of normal processes. Currently, most
protein products are simply administered systemically, for example intrave-
nously, with the intrinsic specificity of the drug expected to provide sufficient
efficacy. If it was possible to actually deliver a protein to a particular site in a
temporally defined manner (sustained release, pulsatile, etc.), the utility of
protein-based pharmaceuticals and vaccines could potentially be significantly
expanded. A major stumbling block in this regard is the usual lack of knowl-
edge concerning exactly how, where and when it would be best to deliver a
specific protein. In general, our knowledge of the pathology, as well as the
normal physiology of the usually complex biology of the situation in which
we wish to intervene, is almost always grossly inadequate. This deficiency is
particularly disappointing with regard to protein pharmaceuticals, since their
D. L. Oxender et al. (eds.), Novel Therapeutics from Modern Biotechnology
© Springer-Verlag Berlin Heidelberg 1999
34 c.R. MIDDAUGH and R. PEARLMAN

exquisite specificity, which is potentially their most unique therapeutic prop-

erty, cannot be fully utilized. Finally, there still exist significant challenges in
the high-resolution analysis of protein pharmaceuticals. This is dramatically
illustrated by the fact that when a new process is devised to produce a protein
pharmaceutical with an already well-established safety and efficacy profile,
it will often be necessary to perform new safety and clinical-efficacy trials to
ensure the identity and quality of the protein derived from the new process.
This is clearly an undesirable situation which, in fact, may result in a significant
impediment to the production of proteins of higher quality and decreased
cost.
In this chapter, we will discuss each of the three issues described above
with the goal of defining the major problems in each area as we view them. In
some cases, potential solutions will also be briefly considered. It is not our
intention to provide a comprehensive review in each of these subjects since
excellent, extensive treatments of each topic already exist. Rather, we will
refer the reader to several of the more comprehensive, recent reviews about·
each subject and will assume some familiarity with each area as described in
these texts.

B. The Analysis of Protein Pharmaceuticals

The ability to accurately define the structure of any protein that is being
contemplated as a drug substance is absolutely essential to the pharmaceutical
development process. Currently, such a characterization is performed by a
series of chromatographic, electrophoretic, spectroscopic, immunological and
biological measurements (JONES 1993). Despite the intrinsically higher accu-
racy and precision of the former four types of assays, the use of a biological
(cell or organism based) method often remains critical, since there is, as of yet,
little confidence in the ability of chemical and physical methods to consistently
detect potentially subtle structural alterations that could be manifested as
decreases in either the safety or efficacy of the product. The major exception
to the need for a biological assay are proteins in which a well-defined enzy-
matic activity comprises the critical pharmaceutical activity. Although such
macromolecules still require the same complex, multifaceted characteriza-
tion as other proteins, a biological assay can usually be replaced by an in vitro
enzymatic assay of superior accuracy and precision. The problem we will
discuss here is the possibility of providing structural analyses of proteins with
sufficient resolution to ensure their functional identity.
A critical question that needs to initially be addressed is the type and
amount of information necessary to unequivocally establish the identity of
any particular protein. A simplistic answer is easily provided: a data array (a
matrix) specifying the three spatial coordinates and identity (C, H, 0, N, S,
etc.) of each atom in a protein uniquely defines any particular molecule. We
will ignore, for the moment, the dynamic, fluctuating nature of proteins and