REGULATIONS

As for chemicals, the development of numerous biopharmaceutical products has been

accompanied during the last 15 years by a sustained and joint effort of the Regulatory Authorities
and the Industry to set up contributory regulatory guidelines.

As shown in the table below, it is interesting to note that the first guideline ever released in 1989
was regarding the hottest topic that is Safety Evaluation; this guideline has been updated in 1997
and remains a robust reference in the matter. It was followed, by the Q6B guideline, on Quality
and pharmaceutical aspects, which equally remains a reference document. It is noticeable that in
all those guidelines, a constant pedagogical attempt is made to define the scope of the products
concerned and to situate the approach in comparison with chemicals.

In addition to these guidelines, there are a number of minutes of ad hoc Biotech Working Parties,
both at the EMEA and at FDA, which are of utility to update and clarify specific topics.

Quality Safety Efficacy

ICH • Q6B :
Test procedures &
acceptance criteria for
biotechnological
& biological products (1999)
• Q5E :
Comparability of
biotechnological/
biological processes
(2004)
EMEA • CPMP/BWP/3207/00 Rev.1
Guideline on Comparability
of Medicinal Products
containing Biotechnology-
derived Proteins as Active
Substance
Quality Issues (2003)
European • Monograph #2031
Pharmacopeia Monoclonal antibodies for human use (2004)
• Points to consider
FDA
in the manufacture & testing of monoclonal antibody product for human use (1997)
• 3BS13a : • M3 :
Preclinical biology Preclinical studies
safety testing on needed for First In
medicinal products Man
derived from
biotechnology (1989)
• S6 :
Preclinical safety
evaluation of
biotechnology-derived
pharmaceuticals (1997)
• CPMP/SWP/1094/04 • CHMP/89249/2004
guideline for the Guidelines on the
evaluation of control clinical
samples in non-clinical investigation of the
safety studies : Pharmacokinetics
checking for of therapeutic
contamination with the proteins
test substance (2004) (2005, draft*)

* In the first semester of 2006, when this article was written, the final version of the Guideline was not
issued.
 PHARMACOKINETICS

The Pharmacokinetics (PK) studies of therapeutic proteins have only recently been addressed in
the EMEA Regulation. Bioanalyses are the key elements in the PK developments of therapeutic
proteins. HPLC and MS are not suitable for the quantification of proteins. Detecting accurately
the structure and the function of an active compound amongst a complex biological medium and
differentiating the therapeutic protein from its endogenous homologous form require a
combination of immunoassays (ELISA, RIA) and in vitro bioassays. The critical points during
PK studies are to develop these specific assays at the beginning of the development, to use the
same assays for all its duration, and to know the concentration time profile of the endogenous
protein.

The general design of the PK studies of therapeutic proteins in Humans is close to that of
chemicals. They consist of single and repeated administrations via the therapeutic routes, and are
performed in healthy volunteers, if possible, and in patients. A series of points that may influence
the PK parameters of the studied protein have yet to be examined. The binding to blood
components is a critical point to be assessed. The presence of soluble receptors in blood may alter
the PK, increase the variability between patients and may result in time-dependent PK. Hence
soluble receptors should be measured by appropriately developed methods. In the same time, the
search for antibodies targeted toward the administered protein should be performed in long term
studies, each subject being its own control, and their potential impact on the PK should be
measured. The impact of the chemical modifications of the protein (e.g. glycosylation,
pegylation) on its PK is also a key element. If the different isoforms of the therapeutical protein
are very different, the PK studies need to be performed for each individual one. In subcutaneous
administration, absorption can be sensitive not only to the site and depth of injection, but may
also be susceptible to local proteolysis.

The elimination of proteins occurs by proteolytic mechanisms that can be predicted by the
molecular weight of the protein. For the proteins of molecular weight up to 50 kD the proteolysis
takes place directly in the plasma and is followed by renal filtration whereas larger proteins
undergo elimination in the liver, via receptor mediated endocytosis. This renders mass balances
useless (metabolic recycling), as well as interaction studies (involvement of cytochrome
dependant mechanisms). The pharmacodynamic activity of the metabolites resulting from the
proteolysis of the therapeutical protein may also need to be measured. Furthermore, the strong
influence of the uptake of the protein’s target receptor on its elimination is to be considered.
Important differences in the elimination of a protein may occur between healthy volunteers and
patients, because of differences in receptor density, e.g. over-expression. Thereof extrapolation of
the data gathered in healthy volunteers to the patients has to be validated.

PK/PD studies are encouraged with a special attention to be paid to the “surrogacy” of
biomarkers and the usual time delay between plasma level and measured effects.
IMMUNOGENICITY

The ability of a therapeutic protein to elicit an immune response is one of the most peculiar issues
for development and crosses the different disciplines of development as it may have various
consequences on pharmacodynamics, PK, toxicity and tolerability. Antibodies occur frequently in
therapies with a considerable inter-individual variability, an unpredictable frequency and with
consequences which depend on the nature of the antibody, especially its neutralizing character,
and which may range from transient appearance of antibodies without any clinical consequences
to severe life threatening conditions. The most outstanding example of immunogenicity issues is
represented by pure red cell aplasia following antibodies induced by EPO treatment which cross-
react with the endogenous cytokines.

The factors of immunogenicity may be grouped in two categories, depending on whether they are
related to the treatment or to the patient. Non-human sequences of amino-acids (e.g.: mouse
mAbs) and impurities related to the product itself or to the manufacturing process belong to the
group of predictable potential immunogenic factors. The dosing regimen and the route of
administration play an important role as well. However, some influencing factors may be more
surprising, such as the drug formulation or even the leachable compounds used during the
container closure process, which can act as adjuvants. In addition the patient-related factors also
play an important role in the immunogenicity of a therapeutic protein and may depend on genetic
variables. In patients with complete hereditary deficiencies for a protein for instance, the deficient
protein may be recognized as a foreign protein by the patient’s immune system. Indeed this
phenomenon explains the occurrence of resistance to factor VIII replacement therapy in patients
with severe hemophilia A.

The immunogenicity of a therapeutical protein should be assessed in a standardized manner and

the anti-drug antibodies should be monitored from the beginning of, and throughout, the
nonclinical and clinical development using immuno-monitoring techniques (e.g.: ELISA
technique). Then, if these anti-drug antibodies are detected, they must be characterized in terms
of activity (neutralizing vs binding), class, complement fixation, clinical relevant threshold and
their potential consequences on PK, efficacy and safety have to be evaluated in appropriate tests.
Indeed, the increased clearance of antigen-antibody immune complexes by the reticulo-
endothelial system and the binding of the antibody on the therapeutical protein may have an
impact on its PK and on its biological activity respectively. In clinical trials, the occurrence of
adverse events related to the immune response to the tested product may cause serious concern
about safety and may even put the patient’s vital prognosis at stake.

This creates the need for the prediction of immunogenicity. Preclinical evaluation of
immunogenic potential in animals is useful but not accurate and often leads to an overestimation
of the occurrence of human immune reactions. Strategies reducing the interspecies natural
immunogenicity may improve the ability to predict it, though. Experimental animals tolerant to
the tested protein (by transgenesis or by the phenomenon of neo natal tolerance) may be used as
well as a surrogate homologous protein of the experimental species (which implies the potential
bias of the differences in the Major Histocompatability Complex between the animal species and
human). However, comparative studies in animals still remain of great interest when assessing the
immunogenicity of a new formulation or the second generation of a therapeutic protein for which
data on immunogenicity in Human is available.