The protein science of biosimilars

Martin Kuhlmann
1
and Adrian Covic
2
1
Department of Internal Medicine Nephrology, Klinikum im Friedrichshain, Berlin, Germany and
2
Dialysis and Transplantation Center, Parhon University Hospital, Romania
Abstract
A sea change is occurring in the off-patent drug
manufacturing industry with a first wave of biotech-
nologically derived products reaching the end of their
patent lives. However, recombinant proteins are in
a different league from their chemical predecessors
in terms of molecular complexity. Small differences
in manufacturing processes can affect the efficacy and
safety of the recombinant proteins in a manner which
is not always measurable using analytical or in vitro
techniques. Thus, comparable clinical profiles do not
automatically follow from physicochemical likeness
and can only be demonstrated through clinical studies.
It is essential for patient safety that both innovator
and biosimilar manufacturers ensure consistency in
their production, by performing rigorous purity and
activity profiling between batches, and implement
tailored pharmacovigilance plans.
Keywords: assays; biopharmaceuticals; biosimilars;
glycosylation; manufacturing; pharmacovigilance;
physicochemistry
Requirements for generic drug manufacturing
A generic drug must meet several criteria to be
approved for market release. Firstly, it has to contain
the same active ingredient as the innovator drug
and be administered by the same route. It must also
have the same formulation, potency and conditions
of use [1].
In the past, medicines have been almost exclusively
low molecular weight compounds. Such chemical
compounds (such as statins, angiotensin-converting
enzyme inhibitors) are relatively easy to synthesize and
precise copies of defined quality can be produced
in a consistent manner. This reliability of synthesis
methods is reflected in the regulatory requirements for
generics manufacturers. They only need to demon-
strate that their compound is physicochemically
identical to the original drug and bioequivalent
(has the same pharmacokinetic profile) in a limited
clinical study for the relevant indication.
A number of biotechnology products are
currently marketed including insulin, growth hormone,
interferon-b and factor VIII. These recombinant
proteins are large molecules, produced from genetically
modified cell lines, and extracted through complex and
lengthy purification procedures. Because of the varia-
bility inherent in such processes, identical copies of
original brands cannot be manufactured and, there-
fore, biogenerics cannot exist. Instead, the term
biosimilars has been coined to describe these off-
patent copies of therapeutic proteins.
Gauging biosimilarity
To clarify the debate on off-patent manufacturing,
a new vocabulary is emerging which dissects different
levels of similarity. Comparability expresses the
impact of changes to a single, established production
process and is measured in terms of physicochemical
properties. However, the goal of manufacturers of off-
patent biologics is to achieve similarity: two products
from different manufacturers are deemed similar
ifimportantlythe same safety and efficacy can be
established.
Biological agents are large and complex
Biological therapeutic agents are much larger
than chemically-synthesized compounds folding
after translation into an elaborate three-dimensional
structure. This molecular structure determines their
function. Table 1 shows the molecular weights for
a representative selection of medicinal agents produced
by biotechnology compared with more classical
therapeutic compounds. It can be seen that biologics
are on average 1001000-fold larger than their
chemical counterparts.
Correspondence and offprint requests to: Martin Kuhlmann,
Klinikdirektor Innere Medizin Nephrologie, Vivantes Klinikum
im Friedrichshain, Landberger Allee 49, 10249 Berlin, Germany.
Email: Martin.Kuhlmann@vivantes.de
Nephrol Dial Transplant (2006) 21 [Suppl 5]: v4v8
doi:10.1093/ndt/gfl474
The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org

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Proteins can undergo complex modifications
Proteins may undergo a number of co- and post-
translational modifications to fine-tune their
activity. These include enzymatic cleavage (which can
lead to activation, for example, hormones), attachment
of lipids (for localization of the protein to a cell
membrane) or glycans (which can affect properties
such as serum half-life). Figure 1 illustrates some
common modifications in an idealized protein and
their possible functional roles.
Post-translational modifications affect
protein activity
Many important therapeutic proteins are glycosylated
including epoetin (EPO), granulocyte macrophage-
colony stimulating factor (GM-CSF) and tissue-
plasminogen activator (t-PA). Glycosylation is
important because it can influence the biological
activity of a protein through different mechanisms.
First of all, glycosylation can affect half-life by
influencing the active clearance of a protein. Certain
pituitary glycoprotein hormones such as lutropin
are rapidly removed from circulation by the liver if
they bear a sulphated GalNAc residue [2]. A second
example is EPO, the serum half-life of which is
dependent on four sialylated N-glycans (sialic acid
is itself a glycan). Poorly glycosylated forms of EPO
are rapidly cleared by filtration in the kidney. In
addition, under-sialylated EPO is rapidly removed by
hepatocytes and macrophages. Thus, increasing the
degree of sialylation and glycosylation decreases the
renal clearance rate [3], and can increase EPO in vivo
activity.
Glycosylation can affect the activity of a recombi-
nant protein more directly. Rituximab, for instance,
is a monoclonal antibody (mAb) used to treat non-
Hodgkins lymphoma through targeting of B-cell
CD20 antigens. Its activity can be assessed with the
antibody-dependent cellular cytotoxicity (ADCC) test,
which measures the ability of an engineered mAb
to lyse target cells. The ADCC of two types of
mAb (one of which was rituximab) was found to
vary inversely according to the mAbs glycosylation
state [4]. Rituximab was produced from Chinese
hamster ovary (CHO) cells with a relatively high level
of glycosylation and was found to be several-fold
less cytotoxic in vitro than another anti-CD20 mAb
(KM3065), which was synthesized with fewer glycan
residues from a rat cell line.
Heterogeneity in biological preparations
Recombinant protein manufacturing processes are
highly elaborate and sophisticated not only due
to the complexity of the molecules themselves,
but also as a consequence of cell-based production.
Furthermore, the therapeutic properties of recombi-
nant proteins are highly dependent on the manufactur-
ing process. Maintaining batch-to-batch consistency
(comparability) is an ongoing challenge and different
production processes yield measurably different
proteins.
Commercial manufacturing processes
are complex
Biological manufacturing processes involve many
steps from cloning the desired gene via selection of
a suitable cell type, fermentation and purification to
formulation of the end product (Figure 2). A variety
of undesired chemical alterations to the drug product
Fig. 1. Idealized protein with typical post-translational
modifications and functional consequences. Cleavage of terminal
amino acids can activate a protein (e.g. in the case of hormones), as
can phosphorylation. Glycosyl groups play a key role in signalling,
and can influence the blood clearance rate (e.g. erythropoietin,
certain pituitary glycoprotein hormones) or biological activity.
Attachment of lipids allows the protein to be anchored in plasma
membranes.
Table 1.
Chemical drug Molecular
weight (Da)
Biological drug Molecular
weight (Da)
Glucophage

166 Neupogen

18 800
Vioxx

314 Roferon-A

19 625
Prozac

346 Humatrope

22 125
Zantac

351 Avonex

22 500
Paxil

375 Epogen

30 400
Zocor

419 Pulmozyme

37 000
Augmentin

420 Enbrel

75 000
Crixivan

712 Zenapax

144 000
Taxol

854 Rituxan

/MabThera

145 000
Factor VIII 264 000
Source: EuropaBio.
Protein science of biosimilars v5

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can occur during this time including oxidation or
deamidation. A frequent problem is also the formation
of protein aggregates.
Glycosylation is notably sensitive to cell growth
conditions. Changes in culture pH, the availability of
precursors and nutrients, and the presence or absence
of various cytokines and hormones can each affect
the extent of glycosylation. In addition, the presence
of sialidases and other glycosidases, either secreted
or released by dead cells, can cause degradation of
a previously intact product.
Differences between EPO preparations
Biological preparations, whether commercial or
from a laboratory can, and do, vary markedly from
one another. One study revealed marked variation
between two laboratory preparations of human
urinary erythropoietin and four preparations of
EPO [5]. The observed differences in isoform profiles
were attributed to differences in glycosylation, as
ascertained by a special binding assay developed by
the investigators. In a second study, which compared
Fig. 2. Flow chart summarizing production processes for biopharmaceuticals. Once the appropriate sequence is cloned, a cell line is selected
which expresses the recombinant protein in sufficient amounts and with the desired post-translational modifications. The cells are grown in
large quantities and the protein harvested through a series of purification steps. The final formulation must allow storage in which the protein
remains stable for extended periods.
v6 M. Kuhlmann and A. Covic

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eight different laboratory EPO preparations by
mass spectrometry, it was shown that each sample
contained a distinct mixture of isoforms with at least
23 observed glycan structures [6]. Complementary
in vivo measurements in mice demonstrated that the
exact N-glycan structure was a major determinant
for EPO biological activity.
Scaled-up commercial production of biologics
suffers from the same problems as laboratory prepara-
tion. Heterogeneity between 12 commercial EPOa
samples manufactured outside of the US and Europe
was demonstrated by isoelectric focusing (IEF) [7]. The
variations in isoform composition can be seen clearly
in Figure 3. Another comparability study of biosimilar
products manufactured outside the US and Europe
revealed that products differed widely in composition,
did not always meet self-declared specifications and
exhibited batch-to-batch variation [8]. These studies
were in turn echoed by a Brazilian study [9], which, in
addition, found unacceptable bacterial endotoxin
levels in three products.
Immunogenicity
Immunogenicity is a critical issue in biotechnologically
derived medicines. The risk of immune responses
against recombinant proteins was made clear by cases
of pure red cell aplasia (PRCA) in patients receiving
a brand of EPO approved in Europe. These cases were
traced to a minor change in the production process,
the consequences of which eluded the rigorous controls
in place at the time [10]. Affected patients experienced
an antibody-mediated neutralization of endogenous
EPO and a complete block in the differentiation of
red blood cells. This incident has been instrumental
in creating awareness of the importance of careful
control of biotechnology drugs, particularly with
regard to immune responses in patients.
Immune responses can be elicited by various
qualities of recombinant protein preparations. For
instance, deglycosylation has previously been asso-
ciated with immunogenicity in the case of interferon-b
by uncovering hydrophobic residues and reducing
solubility [11] or in the case of GM-CSF by exposure
of antigenic sites [12]. Also, misfolding or aggrega-
tion during manufacturing or storage can yield
products which cause an immune response. Finally,
immune responses can be triggered by impurities.
Such impurities may include process-derived chemicals
or antibiotics, or may result from microbial or viral
contamination [13].
Can we manage biopharmaceutical heterogeneity?
Maintaining patient safety and treatment efficacy with
recombinant proteins represents a challenge especially
with the advent of biosimilars. Therapeutic benefit
is critically dependent on a finished drug product of
a very high quality. Both biosimilar manufacturers
and innovators must ensure consistency in their
production. They should have a complete description
of their manufacturing process, monitor batch varia-
tion through impurity profiling, physicochemical and
functional analyses, and maintain a historic database
of in-process control data to assess the impact of any
manufacturing changes.
Currently, neither analytical methods nor in vitro
assays are guaranteed to fully characterize a given
recombinant protein and there are no methods for
predicting clinical efficacy. Animals transgenic for the
human endogenous gene equivalent to a recombinant
protein could provide useful quantitative models to
gauge immunogenicity, but have not yet been validated
[14]. Thus, in order to demonstrate unequivocally
that a biosimilar has the same efficacy and safety as
its branded predecessor, a similarity assessment should
include pre-clinical and clinical data. Current drafts
Fig. 3. Gel of EPO samples from 12 different manufacturers in Latin America and Asia [17].
Protein science of biosimilars v7

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of European guidelines state that the amount of
clinical data required depends on the nature of the
recombinant protein under consideration [15]. Proteins
which have a unique endogenous counterpart with
a critical function or which are particularly large and
complex (such as mAbs) should undergo extensive
clinical testing [16]. Because even phase III trials are
typically not large enough to detect adverse events as
rare as immune responses, tailored pharmacovigilance
plans are also essential.
Conclusion
A change is occurring in the biotechnologically derived
drug industry with the impending arrival of biosimilars
on the market. Experience with brand products
indicates that manufacturing of recombinant proteins
must be supported by stringent controls. Recombinant
proteins are much more complex molecules than
traditional chemical drugs. They require highly
elaborate and sophisticated manufacturing processes,
and their properties are highly dependent on the
process used. Different processes inevitably yield
different products, which cannot always be fully
characterized by analytical methods, yet may have
different clinical safety and efficacy profiles. Current
drafts of European guidelines, still under discussion,
express a requirement that biosimilar manufacturers
provide pre-clinical and clinical data to support
a marketing application, and perform quality checks
as extensively as innovator companies. For the
same reasons, biosimilars should also be accompanied
by a pharmacovigilance plan. Future experience with
biosimilars will determine how these requirements
should be defined.
Conflict of interest statement. M.K.K. has participated in meetings
sponsored by Roche and has received honoraria from Roche and
Ortho Biotech. A.C. has received honoraria and participated in
meetings sponsored by Roche.
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