Nephrology Dialysis Transplantation 2006 21(Supplement 5):v4-v8; 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
The protein science of biosimilars
Martin Kuhlmann1, and
Adrian Covic2
1Department of Internal Medicine – Nephrology, Klinikum im Friedrichshain, Berlin, Germany and 2Dialysis and Transplantation Center, Parhon University Hospital, Romania
Correspondence and offprint requests to: Martin Kuhlmann, Klinikdirektor Innere Medizin – Nephrologie, Vivantes Klinikum im Friedrichshain, Landberger Allee 49, 10249 Berlin, Germany. Email: Martin.Kuhlmann{at}vivantes.de
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Abstract
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A sea change is occurring in the off-patent drug manufacturing
industry with a first wave of biotechnologically 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
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Requirements for generic drug manufacturing
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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 demonstrate 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-ß 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 variability inherent in such processes, identical copies of original brands cannot be manufactured and, therefore, ‘biogenerics’ cannot exist. Instead, the term ‘biosimilars’ has been coined to describe these off-patent copies of therapeutic proteins.
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Gauging biosimilarity
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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 if—importantly—the
same safety and efficacy can be established.
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Biological agents are large and complex
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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
100–1000-fold larger than their chemical
counterparts.
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Proteins can undergo complex modifications
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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.
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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.
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Post-translational modifications affect protein activity
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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 recombinant protein more directly. Rituximab, for instance, is a monoclonal antibody (mAb) used to treat non-Hodgkin's 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 mAb's 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.
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Heterogeneity in biological preparations
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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 recombinant proteins
are highly dependent on the manufacturing process. Maintaining
batch-to-batch consistency (‘comparability’) is
an ongoing challenge and different production processes yield
measurably different proteins.
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Commercial manufacturing processes are complex
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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
can occur during this time including oxidation or deamidation.
A frequent problem is also the formation of protein aggregates.
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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.
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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.
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Differences between EPO preparations
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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 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 preparation. Heterogeneity between 12 commercial EPO
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.
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Immunogenicity
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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 associated with immunogenicity in the case of interferon-ß by uncovering hydrophobic residues and reducing solubility [11] or in the case of GM-CSF by exposure of antigenic sites [12]. Also, misfolding or aggregation 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].
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Can we manage biopharmaceutical heterogeneity?
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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 variation 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 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.
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Conclusion
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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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References
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- Baenziger JU, Kumar S, Brodbeck RM, Smith PL, Beranek MC. (1992) Circulatory half-life but not interaction with the lutropin/chorionic gonadotropin receptor is modulated by sulfation of bovine lutropin oligosaccharides. Proc Natl Acad Sci USA 89:334–338.[Abstract/Free Full Text]
- Misaizu T, Matsuki S, Strickland TW, et al. (1995) Role of antennary structure of N-linked sugar chains in renal handling of recombinant human erythropoietin. Blood 86:4097–4104.[Abstract/Free Full Text]
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Requirements for generic drug...