Manufacturing processes for biosimilars
Biosimilars are produced via a highly complex process requiring several tightly controlled steps that require continued monitoring . Recent developments in manufacturing and analytical technologies have improved the manufacturer and quality control and should reassure prescribers and patients that a biosimilar will provide a similar level of clinical efficacy and safety to the originator product
Alain Astier PharmD PhD French Academy of Pharmacy , Faculty of Pharmacy , Paris , France
Due to their complex structure , both biologics and biosimilars cannot be synthesised in a laboratory and instead are manufactured using recombinant DNA technology and cell culture techniques .
The key steps in the manufacture of biosimilars are described in Table 1 and can be divided into cloning and protein expression and production , purification and validation .
Identification The first stage of developing a biologic is the identification of the target protein that has the particular therapeutic benefit . For biosimilars , the target is the reference biologic product . 1
Defining the structure The three-dimensional structure of the target molecule must be defined and must be replicated as closely as possible in the biosimilar molecule . Determining the amino acid sequence of the protein allows characterisation and replication of the genetic material needed to reproduce the protein . 1
Host cell line selection Gene expression is possible in a number of cell systems including bacteria , yeast , plants and animals . Non-glycosylated biologics are usually manufactured in bacterial cells . Various prokaryotic cells such as Escherichia coli ( E . coli ) can be used as host . 2 E . coli is fast growing , easily cultured and has large production capacities if cultivated at high cell density . Hetero-proteins such as glycoproteins ( monoclonal antibodies , for example ), are best manufactured in eukaryotic cells , often derived from Chinese hamsters ( Chinese hamster ovary ; CHO cells ). 3 Indeed , only mammalian cells are able to carry out post-translational modifications such as glycosylation or some chemical modifications of the native protein , i . e ., lysination or acetylation at its C-terminus . These post-translational modifications , performed in the Golgi apparatus or N-terminus respectively , while crucial , explain the presence of several variants ( isoforms ) that are formed naturally during the production of immunoglobulins , giving rise to a mixture of several closely related entities . CHO cells are commonly preferred because the culture conditions are relatively simple , tolerant to minor variations in the culture media , can carry out glycosylation similar to human cells thereby reducing the potential for immunogenicity and are suitable for large-scale culturing . 3
TABLE 1
Key steps in biosimilar manufacture
• Identifying the target molecule
• Defining the structure of the target molecule
• Inserting the genetic material into a vector for expression
( transfection )
• Selecting a host cell line
• Insertion of the vector into the host cell
• Cell culture
• Isolation and purification of the molecule
• Characterisation and stabilisation .
Transfection Using a living host-cell , a gene fragment that codes for the target protein is introduced as a circular recombinant plasmid , which also contains promoter and regulatory elements . This enables insertion of the gene into the plasmid and a selection marker ( such as resistance genes to ampicillin or tetracycline ) to easily select the transfected clones . The inserted gene induces the synthesis of the corresponding target protein , which will be the same as the corresponding originator .
As biosimilars are more or less identical to the reference product , with the same primary structure , the high order structures ( e . g ., secondary and tertiary ) are also identical , except perhaps for possible variations induced by posttranslational modifications . Because the exact sequence of amino acids of the target protein is known , the building of the plasmid containing the corresponding gene is relatively straightforward , via polymerase chain reaction . Several basic plasmids are also available commercially .
Cell culture Cells that show a stable , high level of expression are selected and cloned to ensure genetic consistency . It is essential to select a clone that is likely to be the most efficient and that best supports the subsequent industrial scale-up . The cell clone chosen forms the basis of the cell bank and its ability to culture the protein of interest is initially established in a small-scale laboratory culture prior to the subsequent scale-up . Moreover , during the therapeutic antibody production via mammalian cells such as CHO , several factors can lead to unwanted modifications , such as variability in the cell line , the number of cell subcultures performed over a time , cell passages over a time , or environmental cell culture conditions . 4
The steps required for cell proliferation require strict
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