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Biosimilars

Biosimilars

A biosimilar (also known as follow-on biologic or subsequent entry biologic) is a biologic medical product that is almost an identical copy of an original product that is manufactured by a different company. Biosimilars are officially approved versions of original "innovator" products and can be manufactured when the original product's patent expires.Reference to the innovator product is an integral component of the approval.

Protein Analytics for Biosimilarity/Comparability Exercise

Protagen Protein Services (PPS) has successfully supported over a dozen different customers in multiple projects to develop biosimilars with state of the art analytical methods. Biosimilars are being developed for a whole number of innovator drugs.

  • List of Molecules
  • Modules
  • Longterm Partnership

Examples for such biosimilar developments analyzed at PPS are:

Abatacept

Orencia®

Abatacept (Orencia®) is a soluble and homodimeric fusion protein for anti-TNF alpha treatment in Rheumatoid Arthritis.
It consists of the Fc part of an IgG1 antibody linked via a disulphide bond to the extracellular domain of the protein receptor CTLA4 that is found on activated T-cell surfaces. It is produced in a CHO cell line. Abatacept binds a so called B7 protein on antigen presenting cells thereby blocking the necessary co-signal to a T-cell. The T-cells cannot be activated and, therefore, T lymphocyte activation is prevented. The protein structure includes N- and O-glycosylation and leads to different glyco- and isoforms by additional N- and C-terminal heterogeneity. Due to the complexity of the protein, such a detailed analysis of the primary structure as well as the glycosylation can only be achieved by a combination of mass spectrometry, bioinformatics, HPLC and electrophoretic methods.
To support the analytical development of Abatacept biosimilars or biobetters, PPS has set up an analysis program in accordance with ICH Q6B guidelines, including method setup, validation of customer-specific methods, and release testing. This package includes:

  • Protein quantification and detemination of the extinction coefficient

Characterization of the protein primary structur including:

  •         Confirming the amino acid sequence
  •         Analysis of N- and C-terminal sequence heterogeneity
  •         Determination of N- and O-glycosylation sites
  •         Quantification of protein modifications
  •         Confirming the disulfide bridging

Characterization of the glycosylation pattern:

  •         N- and O-glycan mapping by mass spectromtry
  •         N- and O-gylcan mapping by HPLC
  •         Quantification of sialic acid levels
  •         Linkage analysis by GC-MS

Characterization of the physicochemical properties:

  •         Intact protein mass by mass spectrometry
  •         Charge isoforms by IEF, cIEF or IEX
  •         Aggregation by AUC or SEC-MALLS

The specific bioassays are performed by a strategic partner:

  •     T-cell proliferation inhibition (Mixed Lymphocyte Reaction)

Adalimumab

Humira®

Adalimumab (Humira ®) is a monoclonal antibody used in the treatment e.g. of Rheumatoid arthritis, Psoriatic arthritis, or Crohn’s disease. Humira was the third TNF-α inhibitor on the market, but the first fully human antibody directed towards this target.
Adalimumab, as a typical IgG1 antibody, is comprised of a tetramer of two heavy and two light chains with one N-glycosylation site per heavy chain.
The extended patent expires end of 2016 and already many companies are developing biosimilar versions of Humira.
We have long-standing experience in the analysis of different variants of Humira originators and biosimilars.
To support the analytical development of Humira biosimilars or biobetters, Protagen has set up an analysis program in accordance with ICH Q6B guidelines, including method setup, validation of customer-specific methods, and release testing. This package includes protein quantification, characterizing the protein primary structure, analysis of N- glycosylation and protein modifications.

Specific BioAssay:

  •     Target: Inhibition of TNF-α signaling Assay type: Proliferation Assay, statistical analysis by 4 parameter fit or PLA

Aflibercept

Eylea®

Aflibercept (Eylea) is a recombinantfusion protein consisting of vascular endothelial growth factor (VEGF)-binding portions from the extracellular domains of human VEGF receptors 1 and 2, that are fused to the Fc portion of the human IgG1 immunoglobulin.

Aflibercept (Eylea) is a biopharmaceuticaldrug invented by Regeneron Pharmaceuticals, approved in the United States and Europe for the treatment of wet macular degeneration under the trade name Eylea, and for metastatic colorectal cancer as Zaltrap. As the active ingredient of Zaltrap, the substance is called ziv-aflibercept in the US.

It is an inhibitor of vascular endothelial growth factor (VEGF).[1][2]

Aflibercept is being co-developed for cancer treatment by Sanofi and Regeneron under a deal signed in 2003, and is being co-developed for eye diseases by Bayer HealthCare and Regeneron under a deal signed in 2006

PPS has generated considerable experience in the analysis of different originator batches of Aflibercept.

We have established an analytical program according to the ICH Q6B guidance, including the method setup, validation of customer specific methods and release testing.

This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification, including the detailed look for the loss of the C-terminal lysine.

 


Belimumab

Benlysta®

Belimumab(Benlysta®) is a human monoclonal antibody that inhibits B-cell activating factor (BAFF) also known as B-lymphocyte stimulator (BLyS). It is approved in the United States, Canada, and Europe to treat systemic lupus erythematosus (SLE). SLE is a rare autoimmune disease (orphan disease) in which organ systems are damaged by a dysregulation of the immune system.

PPS has gained significant experience in the analysis of different originator batches as well as variants of biosimilars of Belimumab.

To support the development of Benlysta biosimilars or biobetters, PPS has set up an analytical development program in accordance with ICH Q6B guidelines, including method setup, validation of customer-specific methods and release testing. This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification.


Bevacizumab

Avastin®

Bevacizumab (Avastin ®) is a humanized monoclonal antibody that inhibits vascular endothelial growth factor A and is commonly used to treat various cancers, including colorectal, lung, breast, kidney, and glioblastomas.
As a typical IgG1 antibody, Bevacizumab is comprised of a tetramer of two heavy and two light chains with one N-glycosylation site per heavy chain.
Bevacizumab came off-patent in 2018 (extended expiry) and already several companies are developing biosimilar versions of Avastin.
Protagen has experience in the analysis of different variants of Avastin originators and biosimilars.
To support the analytical development of Avastin biosimilars or biobetters, Protagen has set up an analysis program in accordance with ICH Q6B guidelines, including method setup, validation of customer-specific methods, and release testing. This package includes protein quantification, characterizing the protein primary structure, analysis of N- glycosylation and protein modifications.

Specific BioAssay:

  •     Target: VEGF Assay type: Neutralization/proliferation Assay

Botulinum Toxin A

Botox, Xeomin

Botulinum toxin (BTX) is a neurotoxic protein produced by the bacterium Clostridium botulinum and related species. It prevents the release of the neurotransmitter acetylcholine from axon endings at the neuromuscular junction and thus causes flaccid paralysis. Infection with the bacterium causes the disease botulism. The toxin is also used commercially in medicine, cosmetics and research.

Botulinum toxin A is an active substance in the group of muscle relaxants used for the temporary treatment of facial wrinkles. It is the deadly poison of the bacterium Clostridium botulinum. Botulinum toxin blocks the release of the neurotransmitter acetylcholine from cholinergic nerve endings, causing flaccid paralysis of skeletal muscle. The drug is injected locally intramuscularly. The effects occur within a few days and usually last for three to six months. The most common potential adverse reactions include injection site reactions and headache. Very rare, serious side effects are possible

There are eight types of botulinum toxin, named type A–H. Types A and B are capable of causing disease in humans, and are also used commercially and medically. Types C–G are less common; Type H is considered the deadliest substance in the world – an injection of only 2 ng can cause death to an adult.

PPS has gained significant experience in the analysis of different originator batches as well as variants of biosimilars of Botulinum Toxin.

To support the development of Botulinum Toxin biosimilars or biobetters, PPS has set up an analytical development program in accordance with ICH Q6B guidelines, including method setup, validation of customer-specific methods and release testing. This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification.

 


Cetuximab

Erbitux®

Cetuximab (Erbitux®) is a chimeric (mouse/human) monoclonal antibody, specific for EGFR (epidermal growth factor receptor), used for treatment of metastatic colorectal cancer and head and neck cancer. Cetuximab inhibits the receptor-associated tyrosine kinase, thus blocking the intracellular signalling pathway that results in cell proliferation.
We have considerable experience in the analysis of different variants of Erbitux originators and biosimilars.
To support the analytical development of Erbitux biosimilars or biobetters, Protagen has set up an analysis program in accordance with ICH Q6B guidelines, including method setup, validation of customer-specific methods, and release testing. This package includes protein quantification, characterizing the protein primary structure, analysis of N- glycosylation and protein modifications.

Specific BioAssay:

  •     Target: EGF-R Assay type: Proliferation Assay, statistical analysis by PLA

 


Daratumumab

Darzalex®

Daratumumab (trade name Darzalex) is an anti-cancer drug. Daratumumab is an IgG1k monoclonal antibody directed against CD38. CD38 is overexpressed in multiple myeloma cells. Daratumumab binds to CD38, causing cells to apoptose via antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity It binds to CD38,[1] which multiple myeloma cells overexpress.[2] Daratumumab was originally developed by Genmab, but it is now being jointly developed by Genmab along with the Johnson & Johnson subsidiary Janssen Biotech, which acquired worldwide commercialization rights to the drug from Genmab.[3]

Daratumumab was given breakthrough therapy drug status in 2013 for multiple myeloma. It was awarded orphan drug status for multiple myeloma, diffuse large B cell lymphoma, follicular lymphoma, and mantle cell lymphoma.[4]

PPS has gained significant experience in the analysis of different originator batches of Daratumumab.

To support the development of Benlysta biosimilars or biobetters, PPS has set up an analytical development program in accordance with ICH Q6B guidelines, including method setup, validation of customer-specific methods and release testing. This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification.


Denosumab

Prolia®

Denosumab (Prolia®) is a fully human IgG2 monoclonal antibody inhibiting the RANK ligand which is an essential factor for the formation, activation and survival of osteoclasts. It is used for the treatment of osterporosis.
PPS has generated considerable experience in the analysis of different originator batches as well as variants of biosimilars of Denosumab.

We have established an analytical program according to the ICH Q6B guidance, including the method setup, validation of customer specific methods and release testing.
This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification.


Eculizumab

Soliris®

Eculizumab (Soliris®) is a humanized monoclonal antibody (IgG 2/4 kappa immunoglobuline) manufactured in NS0. It is used to treat the rare blood disorder Paroxymal nocturnal haemoglobinuria (a genetically derived disease) which leads to haemolysis of the patients. By binding to the human complement protein C5, it blocks the cleavage of the protein itself which leads to the inhibition of the complement mediated intravascular haemolysis.

To support the analytical development of Eculizumab biosimilars or biobetters, PPS has set up an analysis program in accordance with ICH Q6B guidelines, including method setup, validation of customer-specific methods, and release testing. This package includes different assays for protein quantification, characterizing the protein primary structure  including the slight N- and N-terminal heterogeneity, the glycosylation pattern and the analysis of protein modification.


Emicizumab

Hemlibra®

Emicizumab (trade name Hemlibra®) is a humanized monoclonal antibody and drug for the treatment of hemophilia A (with inhibitors). Emicizumab belongs to the so-called bispecific antibodies and binds both to the coagulation factor IXa and to the factor X and mediates their activation. This is usually the function of coagulation factor VIII, which is absent in hemophilia A patients. Emicizumab thus mimics the coagulation factor and thus acts as a factor VIII mimetic.

PPS has generated considerable experience in the analysis of different originator batches of Emicizumab.
We have established an analytical program according to the ICH Q6B guidance, including the method setup, validation of customer specific methods and release testing.
This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification, including the detailed look for the loss of the C-terminal lysine.


Erythropoietin

EPO

Erythropoietin (EPO) is mainly used for the treatment of anemia resulting from different diseases such as chronic kidney disease or from the treatment of cancer or from other critical illnesses. Its main function is promoting red blood cells by protecting these cells from apoptosis.
EPO is a 165 amino acid protein with a high glycosylation ratio of about 40% by weight. The glycosylation consists of N- (three sites) and O-glycans (one site). The glycans are highly branched and carry sialic acids at the branch ends. This degree of sialic acids is an important regulator for the serum half-life and thus for the efficacy of the drug.

Most EPO drug products are recombinantly produced in CHO-cells, but also BHK or in a human fibrosarcoma cell line (HT-1080 derivative). Amgen marketed the first recombinant EPO in 1989 under the trade names Epogen or Epoetin.
Meanwhile, a large number of EPO variants and biosimilars are marketed, such as Darbepoetin, which carries additional glycosylation sites and order to improve the pharmacodynamics.

We have long-standing experience in the analysis of different variants of EPO originators, biosimilars and biobetters.
To support the development of EPO biosimilars or biobetters, Protagen has set up an analysis program in accordance with ICH Q6B guidelines, including method setup, validation of customer-specific methods, and release testing. This package includes protein quantification, characterizing the protein primary structure, analysis of N- and O-glycosylation, sialic acid content, antennarity and protein modifications.

Specific Bioassay:

  • Proliferation Assay, statistical analysis by PLA

Etanercept

Enbrel®

Etanercept (Enbrel ®) is a receptor fusion protein used in the treatment of Rheumatoid Arthritis, Polyarticular Juvenile Idiopathic Arthritis, Psoriatic Arthritis, Ankylosing Spondylitis and Plaque Psoriasis. Etanercept is a homodimer made of two chains of 75kDa each, binding to TNF-α. The protein structure is complicated by the high degree of N- and O-glycosylation. Also, a N- and C-terminal sequence heterogeneity adds to the complexity of the protein analysis.
The extended patent expired end of 2012. Several companies are developing biosimilar versions of this drug.
We have long-standing experience in the analysis of different variants of Enbrel originators and biosimilars. Due to the complexity of the protein, such a detailed analysis of the primary structure as well as the glycosylation can only be acheived by a combination of mass spectrometry, bioinformatics, HPLC and electrophoretic methods.
To support the development of Enbrel biosimilars or biobetters, Protagen has set up an analysis program in accordance with ICH Q6B guidelines, including method setup, validation of protein-specific methods, and release testing. This package includes

Protein quantification and determination of the extinction coefficient

Characterization of the protein primary structure including

  •     confirming the amino acid sequence,
  •     analysis of N- and C-terminal sequence heterogeneity,
  •     determination of N- and O-glycosylation sites
  •     quantificaiton of protein modifications,
  •     confirming the disulfide bridging.

Characterization of the glycosylation pattern

  •     N- and O-glycan mapping by mass spectromtry
  •     N- and O-gylcan mapping by HPLC
  •     Quantification of sialic acid levels
  •     Linkage analysis by GC-MS

Characterization of the physicochemical properties

  •     Intact protein mass by mass spectrometry
  •     Charge isoforms by IEF, cIEF or IEX
  •     Aggregation by AUC or SEC-MALLS

Factor VIII

Coagulation Factor VIII is a heavily glycosylated heterodimeric plasma protein that consists of a heavy (domains A1-A2-B) and light chain (domains A3-C1-C2). Factor VIII participates in the intrinsic pathway of blood coagulation. Defects in the Factor VIII gene result in haemophilia A. Both variants, plasma-derived and recombinant Factor VIII products are available on the market. With its high degree of complex glycosylation (25 N-glycosylation sites and 6 O-glycosylation sites) and a size of ca. 265 kDa (2351 amino acids) this molecule is very challenging from an analytical point of view.
To support the development of Factor VIII biosimilars or biobetters, Protagen Protein Services has set up an analysis program in accordance with ICH Q6B guidelines, including method setup, validation of customer-specific methods, and release testing.
This package includes protein quantification, characterizing the protein primary structure, analysis of N- glycosylation and protein modifications. The analysis also allows to monitor the Tyrosine-sulfation levels, which are important for the functionality of Factor VIII.

The following analytical methods can be provided for Factor VIII:

Quantity and Extinction Coefficient:

  •         Amino acid analysis: determination of protein content and amino acid composition
  •         Determination of extinction coefficient

Structural Characterization and Confirmation – Amino Acid Sequence:

  •         Peptide mapping
  •         Deamidation, methionine oxidation and tyrosine sulfation levels
  •         Disulfide bridge analysis by mass spectrometry
  •         N/C terminal sequencing: MALDI-ISD
  •         Total molecular weight of each subunit

Structural Characterization and Confirmation – Glycosylation:

  •         Monosaccharide composition
  •         Sialic acid content
  •         N- and O-Glycan mapping (MALDI MS) and Linkage analysis by GC-MS
  •         N- and O-Glycosylation site determination

Physicochemical Properties:

  •         Isoform pattern (IEF)
  •         Liquid chromatographic pattern: IEX
  •         Spectroscopic profiles: UV/VIS Spectra
  •         Spectroscopic profiles: CD
  •         SEC-MALLS for aggregation

Filgrastim (G-CSF)

Neupogen®

Filgrastim is a recombinant form of the human granulocyte colony-stimulating factor (G-CSF). It is a 175-amino-acid polypetide with two disulfide bridges, which is expressed in E.coli. Lenograstim, a variant expressed in Chinese hamster ovary (CHO) cells, represents a recombinant glycoprotein. There are already a number of Filgrastim biosimilars and biobetters on the market. Pegfilgrastim is a variant coupled to a polyethylene glycol (PEG) molecule.

We have long-standing experience in the analysis of different variants of G-CSF originators, biosimilars and biobetters.

To support the development of filgrastim biosimilars or biobetters, Protagen has set up an analysis program in accordance with ICH Q6B guidelines, including method setup, validation of customer-specific methods, and release testing.

This package includes:

  • Volume determination
  • Bacterial endotoxin testing
  • Sterility testing
  • pH determination, opalescence and coloration
  • Biological activity testing
  • Protein identification by western blotting
  • Protein identification by peptide mapping
  • Protein concentration
  • IEF

 Compendial Bioassay and additional analysis:

  • Proliferation Assay, statistical analysis

Glatiramer acetate

Copaxone®

Glatiramer acetate (Copaxone®) is the acetic acid salt (acetate) of a synthetically produced polypeptide containing the four naturally occurring amino acids L-alanine, L-glutamic acid, L-lysine and L-tyrosine in random order. The immunomodulatory active substance is used as a drug for the treatment of relapsing forms Multiple Sclerosis (MS). The exact mechanism of action is unknown. Because the composition of glatiramer resembles the components of nerve cell isolation, it is believed to reduce the central nervous system inflammatory reactions that occur in MS

It is a mixture of random-sized peptides that are composed of the four amino acids found in myelin basic protein, namely glutamic acid, lysine, alanine, and tyrosine. Myelin basic protein is the antigen in the myelin sheaths of the neurons that stimulates an autoimmune reaction in people with MS, so the peptide may work as a decoy for the attacking immune cells.

PPS has generated considerable experience in the analysis of different originator batches of Copaxone.

We have established an analytical program according to the ICH Q6B guidance, including the method setup, validation of customer specific methods and release testing.

This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification, including the detailed look for the loss of the C-terminal lysine.


Golimumab

Simponi®

Golimumab (Simponi®) is a human IgG1 monoclonal antibody and binds with high affinity and specificity to both soluble and transmembrane forms of TNFa, which prevents the binding of TNFa to its receptors, thereby neutralizing its biological activity. It is used among others for the treatment of Rheumatoid arthritis and Psoriatic arthritis. PPS has gained significant experience in the analysis of different originator batches as well as variants of biosimilars of Golimumab.

We have established an analytical program according to the ICH Q6B guidance, including the method setup, validation of customer specific methods and release testing.
This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification, including the detailed look for the C-terminal lysine heterogeneity of the heavy chain.


Infliximab

Remicade®

Infliximab (Remicade®) is a chimeric (mouse/human) IgG1 monoclonal antibody. Like Etanercept and Adalimumab, Infliximab's is a TNFa blocker. Infliximab is used for treatment of a variety of autoimmune diseases, e.g. Crohn's disease, ulcerative colitis and rheumatoid arthritis. It is composed of human constant and murine variable regions with an approximate molecular weight of 149,1 kDa. The patent for the monoclonal TNF-alpha antibody Infliximab (Remicade®) expired at the beginning of 2015.

Protagen has considerable experience in the analysis of different variants of Remicade originators and biosimilars.
To support the analytical development of Remicade biosimilars or biobetters, Protagen has set up an analysis program in accordance with ICH Q6B guidelines, including method setup, validation of customer-specific methods, and release testing. This package includes protein quantification, characterizing the protein primary structure, analysis of N- glycosylation and protein modifications.

Specific BioAssay:

  • Target: Inhibition of TNF-α signaling   
    Assay type: Proliferation Assay, statistical analysis by 4 parameter fit or PLA

Insulin

Lantus®

Insulin(Insulinum glarginum, trade name Lantus®, manufacturer Sanofi) is a drug from the group of insulin analogues, which is used for the treatment of diabetes mellitus (diabetes mellitus), and is one of the basal insulins. The active ingredient is a recombinant protein produced from genetically modified microorganisms. It differs slightly from the body's own insulin. Insulin glargine has the amino acid glycine in the amino acid sequence compared to human insulin at position A21 (Asn21) instead of asparagine. The B chain is extended by two arginine units. Compared with other retinal insulins, it has a longer half-life, which results in it being absorbed more slowly and more evenly by the body after injection, allowing once daily dosing.

Since 2015, insulin glargine is also available as the first insulin biosimilar (trade name Abasaglar), since 2017 as another insulin biosimilar (trade name Lusduna).

PPS has generated considerable experience in the analysis of different originator batches of Lantus.

We have established an analytical program according to the ICH Q6B guidance, including the method setup, validation of customer specific methods and release testing.

This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification, including the detailed look for the loss of the C-terminal lysine.


Interferon alpha-2a

Roferon®

Interferon alfa-2a (Roferon®) is a recombinant protein obtained by biotechnological methods from an Escherichia coli strain. It consists of 165 amino acids and has a molecular weight of about 19 kDa.

Interferon alfa-2a is an antitumor and antiviral agent in the interferon group used to treat cancers and hepatitis B and C. It is usually given subcutaneously daily or three times a week and has a shorter duration of action than the pegylated peginterferon alfa-2a.

Interferon alfa-2a has antiviral, antitumoral, immunomodulating and antiproliferative properties. The effects are due to binding to interferon alfa receptors on the cell surface, resulting in gene transcription. Unlike peginterferon alfa-2a, interferon alfa-2a has a short half-life of about 5 hours.

PPS has generated considerable experience in the analysis of different originator batches of Roferon.

We have established an analytical program according to the ICH Q6B guidance, including the method setup, validation of customer specific methods and release testing.

This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification, including the detailed look for the loss of the C-terminal lysine.


Interferon beta-1a

Avonex®

Interferon beta-1a (Avonex®) is a recombinant protein that is biotechnologically derived from CHO cells. It consists of 166 amino acids, has the same amino acid sequence as the natural interferon beta and is glycosylated like this.

  • Interferon beta upregulates the expression of MHC I proteins, allowing for increased presentation of peptides derived from viral antigens. This enhances the activation of CD8+ T cells that are the precursors for cytotoxic T lymphocytes (CTLs) and makes the macrophage a better target for CTL-mediated killing.
  • Interferon beta binds to type I interferon receptors (IFNAR1 and IFNAR2c) which, upon dimerization, activate two Jak (Janus kinase) tyrosine kinases (Jak1 and Tyk2). These transphosphorylate themselves and phosphorylate the receptors. The phosphorylated INFAR receptors then bind to Stat1 and Stat2 (signal transducers and activators of transcription) which dimerize and activate multiple (~100) immunomodulatory and antiviral proteins. Interferon beta binds more stably to type I interferon receptors than interferon alpha.

PPS has generated considerable experience in the analysis of different originator batches of Avonex.

We have established an analytical program according to the ICH Q6B guidance, including the method setup, validation of customer specific methods and release testing.

This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification, including the detailed look for the loss of the C-terminal lysine.


Interferon beta-1b

Betaserone®

Interferon beta-1b (Betaserone®) is a recombinant protein of 165 amino acids with a molecular weight of about 18,500 Da. It is derived from an E. coli strain and, unlike interferonum beta-1a, is not glycosylated. It therefore corresponds to the natural protein slightly less than interferon beta-1a.

Interferon beta-1b is an active substance in the interferon group for the treatment of multiple sclerosis. The solution for injection is injected subcutaneously every other day. The medicine reduces the number of relapses, their severity and slows the progression of the disease. Common adverse events include flu-like symptoms, abdominal pain, elevated liver enzymes, blood disorders, skin rash, and application site reactions. Interferon beta-1b, unlike interferon beta-1a, is derived from E. coli and not from CHO cells and is not glycosylated.

PPS has generated considerable experience in the analysis of different originator batches of Betaserone.

We have established an analytical program according to the ICH Q6B guidance, including the method setup, validation of customer specific methods and release testing.

This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification, including the detailed look for the loss of the C-terminal lysine.


Interleukin-2

Proleukin®

Interleukin-2 (Proleukin) a lymphokine, is produced by recombinant DNA technology using a genetically engineered E. coli strain containing an analog of the human interleukin-2 gene. Genetic engineering techniques were used to modify the human IL-2 gene, and the resulting expression clone encodes a modified human interleukin-2. This recombinant form differs from native interleukin-2 in the following ways: a) Aldesleukin is not glycosylated because it is derived from E. coli; b) the molecule has no N-terminal alanine; the codon for this amino acid was deleted during the genetic engineering procedure; c) the molecule has serine substituted for cysteine at amino acid position 125.

Used to treat renal cell carcinoma, Interleukin-2 induces the enhancement of lymphocyte mitogenesis and stimulation of long-term growth of human interleukin-2 dependent cell lines, the enhancement of lymphocyte cytotoxicity, the induction of killer cell (lymphokine-activated (LAK) and natural (NK)) activity; and the induction of interferon-gamma production. IL-2 is normally produced by the body, secreted by T cells, and stimulates growth and differentiation of T cell response. It can be used in immunotherapy to treat cancer. It enhances the ability of the immune system to kill tumor cells and may interfere with blood flow to the tumor.

Aldesleukin binds to the IL-2 receptor which leads to heterodimerization of the cytoplasmic domains of the IL-2R beta and gamma(c) chains, activation of the tyrosine kinase Jak3, and phosphorylation of tyrosine residues on the IL-2R beta chain. These events led to the creation of an activated receptor complex, to which various cytoplasmic signaling molecules are recruited and become substrates for regulatory enzymes (especially tyrosine kinases) that are associated with the receptor. These events stimulate growth and differentiation of T cells.

PPS has generated considerable experience in the analysis of different originator batches of Proleukin.

We have established an analytical program according to the ICH Q6B guidance, including the method setup, validation of customer specific methods and release testing.

This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification, including the detailed look for the loss of the C-terminal lysine.


Ipilimumab

Yervoy®

Ipilimumab (Yervoy ®) is a fully human anti human IgG1 monoclonal antibody and an immune-modulatory agent that has been developed for the treatment of melanoma, an aggressive form of skin cancer. Ipilimumab targets the protein CTLA-4. The monoclonal antibody influences the modulation of the immune response, weakens the inhibitory signal of CTLA-4 and therefore acts as an enhancer of T cell function.
The patent expired end of 2016, the extended patent protection last until 2022 and already several companies are developing biosimilar versions of Ipilimumab. PPS has generated considerable experience in the analysis of different originator batches as well as variants of biosimilars of Ipilimumab. We have established an analytical program according to the ICH Q6B guidance, including the method setup, validation of customer specific methods and release testing.

This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification.


Lenograstim

Granocyte®

Lenograstim (marked under  the brand name Granocyte). is a recombinantgranulocyte colony-stimulating factor which functions as an immunostimulator.[1][2]

Lenograstim is the glycosylated recombinant form of human granulocyte colony stimulating factor (G-CSF). factor used in cytostatic neutropenia. Lenograstim accelerates neutrophil recovery significantly after chemotherapy, with beneficial effects on clinical end-points such as incidence of laboratory-confirmed infection and length of hospital stay. Chemotherapy dose intensity has also been increased in patients receiving lenograstim, notably those with breast or small cell lung cancer, although improvements in tumour response and survival have not been demonstrated. Lenograstim also assists neutrophil recovery in patients undergoing bone marrow transplantation, and stimulates the production of peripheral blood stem cells (PBSCs) for autologous transfusion after aggressive chemotherapy.

PPS has generated considerable experience in the analysis of different originator batches of Lenograstim.

We have established an analytical program according to the ICH Q6B guidance, including the method setup, validation of customer specific methods and release testing.

This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification, including the detailed look for the loss of the C-terminal lysine.


Mepolizumab

Nucala®

Mepolizumab (trade name Nucala) is a humanizedmonoclonal antibody used for the treatment of severe eosinophilic asthma. It recognizes and blocks interleukin-5 (IL-5), a signalling protein of the immune system. Mepolizumab binds to IL-5 and prevents it from binding to its receptor, more specifically the interleukin 5 receptor alpha subunit, on the surface of eosinophil white blood cells. While eosinophils play a role in inflammation associated with asthma, the exact mechanism of mepolizumab is unknown.[6]

PPS has gained significant experience in the analysis of different originator batches of Mepolizumab.

To support the development of Benlysta biosimilars or biobetters, PPS has set up an analytical development program in accordance with ICH Q6B guidelines, including method setup, validation of customer-specific methods and release testing. This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification.


Natalizumab

Tysabri®

Natalizumab (Tysabri ®) is a humanized monoclonal antibody against the cellular adhesion molecule α4-integrin. Natalizumab is used in the treatment of multiple sclerosis and Crohn's disease.

Natalizumab is expected to come off-patent in 2015 and already many companies are developing biosimilar versions of Tysabri.

We have long-standing experience in the analysis of different variants of Tysabri originators and biosimilars.

To support the analytical development of Tysabri biosimilars or biobetters, Protagen has set up an analysis program in accordance with ICH Q6B guidelines, including method setup, validation of customer-specific methods, and release testing. This package includes protein quantification, characterizing the protein primary structure, analysis of N- glycosylation and protein modifications.

Specific BioAssay processed for Natalizumab :

  • Target: α4-integrin Assay type: Binding Assay (flow cytometry), statistical analysis by PLA


Omalizumab

Xolair®

Omalizumab, sold under the trade name Xolair®, is a medication originally designed to reduce sensitivity to allergens. It has been used to try to control severe allergic asthma, which does not respond to high doses of corticosteroids and less widely for chronic spontaneous urticaria.

Omalizumab is a recombinant DNA-derived humanized IgG1k monoclonal antibody that specifically binds to free human immunoglobulin E (IgE) in the blood and interstitial fluid and to membrane-bound form of IgE (mIgE) on the surface of mIgE-expressing B lymphocytes. Unlike an ordinary anti-IgE antibody, it does not bind to IgE that is already bound by the high affinity IgE receptor (FcεRI) on the surface of mast cells, basophils, and antigen-presenting dendritic cells.

PPS has gained significant experience in the analysis of different originator batches as well as variants of biosimilars of Omalizumab.

To support the development of Benlysta biosimilars or biobetters, PPS has set up an analytical development program in accordance with ICH Q6B guidelines, including method setup, validation of customer-specific methods and release testing. This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification.

 


PEG-Interferon alpha-2a

Pegasys®

Peginterferon alfa-2a is an antiviral agent in the interferon group for the treatment of chronic hepatitis B and chronic hepatitis C. It is pegylated and therefore has a much longer half-life than common inferferon alfa-2a.

Peginterferon alfa-2a is a covalent conjugate of the recombinant protein interferon alfa-2a and a branched monomethoxy-polyethylene glycol (PEG). It has a molecular weight of about 60 kDa and is obtained by biotechnological methods from Escherichia coli.

Peginterferon alfa-2a has antiviral and antiproliferative properties. The effects are due to binding to interferon alfa receptors on the cell surface, resulting in gene transcription. Thanks to pegylation, the half-life of 160 hours is significantly longer than that of interferon alfa-2a (5 hours).

PPS has generated considerable experience in the analysis of different originator batches of Pegasys.

We have established an analytical program according to the ICH Q6B guidance, including the method setup, validation of customer specific methods and release testing.

This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification, including the detailed look for the loss of the C-terminal lysine.


Pertuzumab

Perjeta®

Pertuzumab (also called 2C4, trade name Perjeta®) is a monoclonal antibody used in combination with trastuzumab and docetaxel for the treatment of metastatic HER2-positive breast cancer; it also used in the same combination as a neoadjuvant in early HER2-positive breast cancer.[2]

Side effects in more than half the people taking it include diarrhea, hair loss, and loss of neutrophils; more than 10% experience loss of red blood cells, hypersensitivity or allergic reaction, infusion reactions, decreased appetite, insomnia, distortions in the sense of taste, inflammation of the mouth or lips, constipation, rashes, nail disease, and muscle pain.[3] Women who are pregnant or planning on getting pregnant should not take it, it was not studied in people with certain heart conditions and should be used in caution in such people, and it should not be used with an anthracycline.[3] It is unknown if pertuzumab interacts with doxorubicin.[3]

It is the first-in-class of a kind of drug called a "HER dimerization inhibitor" — it inhibits the dimerization of HER2 with other HER receptors, which prevents them from signalling in ways that promote cell growth and proliferation.

PPS has generated considerable experience in the analysis of different originator batches of Pertuzumab.

We have established an analytical program according to the ICH Q6B guidance, including the method setup, validation of customer specific methods and release testing.

This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification, including the detailed look for the loss of the C-terminal lysine.


Ranibizumab

Lucentis®

Ranibizumab (Lucentis®) is a non-glycosylated monoclonal antibody fragment (Fab) created from the humanized Bevazizumab (Avastin®) antibody but expressed in e.coli. There are slight differences in the amino acid sequence in the VEGF-binding region. VEGF-A Protein is considered an activator of blood vessel growth which can lead to vision loss in the eye, especially in the disease that is called wet type of age related macular degeneration (AMD).

The solution is directly injected into the eye so that the fragment binds to the VEGF-A protein to inhibit angiogenesis and therefore hinder further vision loss.

The patent expired in 2017. PPS has experience in the analysis of different variants of Ranibizumab originators and biosimilars.

To support the analytical development of Ranibizumab biosimilars or biobetters, PPS has set up an analysis program in accordance with ICH Q6B guidelines, including method setup, validation of customer-specific methods, and release testing. This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification

The specific for Ranibizumab bioassays are:

  • Target: VEGF Assay type: Neutralization/proliferation Assay


Rituximab

Rituxan®, MabThera®

Rituximab (Rituxan ®, MabThera ®), is a chimeric monoclonal antibody against the protein CD20, which is primarily found on the surface of B cells. Rituximab is used in the treatment of many lymphomas, leukemias, transplant rejection and some autoimmune disorders. Rituximab is also used off-label to treat difficult cases of multiple sclerosis, systemic lupus erythematosus and autoimmune anemias.

Rituximab destroys both normal and malignant B cells that have CD20 on their surfaces, and is therefore used to treat diseases which are characterized by having too many B cells, overactive B cells or dysfunctional B cells.

Rituximab comes off-patent in 2012 (Europe) and 2015 (US), respectively and already many companies are developing biosimilar versions of Rituxan / MabThera.

We have long-standing experience in the analysis of different variants of Rituxan / MabThera originators and biosimilars.

To support the analytical development of Rituxan / MabThera biosimilars or biobetters, Protagen has set up an analysis program in accordance with ICH Q6B guidelines, including method setup, validation of customer-specific methods, and release testing. This package includes protein quantification, characterizing the protein primary structure, analysis of N- glycosylation and protein modifications.

Specific BioAssays processed for Rituximab:

  • Target:CD20 Assay type: Binding Assay (based on flow cytometry), statistical analysis by PLA

  • Target:CD20 Assay type: ADCC Assay

  • Target:CD20 Assay type: CDC Assay


Somatropin

Neutropin

Recombinant human growth hormone (somatotropin) 191 residues, MW 22.1 kD, synthesized in E. coli

Somatropin (Neutropin) is a recombinant human growth hormone (hGH) with 191 residues, MW 22.1 kD, synthesized in E. coli that corresponds to the natural pituitary-derived human growth hormone (STH). Somatropin is produced by biotechnological methods. It stimulates bone and body growth and is used to treat dwarfism and growth failure, growth hormone (hGH) stimulates skeletal growth in pediatric patients with growth failure due to a lack of adequate secretion of endogenous GH. Skeletal growth is accomplished at the epiphyseal plates at the ends of a growing bone. Growth and metabolism of epiphyseal plate cells are directly stimulated by GH and one of its mediators, IGF-I (insulin-like growth factor).

hGH binds to the human growth hormone receptor (GHR). Upon binding, hGH causes dimerization of GHR, activation of the GHR-associated JAK2 tyrosine kinase, and tyrosyl phosphorylation of both JAK2 and GHR. These events recruit and/or activate a variety of signaling molecules, including MAP kinases, insulin receptor substrates, phosphatidylinositol 3' phosphate kinase, diacylglycerol, protein kinase C, intracellular calcium, and Stat transcription factors. These signaling molecules contribute to the GH-induced changes in enzymatic activity, transport function, and gene expression that ultimately culminate in changes in growth and metabolism.

PPS has generated considerable experience in the analysis of different originator batches of Neutropin.

We have established an analytical program according to the ICH Q6B guidance, including the method setup, validation of customer specific methods and release testing.

This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification, including the detailed look for the loss of the C-terminal lysine.


Tocilizumab

RoActemra®

Tocilizumab (RoActemra®) is a humanized anti human IgG1 monoclonal antibody against the interleukin-6 receptor (IL-6R). Interleukin 6 (IL-6) is a cytokine that plays an important role in immune response and is implicated in the pathogenesis of many diseases, such as autoimmune diseases, multiple myeloma and prostate cancer.

PPS has generated considerable experience in the analysis of different originator batches as well as multiple variants of biosimilars of Tocilizumab.

We have established an analytical program according to the ICH Q6B guidance, including the method setup, validation of customer specific methods and release testing.

This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification, including the detailed look for the C-and N-terminal heterogeneity of the heavy chain and the incomplete cleavage of the signal sequence from the N-terminus if the light chain.


Trastuzumab

Herceptin®

Trastuzumab (Herceptin) is a humanized monoclonal antibody that interferes with the HER2/neu receptor. Trastuzumab is used to treat certain breast cancers.

As a typical IgG1 antibody, Trastuzumab is comprised of a tetramer of two heavy and two light chains with one N-glycosylation site per heavy chain.

Protagen has experience in the analysis of different variants of Herceptin originators and biosimilars.

To support the analytical development of Herceptin biosimilars or biobetters, Protagen has set up an analysis program in accordance with ICH Q6B guidelines, including method setup, validation of customer-specific methods, and release testing. This package includes protein quantification, characterizing the protein primary structure, analysis of N- glycosylation and protein modifications.

Specific BioAssay processed Trastuzumab for:

  • Target: HER2, ErbB2 Assay type: Proliferation Assay, statistical analysis by PLA


Ustekinumab

Stelara®

Ustekinumab (Stelara®) is a human IgG1κ monoclonal antibody, directed against interleukin 12 and interleukin 23, naturally occurring proteins that regulate the immune system and immune-mediated inflammatory disorders. With this function it works immunosuppressive and anti-inflammatory.

PPS has generated considerable experience in the analysis of different originator batches as well as multiple variants of biosimilars of Ustekinumab.

We have established an analytical program according to the ICH Q6B guidance, including the method setup, validation of customer specific methods and release testing.

This package includes different assays for protein quantification and characterizing the protein primary structure and secondary structure, as well as detailed analysis of N-glycosylation and protein modification, including the detailed look for the loss of the C-terminal lysine.


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Protagen Protein Services (PPS) is a world leading CRO and recognized expert for analytical services in protein science. More than 20 years of market experience and the comprehensive spectrum of validated analytical methods ensure the highest quality for customers in the pharmaceutical, biotech and life science industry.

PPS supports Biosimilar developers with a broad range of analytical methods and consulting in achieving and demonstrating Biosimilarity