Infliximab

1

به نام خدا

2

Infliximab Manufacturing

[email protected]

3

Infliximab is a recombinant chimeric murine monoclonal antibody glycoprotein

• Binds specifically to human TNF-alpha • Trade name: Remicade, Remsima, Inflectra• TNF-alpha: cytokine that is involved in normal inflammatory and immune

responses.

4

Infliximab was approved by the FDA for the treatment of 6 situations

Crohn's disease

Ulcerative colitis

Psoriasis

Psoriatic arthritis

Ankylosing spondylitis

Rheumatoid arthritis

5

Infliximab is administered by intravenous infusion, typically at 6-8 week intervals, at a clinic or hospital.

It can not be administered orally because the digestive system would destroy the drug

Root of administration

6

History

• Infliximab was developed by Junming Le and Jan Vilcek at New York University School of Medicine and developed by Centocor, (now Janssen Biotech, Inc.)

• The role of TNF as a key player in the development of rheumatoid arthritis was originally demonstrated by George Kollias and colleagues in proof of principle studies in transgenic animal models.

• Remicade® (infliximab) has been approved for the treatment of Crohn’s disease (1998), rheumatoid arthritis (1999), ankylosing spondylitis (2004), psoriatic arthritis (2005), and ulcerative colitis (2006).

• In September 10, 2013, the European Commission issued the first ever approval for a biosimilar antibody.

7

Manufacturers

Manufacturer of the active

substance

Manufacturer responsible

for batch release

CELLTRION Inc

13-6 Songdo-dong

Yeonsu-gu, Incheon, 406-840

Republic of Korea

Biotech Services International Limited

Biotec House,Central Park, Western Avenue

Bridgend Industrial Estate

Bridgend, CF31 3RT

United Kingdom

8

•Johnson & Johnson •Merck & Co •Celltrion •Centocor, Inc •Janssen Biotech, Inc, •Hospira, Inc •Nippon Kayaku •BioXpress Therapeutics (Swiss ) •Mitsubishi Tanabe Pharma Corporation •Samsung Bioepis

http://www.frontresearch.com/news/infliximab-market-of-innovator-substituted-by-biosimilars-companies/

9

Marketing• Remicade is marketed by

Janssen Biotech, Inc. (formerly Centocor Biotech, Inc.) in the USA Mitsubishi Tanabe Pharma in Japan Xian Janssen in China Schering-Plough (now part of Merck & Co) elsewhere

In 2013, two biosimilars were submitted for approval in Europe, by Hospira and Celltrion Healthcare.

13

http://shafaonline.ir/fa/news/66348/

http://www.pezeshkan.org/?p=20170 هزار نفر مبتال به بیماری 10در ایران •کولیت اولسراتیو و کرون

میلیون نفر مبتال به آرتریت روماتوئید 3•در ایران

به درصد از افراد مبتال ۳۰حدود • ورم مفاصل پسوریاتیک بهپسوریازیس

مبتال می شوند.

به در ايران آمار مبتاليان به پسوريازيس•يک ميليون و پانصد هزارنفر.

شیوع اسپوندیلیت انکیلوزان حدود یک نفر • 80از هر هزاز نفر است. )در ایران حدودا

هزار(

psoriasiss.blogfa.com/post/145

www.tebbook.com/forum

http://www.iranorthoped.ir/fa/news/781

در ایرانکل بیماران نیازمند به اینفیلیکسیماب10,000+3million+1.5million+80,000

At least 4.5million patients

In the US, Infliximab can cost $19,000 to $22,000 a year per patient

14

Amarnameh 94 (first 6 month)

16

http://www.pmlive.com/top_pharma_list/Top_50_pharmaceutical_products_by_global_sales

17

Infliximab is required to be produced on a commercial large scale set up

• Purifying MAbs for therapeutic use requires highly selective and robust technologies to achieve the very high purity required for biopharmaceuticals.

• Processing such valuable product streams at large-scale while manufacturing to such high standards requires careful technology design and execution

• This is because supernatant containing the target MAbs are most often obtained from the culture of live cells and are variable in both product content and composition.

18

CT-P13

19

Active Substance• Like other IgG subclasses, CT-P13 is a glycoprotein with one N-linked

glycosylation site (Asn 300) in the CH2 domain of each heavy chain.

• The detected oligosaccharides are mostly G0F (absence of terminal galactose) and G1F (one terminal galactose) structures.

• Each heavy chain consists of 450 amino acids with 11 cysteine residues, and each light chain consists of 214 amino acids with 5 cysteine residues.

• All cysteines in heavy and light chain are involved in either intra- or inter-disulphide bonding.

20

Development genetics• The Sp2/0-Ag14 cell line was formed by fusing BALB/c

spleen cells (from mouse immunised with sheep red blood cells) with the P3X63Ag8 myeloma.

• Generation of the CT-P13 cell substrate was accomplished using standard cell line transfection and methotrexate amplification protocols for the expression vector.

• The vector encodes both the heavy and light chains and the dihydrofolate reductase expression system.

21

Cell banking system

• A two-tiered cell banking system of Master Cell Bank (MCB) and Working Cell Bank (WCB) is developed from a pre-MCB and maintained in accordance to current GMP and ICH guidelines.

• An end-of-production cells bank (EPCB) derived from the WCB is also generated with cells used for commercial production of CT-P13.

• An extensive range of tests os performed for their characterisation, in accordance to ICH guidelines, including

Identity Viability Stability Presence of adventitious agents

22

Fermentation process• The CT-P13 manufacturing process is initiated using a single vial of the

WCB.

• The cells are expanded to sufficient numbers to seed the production bioreactor.

• After fixed production duration,the cell culture media is harvested and filtered.

• The harvested cell culture fluid (HCCF) is then stored until initiation of downstream operations.

23

Perfusion bioreactors are being used more commonly in biopharmaceutical manufacturing including the manufacturing of Remicade, from Centocor and Genzyme (Sanofi) and Centocor (J&J/Janssen ).

24

Affinity chromatography

Anion exchange chromatography

Cation exchange chromatography(it is more specifically a polishing step)

Anion exchange and cation exchange chromatography are used for removal of process related impurities like host cell protein

and host cell DNA

25

The cell culture harvest is clarified by using a cellulose disposable filter.

This filter is expressly customized with the effective filtration area being 650-1000 cm2 and an operating pressure of up to 30 psi.

The filtrate is checked for turbidity and target protein content.

Filteration

26

Affinity chromatography

• Affinity chromatography is used in binding and elution mode with column of 32 mm diameter for capturing; with Tris buffer pH 7.2 - 7.6 as equilibration buffer.

• After the sample is loaded on to the column, it is washed with equilibration buffer followed by 50 mM Tris-Cl.

• Then the second wash is performed with 50 mM Tris-Cl containing 100-400 mM NaCl pH 7-8 buffer solution.

27

The protein of interest is eluted with citrate

buffer pH 3.0-3.8 .

The eluate is held for 45 - 60 min at acidic

pH at room temperature for virus

inactivation and later neutralized.

28

Anion exchange chromatography• Anion exchange chromatography in negative binding mode is

then carried out at an operational flow rate of 140 cm/hr.

• The column has been equilibrated with Tris buffer pH 6.8 - 7.2.

• Protein of interest is collected in flow through.

• This step is used for the removal of process related impurities like leachate protein A, host cell DNA and host cell protein.

29

After anion exchange the flow through is

filtered for virus removal using viral removal

filter having an effective filtration area of

0.01 m2.

30

The filtrate is then buffer exchanged using a 50 kDa TFF membrane.

• The buffer used for the diafiltration process is Tris buffer ph 6.8-7.2

32

Cation exchange chromatography• Cation exchange chromatography is carried out with the

diafiltered protein solution after equilibrating the column with Tris buffer pH 6.8-7.2.

• The protein of interest is eluted with elution buffer using NaCl salt gradient.

• This step is used for the removal of process related impurities like host cell DNA and host cell protein, which are there in traces

33

Buffer exchanging and filtration • The eluate is buffer exchanged and concentrated using a 50 kDa TFF

membrane at a Trans Membrane Pressure (TMP) of 5 - 10 psi.

• The buffer exchanged protein solution is filtered using 0.2μm filter.

34

Characterizations for biosimilar

• A) Elucidation of structure and other characteristics• A1) Physicochemical characterization1. Elucidate the primary structure2. Elucidation of higher order structures3. In relation to purity/impurity4. Glycosylation• A2) Biological characterization

• B) Variants and impurities• B1) Product-related variants and impurities• B2) Process-related impurities

35

Elucidation the primary structure

• Amino acid analysis: content analysed by acid hydrolysis, derivatisation, reversed phase high performance liquid chromatography (RP-HPLC) and fluorescence detection.

• Peptide mapping: active substance and finished product were analysed by liquid chromatography mass spectrometry (LC-MS) peptide mapping

• Analysis of post-translational modifications.

• N-terminal and C-terminal sequencing by peptide mapping in combination with tandem mass spectrometry (MS/MS).

• Heavy and light chain mass determination: by liquid chromatography electrospray ionization mass spectrometry (LC-ES-MS)

36

Elucidation of higher order structures

• Determination of disulphide bond location by native and reduced peptide mapping.

• Determination of the amount of free sulfhydryl groups using Ellman’s reagent.

• Secondary structure analysis using Fourier transform infrared spectroscopy (FTIR) and circular dichroism (far UV).

• Tertiary structure analysis using circular dichroism (near UV).

• Differential scanning calorimetry (DSC) to assess the thermal stability and general higher order structure of CT-P13.

37

In relation to purity/impurity

• Aggregate content and monomeric purity determination : by size exclusion chromatography (SEC-HPLC) under non-denaturing conditions.

• Determination of electrophoretic mobility and purity : by using capillary electrophoresis sodium dodecyl sulphate (CE-SDS). Non-reducing conditions were used for determination of levels of intact IgG (H2L2) and any detectable non-assembled antibody species. Reducing CE-SDS was performed for determination of purity.

• In order to characterise the CT-P13 IgG fragments, CT-P13 finished product was assessed by means of SDS polyacrylamide gel electrophoresis (SDS-PAGE). The bands identified were excised and subject to LC-MS analysis.

38

The CT-P13 charged variants analysis

Isoelectric focusing (IEF)

Ion exchange chromatography (IEC-HPLC) combined with tryptic peptide mapping

(charge isoform distribution)

LC-MS peptide mapping (oxidised molecular variants).

39

Glycosylation

• Site-specific glycosylation analysis (LC-MS peptide analysis and oligosaccharide profiling)

• To determine the range of glycan structures displayed by CT-P13 at Asn300.

• Monosaccharide analysis of neutral and amino sugars • Type/amount of sialic acids capping the glycan structures

40

• N-linked glycan analysis for determination of oligosaccharide structures, attachment sites and distribution.

• Oligosaccharide profilingTo further characterise the glycan micro-heterogeneity high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) was used to resolve and reveal the oligosaccharide structures

• Monosaccharide analysis determination and quantitation of neutral and amino sugar composition by acid hydrolysis followed by chromatography to separate the monosaccharides

• Sialic acids analysis by HPAEC-PAD analysis Sialic acid was detected in the form of N-glycolylneuraminic acid (NeuGc) in all samples, with no other forms detected.

41

Summary of Physicochemical Test Methods for Comparability of CT-P13 Active Substance and Finished Product with Remicade

42

Summary of Physicochemical Test Methods for Comparability of CT-P13 Active Substance and Finished Product with Remicade

43

Summary of Studies Comparing Biological Activity between Remsima and Remicade

44

The drug substance was characterized as per the specifications

• The bioassay is the only analytical method that directly measures the biological activity of Remicade .

• One of the most important assessments that should be conducted as part of product characterisation, lot release and stability programs.

45

Biological activity that describes the specific ability or capacity of a product to achieve a defined biological effect.

• As with the majority of therapeutic monoclonal antibodies, Remicade has a number of different mechanisms of action and therefore a range of bioassays are required to measure each of these mechanisms in order to fully characterise the product and demonstrate comparability with the Originator/Innovator material.

• Remicade TNF-Alpha Neutralisation of cell death (L929/U937 cells)• Remicade TNF-Alpha Neutralisation of apoptosis (U937 cells)• Remicade TNF-Alpha Neutralisation of adhesion molecules (HUVEC cells)• Remicade complement dependent cytotoxicity (CDC)• Remicade antibody dependent cytotoxicity (ADCC)

46

Formulation • The Drug Substance (Active Pharmaceutical Ingredient) is

formulated using:Formulation buffer pH 7.2 500 mg sucrose 0.5 mg polysorbate 80 2.2 mg monobasic sodium phosphate Monohydrate 6.1 mg dibasic sodium phosphate

• Dihydrate yielding a concentration of 10mg/ml of Infliximab.

• No preservatives are present.

47

References

49

http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Public_assessment_report/human/002576/WC500151486.pdf

52

Thank you for your attention

Infliximab

Technology

Transcript of Infliximab