6-7 The drug manufacturing process Analysis of final productweb.xidian.edu.cn/yqxia/files/20170331_145235.pdf · 2017. 3. 31. · Product potency. 20. Bioassay. Analysis in a biological

By Yuqiong Xia3/31/2017

药物制造过程：终产品分析The drug manufacturing process: Analysis of

final product

Pharmaceutical Biotechnology 西安电子科技大学夏玉琼

Outline

2

Protein structure Primary structure Secondary structure Tertiary structure

Analysis of final product Removal and Detection of contaminates

西安电子科技大学夏玉琼

3

Amino acid 西安电子科技大学夏玉琼

4

Nonpolar amino acids 西安电子科技大学夏玉琼

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Charged amino acids 西安电子科技大学夏玉琼

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Polar

Tryptophan


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Primary structure 西安电子科技大学夏玉琼

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G-CSF （granulocyte-colony stimulating factor）西安电子科技大学夏玉琼

Outline

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Secondary structures

http://quizlet.com/13505003/amino-acids-carbohydrates-lipids-structure-and-trivia-flash-cards/


α-helix

11

Stablized by the electric dipole moment interaction between the C=O of i amino acid and the NH of i+3 amino acid

Positive partial charge at N terminal and negative at C terminal


12

Helical wheel of a peptideplot every 360o/3.6 = 100o around a circle

http://sbb.uvm.edu/~sje/351/sub2.1.html


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β-sheet西安电子科技大学夏玉琼

β-turn and loop

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β-turn Proline and glycine

Long loops hydrophilic

http://ww2.chemistry.gatech.edu/~lw26/structure/protein/secondary_structure/beta_turn/index.html


15

Structure of G-SCF: predicted by software

alpha

betaturncoil


Outline

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17

Tertiary and Quaternary structures

http://www.umass.edu/molvis/workshop/imgs/protein-structure2.png


Outline

18


Analysis of final product Product potency Concentration determination Structure characterization Immunogenicity

Removal and Detection of contaminates


Why is analysis of final product so important

19

Proteins are much larger and more structurally complex than traditional low molecular mass drugs

Far broader range of potential contaminants due to their production in biological systems

https://web.stanford.edu/class/cs279/lectures/lecture2.pdf


Product potency

20

Bioassay Analysis in a biological system

Immunoassays

http://res.cloudinary.com/dk-find-out/image/upload/q_80,w_960/MA_00766318_t4yw5n.pnghttps://openclipart.org/image/800px/svg_to_png/161839/cell-culture2.pnghttp://media.opencurriculum.org/articles_manual/ck12_biology/immune-response/4.png


Bioassay

21

Steps Add a known quantity of product to a biological

system The system responds in some way to the product. The response is measured quantitatively to estimate

product potency

Biological system

Add drug Measure response


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Bioassay of EPO

https://edc2.healthtap.com/ht-staging/user_answer/reference_image/10091/large/Syringe.jpeg?1386670665http://res.cloudinary.com/dk-find-out/image/upload/q_80,w_960/MA_00766318_t4yw5n.pnghttps://img.clipartfox.com/fc3241695d2bfa1f170d65b78e91c431_blood-cells-blood-corpuscles-red-blood-cells-clipart_800-587.jpeg

1. Inject EPO labeled with 57Fe

2. Measure the rate of incorporation of radioactivity into proliferation RBCs


Bioassay of interferon

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‘cytopathic effect inhibition assay’ (细胞病变效应抑制分析) based upon the ability of interferons to render animal cells

resistant to viral attack

http://ecx.images-amazon.com/images/I/31ooqjcBXgL.jpghttps://openclipart.org/image/800px/svg_to_png/161839/cell-culture2.pnghttp://www.moleculardevices.com/sites/default/files/product-images/hcs-sw-images/metaxpress_appmod_livedead_450.jpg

1. Add interferon (drug)

2. Add virus

3. Measure survived cells by neutral red dyes

Green: liveRed: dead


Drawbacks of bioassay

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Lack of precision. The biological system is influenced by many factors.

Time Most bioassays take days, and in some cases weeks, to run

Cost Most bioassay systems are extremely expensive to undertake


Immunoassays

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Employ monoclonal or polyclonal antibody preparations to detect and quantify the product

Two types Enzyme immunoassay (EIA) and radioimmunoassay

(RIA)

http://media.opencurriculum.org/articles_manual/ck12_biology/immune-response/4.png


26

Enzyme Immunoassay (EIA)

Add sample containing antibody

Add antigen specific for the drug

Add enzyme-labeled antibody specific for antibody in the drug

Add substrateShow color


27

Radioimmunoassay (RIA)

Add sample containing antibody

Add antigen specific for the drug

Add radio-labeled antibody specific for antibody in the drug

Detect radioactivity

125I 125I


Immunoassays

28

Advantage rapid (in minutes to hours), inexpensive and easy

Limitation Immunological reactivity cannot be guaranteed to

correlate directly with biological activity

http://media.opencurriculum.org/articles_manual/ck12_biology/immune-response/4.png


Outline

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Amino acid and peptide chain

https://web.stanford.edu/class/cs279/lectures/lecture2.pdf

Amino acid

Peptide chain

Protein


Most simple methods: UV280 and UV205

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Absorbance at 280 nm (A280; UV method) The side chain of selected amino acids

(particularly tyrosine and tryptophan) absorbs UV at 280nm

A fast but insensitive method

http://elte.prompt.hu/sites/default/files/tananyagok/IntroductionToPracticalBiochemistry/ch04s06.html


Most simple methods: UV280 and UV205

32

Absorbance at 205 nm (far UV method) Peptide bonds absorb UV at 190–220 nm A more sensitive method

http://elte.prompt.hu/sites/default/files/tananyagok/IntroductionToPracticalBiochemistry/ch04s06.html


Most common method: Bradford method

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Bradford reagent contains the dye, Coomassie blue (考马斯亮蓝) G-250, in an acidic solution.

The dye binds to protein with basic and aromatic side chains, yielding a blue color which absorbs maximally at 595 nm

考马斯亮蓝G-250

http://www.qcbio.com/pierce/23236.htm

Proteins containing lysine, arginine, tyrosine, tryptophan, phenylananine


Most common method: Bicinchonic acid (双金鸡纳酸 BCA) method

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Copper-containing reagent which, when reduced by protein, reacts with bicinchonic acid yielding a complex that displays an absorbance maximum at 562 nm

http://microamaze.blogspot.com/2015/10/bicinchoninic-acid-bca-assay-for.html

Proteins containing cysteine, cysteine, tyrosine, tryptophan


Outline

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Structural characterization of product

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Characterizing primary structure Amino acid analysis Peptide mapping N-terminal sequencing

Characterizing secondary structure CD FT-IR

Characterizing tertiary structure XRD


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Amino acid analysis

Protein hydrolysis6N HCl, 110 oC, vacuum, 12-24 h

Protein

Separation of amino acids by IEC

Quantified by

Learning the quantity of each type of amino acid

Limited application due to 1. Protein hydrolysis may modify/destruct amino acids2. Semi-quantitative 3. Low sensitivity

https://moodle.beverleyhigh.net/pluginfile.php/7490/mod_resource/content/0/assets/images/ks4/protein.gifhttp://www.chem.ucalgary.ca/courses/350/Carey5th/Ch27/ch27-3-3.html

ninhydrin (茚三酮)


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Peptide mapping

Cleaved by cyanogen bromide (BrCN) or protease V8 with trypsin

Protein

Separation of peptide fragmentsby gel electrophoresis or HPLC

Peptide fragments

Marker

Peptide fragments

Peptide map

Correct protein structure should have correct peptide map

t

A


Peptide mapping: how is a protein cleaved

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Protein hydrolysis at specific points (7-14 AA best) BrCN: cleave at the carboxylic side of Met V8 protease with trypsin: V8 protease cleaves at

carboxylic side of Asp and Glu, while trypsin cleaves that of Arg and LysNH2-RLVKEVMLGVKIGRFMGKAGDRNTRV-COOH

-RLVKEVMLGVKIGRFMGKAGDRNTRV-

BrCN

-RLVKEVMLGVKIGRFMGKAGDRNTRV-

V8 protease Trypsin NH2-RLVKEVMLGVKIGRFMGKAGDRNTRV-


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N-terminal sequencing (accurate) by Edman degradation

phenylisothiocyanate异硫氰酸苯酯

6N HCl

6N HCl

Analyze the amino acid

Analyze the amino acid


Outline

41


Analysis of final product Product potency Concentration determination Structure characterization Immunogenicity analysis

Removal and Detection of contaminates Removal and detection of protein contaminates Removal and detection of LPS Removal and detection of DNA Removal and detection of virus


Two types of immune response

Breaking B-cell tolerance

Vaccine-type response

(classical immune response)

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Takes in 6 to 12 months

Produce binding antibodies with no biological effect

Antibodies disappear shortly after stopping treatment

No memory effect

Occur within weeks

A single injection will induce antibody response

Induce high level of neutralizing antibodies

Memory effect


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Mechanism of breaking B-cell tolerance

B-cells are only activated when their receptors bind to repeated peptide/protein structures. In nature, only some bacteria and viruses have repeated peptide/protein structures.


Mechanism of classical immune response

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Ingestion and cleaving of the proteins into peptides by macrophages and dendritic cells

Presentation of peptides by the MHC-II system

Activation of B-cells Boosting and affinity

maturation and isotype switching of the B-cells by helper T-cells

humoral immunity

Microorganisms/foreign proteins

B-cell

Secrete antibodies

neutralization

phagocytosis


Influence factors of immunogenicity

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Structural factors Sequence variation glycosylation

Impurities Formulation Route of Administration Dose Patient features


Structural factors: sequence variation

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The type of proteins The mutation of proteins For insulin, some single mutations lead to

immunogenicity, while other mutations do not


Structural factors: glycosylation

47

Glycosylation reduces immune response Glycosylated β–interferon is less immunological than the

non-glycosylated Normally glycosylated epoetin had a lower affinity to

antibodies than the non-glycosylated


48

impurities

http://en.wikipedia.org/wiki/File:Anthrax_culture.jpghttp://en.wikibooks.org/wiki/Structural_Biochemistry/Proteins/Purification/Affinity_chromatographyhttp://www.glutenfreehelp.info/wp-content/uploads/2010/08/EnzymeExample.jpghttp://www.molprofiles.com/images/hero/FormulationDev.jpghttp://www.hemltd.ru

Cell debris

Enzyme to activate proteins

Formulation Resin Leak from container and sealing


formulation

49

Case 1: A freeze-dried drug (human serum albumin as stabilizer) at room temperature induces immune response Proteins are oxidized and from aggregates at r.t.

Case 2: Epoetin-α (Tween-80 as stabilizer) induces immune response, and causes anemia (贫血) While human serum albumin stablized Epoetin is fine Reason could be Tween-80 stablized formulation is less stable and has aggregates


route of administration

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SC route induces most serious immune response IV route induces least immune response Any route can induce immune response


dose

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The effect of dose is not quite clear Highest dose could lead to lowest immune

response


patient features

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Other treatment of patients Underlying disease of patients


Assay for antibodies

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Different assays on the same sera can have 50-fold difference

Lack of standardization of assay methodology


Assays for antibodies

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Many new assays need to be used together Two-tier (两层) approach Screening to Identify antibody positive sera Further analysis to determine If antibodies are

neutralizing and then titer, affinity and isotype.


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First tier: ELISA

Sandwich Ab-Ag-Ab


First tier for new proteins

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Define an absolute sensibility is impossible Set the cut-point for the assay at a 5% false-

positive level using a group of normal human seraTest say you don’t have it

Test say youdo have it

You really don’t have it

You really do have it

100dialysis.wordpress.com


Second tier: detect neutralizing antibodies

57

False negative: factors stimulating the bioassay may compensate for neutralization activity

Patient serum with and without IgG should be used as control To determine if neutralizing factors are from drugs or serum

Modified from http://www.tebu-bio.com/userfiles/image/ilite%20scheme.jpg

Add serum Add protein drugDetect neutralizingantibodies


Predicting immunogenicity is difficult

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No constant “sequence variation”-immunogenicity relationship A single amino acid change could make a protein

immunogenic Substantial divergence from the natural sequence

may have no effect


Method of predicting immunogenicity

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Simulation and computational models Only give limited information

Binding tests T-cell proliferation assay have false positive results

Many antibodies can activate T-cell or inhibit cell proliferation


Method of predicting immunogenicity

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For human homologues Check aggregates or impurities

Animal studies are not suitable All human proteins are in principle immunogenic to

animals Transgenic mice are OK


Reducing immunogenicity

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Change the amino acid sequence Link proteins to polymers such as PEG and low

MW dextran Immunosuppressive treatments Tolerance induction


Outline

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Contaminants

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Potential impurities present in biopharmaceutical products destined for parenteral administration.

http://tse3.mm.bing.net/th?id=OIP.M261ff433d94625c9750f4f2466804adfo0&pid=15.1http://ecoki.com/wp-content/uploads/virus.jpghttp://cn.bing.com/images/search?view=detailV2&ccid=JVOxyEXX&id=59D759567DF3467DE6C81B4880CC9197C9E83039&q=DNA&simid=608056199566461642&selectedIndex=0&ajaxhist=0http://tse3.mm.bing.net/th?id=OIP.wA0JpCCODW8d066ubwxMCAEsEs&pid=15.1


Source of protein contaminants

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From the source material (E. coli, CHO cells) From cell medium, such as bovine serum/fetal calf

serum (2–25%) and regulatory proteins From downstream processing, endonuclease to

break DNA From production personnel

http://munford5th.weebly.com/uploads/9/1/9/4/9194989/3917932_orig.jpghttp://science.howstuffworks.com/life/evolution/evolution2.htmhttps://openclipart.org/image/800px/svg_to_png/161839/cell-culture2.pnghttp://www.nature.com/nrm/journal/v2/n4/images/nrm0401_237a_i2.gif

E. coli CHO cells Cell medium endonuclease


Clinical significance of removing protein-based impurities

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Avoid potential biological activities Avoid antigenicity Reduce immune response


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Removal of protein contaminates

http://www.tissuegroup.chem.vt.edu/chem-ed/sep/lc/graphics/size-exc.gifhttps://www.labome.com/method/Protein-Purification.html

GPC

Remove 1. Aggregates of the product 2. proteolyzed forms of the

product

remove proteins1. with deamidation and oxidation2. With incorrect disulfide bond

formation3. With partial denaturation and

limited proteolysis

IEC Cationic Anionic

Less positively charged

More positively charged

Less negatively charged

More negatively charged


SDS-PAGE

67

Separation of proteins by SDS–PAGE (detection limit 100 ng protein)


2D gel electrophoresis

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Separate protein with same MW by 2D Gel electrophoresis

http://en.wikibooks.org/wiki/Proteomics/Protein_Separations-_Electrophoresis/Two_Dimensional_Polyacrylamide_Gel_Electrophoresis(2D-PAGE)


Western blot: sample transfer

69

Western blot Transfer proteins from gel to membrane

Emembrane

gel


Western blot: detecting the sample by antibody

70

Binding of the antibody of product to the ‘contaminant’ bands suggests that they are variants of the product

Ag antigen

Ab antibodyB biotin

SA streptavidin

E enzyme

S substrate

P product


Capillary Electrophoresis (CE)

71

separation occurs within a capillary tube

http://theses.ulaval.ca/archimede/fichiers/24330/apa.html

Diameter 20-50 mmLength: up to 1 m

in 15–30 min Advantage: the speed, sensitivity, high degree of automation and ability to directly quantify proteinbands


High-pressure liquid chromatography (HPLC)

72

Reverse phase, size exclusion and ion-exchange-based HPLC

http://www.laboratory-journal.com/applications/analytics/hplc-analysis

• excellent fractionation speeds (minutes per sample);

• superior peak resolution;• high degree of automation;• commercial available.


High-pressure liquid chromatography (HPLC)

73

Reverse phase HPLC (RP-HPLC) Mostly used HPLC to analyze proteins Principle: separate protein based on hydrophobicity Application: detect deamidated proteins and proteins with

only one amino acid substitution or removal Limitation: cause protein to denature due to the column

silica or polymer Size exclusion and ion-exchange-based HPLC Similar to RP HPLC Different separating column


Mass spectrometry

74

Measure molecular weight of protein Can detect proteins isoforms with only one amino acid difference Glycoproteins, having extremely complex spectra, are hard to analyze

Singly charged

doubly charged

accuracy ±0.01%


Immunological approaches to detection of contaminants

75

Nonself-protein from microbial or mammalian cell lines cause immune response

‘Blank run approach’

https://openclipart.org/image/800px/svg_to_png/161839/cell-culture2.pnghttp://res.cloudinary.com/dk-find-out/image/upload/q_80,w_960/MA_00766318_t4yw5n.png

Cells without target gene

Cell culture

Extraction and purification except last step of purification

Protein impurities

Inject to animals

Antibodies

Use to detect protein impurities


Outline

76





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Lipopolysacchraride (LPS)

http://microbeonline.com/lipopolysaccharide-lps-of-gram-negative-bacteria-characteristics-and-functions/

LPS in Gram negative bacteria causes fever (pyrogenicity), which is hard to cure

It is important to remove LPS from biopharmaceuticals

O-antigen is different for different Gram negative bacteria

Core polysaccharide is similar for different bacteria

Lipid A is associated with the pyrogenicity


Removal of LPS

78

GPC LPS in water form aggregates, with MW > 100-1000 kDa While most biopharmaceuticals have MW less than 100 kDa

IEC Since LPS is negatively charged

http://www.tissuegroup.chem.vt.edu/chem-ed/sep/lc/graphics/size-exc.gifhttps://www.labome.com/method/Protein-Purification.html

GPC IEC


Detection of LPS

79

Rabbit pyrogen test Measure the temperature change in three rabbits

after injection of test drug Negative (<1.15 oC), positive (>2.65 oC)

http://st.depositphotos.com/1765308/1274/i/950/depositphotos_12743051-Three-brown-rabbits.jpg

Inject drugs Measure temperature Negative <1.15 oCPositive >2.65 oC


Detection of LPS

80

The limulus amoebocyte lysate test (马蹄蟹变形细胞溶解物) Principle: LPS can result in the coagulation of the blood

cells (amoebocyte) of limulus Method: incubate limulus amoebocyte lysate with test

drug and check clotlimulus amoebocyte lysate

Inject drugs Check blotBlot, positiveWithout blot, negative

http://tse4.mm.bing.net/th?id=OIP.bCmj6sExiT-F71c0w_qEagEsEs&pid=15.1


Outline

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Removal of DNA contamination

82

Why is it necessary? Active onco-genes in producer cells (hybridoma cell

lines) may cause cancer Contaminated DNA should be less than 10 pg per

therapeutic dose

DNA contamination is little Nuclease will digest DNA during cell lysis IEC will remove residual DNA


83

Detection of DNA contamination: Dot-blot assay

https://openclipart.org/image/800px/svg_to_png/161839/cell-culture2.png

DNA extraction (phenol/chloroform extraction and ethanol precipitation)

DNA

Transfer to nitrocellulose filter paper

Denaturation

Labeled with 32P

32P 32P 32P

Measure radioactivity

Drugs


Outline

84





Removal of virus contamination

85

GPC MW of virus is different from biopharmaceuticals

Filtration 0.1 μm filtration

Ultrafiltration

Heat 40-60 oC for several hours

UV irradiation

http://www.milian-usa.com/images/normal/0B-20-04.jpg


Detection of virus contamination

86

Immunoassays

Assays based on viral DNA probes

Inject to animalsUse to detect virus

DNA extractionDenaturation

Virus DNA probe 32P

Drugs

32P32P

Negative

Positive


Detection of virus contamination

87

Bioassays

Drugs

Inject to animals

No antibodies Negative

Positive


Summary

88

Protein structure: primary structure, secondary structure and tertiary structure

Protein drug analysis: product potency, concentration determination, structure characterization, immunogenicity analysis

Contaminants: other proteins, LPS, DNA, virus


Documents

6-7 The drug manufacturing process Analysis of final productweb.xidian.edu.cn/yqxia/files/20170331_145235.pdf · 2017. 3. 31. · Product potency. 20. Bioassay. Analysis in a biological