05 - 07 Elelyso (Taliglucerase Alpha) Final Presentation

ALS 419: Chemistry, Manufacturing, Controls
Elelyso (Taliglucerase alfa)

Team 1
Alex Acosta, Uchechukwu Anyaduba, Cathy Pham, Diti Shah
May 07, 2020
1
Meet Regulatory the Team
Alex Acosta Uchechukwu Cathy Pham Diti Shah

Master of Business Anyaduba Master of Business Master of Business
and Science ‘20 Master of Business and Science ‘21 and Science ‘21
and Science ‘21
2
Executive Summary
Regulatory CMC
01 02 03 04
Introduction Drug Drug Product Appendices

Substance
Facilities and
About Protalix Characterization Manufacture
Equipments
About Gaucher’s Agents and

Manufacture Analytical
Disease Recipients
Information and
About Elelyso Analytical Stability References
POST- APPROVAL CHANGE 3

Executive Summary
Regulatory CMC
01
Introduction
About Protalix
About Gaucher’s
Disease
About Elelyso
4
About Protalix
Biopharmaceutical company Pipeline consists of

focused on the development, proprietary, potentially
production and commercialization clinically superior versions of
of recombinant therapeutic recombinant therapeutic
proteins produced by our proteins that target
proprietary ProCellEx® plant cell- established pharmaceutical
based protein expression system markets
5
● Genetic disorder in which
Gaucher’s glucocerebroside accumulates in cells
Disease (Gaucher cells) and specific organs
● Life long disease
● Diet and exercise combined with ERT,

patients live normal lives
6
● Drug type: Enzyme replacement
therapy (ERT)
Elelyso ● Indication: Gaucher's Disease
● Route of Administration: IV
7
Executive Summary
Regulatory CMC
01 02
Introduction Drug
Substance
About Protalix Characterization
About Gaucher’s
Manufacture
Disease
About Elelyso Analytical
8
Drug Substance
9
3.2.S. DRUG SUBSTANCE
3.2.S.1.1 GENERAL INFORMATION (Nomenclature)
3.2.S.1.2 GENERAL INFORMATION (Structure)
3.2.S.1.3 GENERAL INFORMATION (General Properties)
3.2.S.2.1 MANUFACTURE (Manufacturers)
3.2.S.2.2 MANUFACTURE (Description of Manufacturing and Manufacturing Controls)
10
3.2.S.1.1 GENERAL INFORMATION (Nomenclature)
● Taliglucerase alfa:
β-Glucocerebrosidase: β-D-glucosyl-N-acylsphingosine glucohydrolase, E.C. 3.2.1.45
● Trade Name- “Elelyso”
https://www.ema.europa.eu/en/documents/assessment-report/elelyso-epar-public-assessment-report_en.pdf
11
3.2.S.1.2 GENERAL INFORMATION (Structure)
● Protein chemical formula (Structure)
C2580H3918N680O727S17
● Purified taliglucerase alfa is a monomeric glycoprotein containing 4 N linked

glycosylation sites (Mr = 60,800)
● Taliglucerase alfa differs from native human glucocerebrosidase by two amino

acids at the N-terminal and 7 amino acids at the C-terminal. The molecular weight
of taliglucerase alfa protein is approximately 60,800 Daltons.
https://www.drugbank.ca/drugs/DB08876
https://www.ema.europa.eu/en/documents/assessment-report/elelyso-epar-public-assessment-report_en.pdf 12
:
Predicted Amino Acid Sequence for taliglucerase alfa:
https://www.tga.gov.au/sites/default/files/auspar-taliglucerase-alfa-rpc-141014-pi.pdf
13
Glycan Structures on Recombinant GCD enzyme Comparison of Glycans expressed in Mammalian cell line and Plant cell line
https://www.tga.gov.au/sites/default/files/aus https://link.springer.com/chapter/10.1007/10_2018_76
par-taliglucerase-alfa-rpc-141014-pi.pdf
14
3.2.S.1.3 GENERAL INFORMATION (General Properties)
● Accession Number
DB08876 (DB06667)
● Taliglucerase alfa is supplied as a sterile, non-pyrogenic, lyophilized product
● Taliglucerase alfa is a white to off-white lyophilised powder that may form a

cake.
https://www.tga.gov.au/sites/default/files/auspar-taliglucerase-alfa-rpc-141014-pi.pdf 15
3.2.S.2.1 MANUFACTURE (Manufacturers)
16
http://protalix.com/technology/procellex-platform/
https://www.elelyso.com/how-is-elelyso-mad
e 17
3.2.S.2.2 MANUFACTURE (Description of Manufacturing and Manufacturing

Controls)
18
https://www.ncbi.nlm.nih.gov/pubmed/26102075
https://www.ncbi.nlm.nih.gov/pubmed/21236330 19
Upstream Downstream Purification
Initial Production Step Final Steps
Process Process Process
Taliglucerase alfa is Expansion of the Extraction, The standard Filtered using a 0.2
purified from recombinant carrot Clarification and techniques for μm filter and filled
genetically modified cells and expression Purification of the protein purification into bottles with
carrot cells of taliglucerase alfa; taliglucerase alfa by chromatography HDPE screw
protein. on multiple columns closures, pre-
sterilized with
gamma irradiation
Prior to final formulation, the

bulk active substance may be
stored frozen for up to 24
months at -20 ± 5°C.
Taliglucerase alfa AS will be
shipped at not more than -15°C 20
Taliglucerase alfa is
purified from
genetically modified
carrot cells
21
https://europepmc.org/article/med/25621170
https://plantcellbiology.masters.grkraj.org/html/Genetic_Engineering4D-Transformation-Plant_Cells.htm 22
INITIAL CELL LINE
PRODUCTION
23
Expansion of the
recombinant carrot
cells and expression
of taliglucerase alfa;
24
25
CELL LINE
EXPANSION
OSMOTEK BAG BIOREACTOR
● Made up of a simple culture bag without any exterior
supporting vessel
● This system was developed originally by Osmotek LTD

(Rehovot, Israel) to grow tissues
● More recently has been modified by Protalix to

accommodate large-scale plant cell cultures by employing
an external open wire cage around the bag
● FDA-approved and successfully commercialized
27
Extraction, The standard

Clarification and techniques for
Purification of the protein purification
taliglucerase alfa by chromatography
protein. on multiple columns
28
29
Polishing
Size Exclusion Chromatography;
05 Ion Exchange Chromatography
Harvesting Cells
Cell Separator - separates cells and media
Intermediate Purification
.
01 04 Ion Exchange Chromatography
Hydrophobic Interaction Chromatography
Affinity Chromatography
02 03 Clarification, Pre-
Cell Disruption purification and Capturing
Crude Pressing-Homogenizer Ion Exchange Chromatography/
. Hydrophobic Interaction Chromatography
Filtered using a 0.2

μm filter and filled
into bottles with
HDPE screw
closures, pre-
sterilized with
gamma irradiation
Prior to final formulation, the

bulk active substance may be
stored frozen for up to 24
months at -20 ± 5°C.
Taliglucerase alfa AS will be
shipped at not more than -15°C 31
3.2.S.2.3 MANUFACTURE (Control of Materials)
3.2.S.2.4 MANUFACTURE (Control of Critical Steps and Intermediates)
3.2.S.2.5 MANUFACTURE (Process Validation and-or Evaluation)
3.2.S.3.1 CHARACTERIZATION (Elucidation of Structure and other Characteristics)
3.2.S.3.2 CHARACTERIZATION (Impurities)
3.2.S.4.1 CONTROL OF DRUG SUBSTANCE (Specifications)
32
3.2.S.2.3 MANUFACTURE (Control of Materials)
The corrective actions for

most parameters is described
CPPs/KPPs/IPCs
are well defined
Large number of parameters aimed

at preventing microbiological
contamination (for which the
corrective action is ‘rejection of
batch’)
33
3.2.S.2.4 MANUFACTURE (Control of Critical Steps and Intermediates)
Two
Critical
Process
Parameters
3.2.S.2.5 MANUFACTURE (Process Validation and-or Evaluation)

● Process validation studies:
- Cell Culture
-Harvesting
-Purification Operations of three consecutive taliglucerase
alfa active substance batches at commercial size.
● Improved validation studies for better control of the bioburden.
● The pool hold times of intermediates were validated.
3.2.S.3.1 CHARACTERIZATION (Elucidation of Structure and other Characteristics)
The structure had been evaluated using

● X-ray crystallography
● amino acid sequencing
● peptide mapping
● disulfide bond
● free sulfhydryl content analysis.
36
3.2.S.3.2 CHARACTERIZATION (Impurities)
● Product related substances (variants and impurities) and process

related impurities.
● Electrophoresis, RP-HPLC, capillary IEF, and Size

Exclusion Chromatography-purity of taliglucerase alfa and
characterize the product-related impurities.
● Process-related Impurities -Host Cell Protein and Host Cell

DNA are controlled at release
3.2.S.4.1 CONTROL OF DRUG SUBSTANCE (Specifications)
Property Method
Appearance, pH European Pharmacopoeia Standards
Identification Peptide Map, SDS-PAGE – Western blot analysis
Purity RP-HPLC, host cell protein by ELISA, automated electrophoresis,Capillary Isoelectric Focusing
Taliglucerase alfa content RP-HPLC
Specific Activity Enzymatic activity and taliglucerase alfa assessment determination by RP-HPLC absorption at 280 nm
Enzymatic Kinetics Michaelis-Menten kinetics
Glycan Structure Analysis NP-HPLC combined with glycosidase digestion
Cellular Uptake Cell based assay
Mannose Content Mass spectrometry
Total Aerobic Count ,Bacterial Endotoxin European Pharmacopoeia Standards
Residual DNA and Free sulfhydryl content Ellman’s reaction for thiol determination)
38
3.2.S.4.2 CONTROL OF DRUG SUBSTANCE (Analytical Procedures)
3.2.S.4.3 CONTROL OF DRUG SUBSTANCE (Validation of Analytical Procedures)
3.2.S.4.4 CONTROL OF DRUG SUBSTANCE (Batch Analyses)
3.2.S.4.5 CONTROL OF DRUG SUBSTANCE (Justification of Specification)
3.2.S.5 REFERENCE STANDARDS
3.2.S.6 CONTAINER CLOSURE
3.2.S.7.1 STABILITY (Summary and Conclusions)
3.2.S.7.2 STABILITY (Post Approval Stability Protocol and Stability Commitment)
3.2.S.7.3 STABILITY (Stability Data)

39
3.2.S.4.2 CONTROL OF DRUG SUBSTANCE (Analytical Procedures)
Principle/Sc Apparatus/Eq Operating Reagents/Sta Sample

ope uipment Parameters ndards Preparation
Standards System Data

Control Procedure Calculations
Suitability Reporting
Solution
Preparation 40
https://www.fda.gov/files/drugs/published/Analytical-Procedures-and-Methods-Validation-for-Drugs-and-Biologics.pdf
3.2.S.4.3 CONTROL OF DRUG SUBSTANCE (Validation of Analytical Procedures)
Typical validation characteristics are:

Linearity Accuracy
Specificity
Precision Range Quantitation limit
Detection limit
41
https://www.fda.gov/files/drugs/published/Analytical-Procedures-and-Methods-Validation-for-Drugs-and-Biologics.pdf
3.2.S.4.4 CONTROL OF DRUG SUBSTANCE (Batch Analyses)
128 batches.
128
Batches were analyzed
42
3.2.S.4.5 CONTROL OF DRUG SUBSTANCE (Justification of Specification)
Acceptable Specifications Tightened
Reviewing
43
3.2.S.5 REFERENCE STANDARDS
Elelyso
44
3.2.S.6 CONTAINER CLOSURE
3.2.S.7.1 STABILITY (Summary and Conclusions)
Reconstituted
taliglucerase alfa
finished product diluted
into infusion bags is
considered stable for at
least 18 hours at 2-8°C
Storage or temperature excursions of taliglucerase alfa

The finished product powder for Drug Product to -20°C,
solution for injection is stable when
stored at 5 ± 3°C for the shelf life of 24 the labelling will include the precaution of “Do not
months freeze”.
46
3.2.S.7.2 STABILITY (Post Approval Stability Protocol and Stability Commitment)
The post-approval stability protocol and the stability commitment have been provided and are considered
satisfactory. In accordance with EU GMP guidelines, any confirmed out-of-specification result of significant
negative trend should be reported to the rapporteur and EMA.
Important Identified Safety Concerns Important Potential Safety Concerns

● Hypersensitivity ● Immunogenicity
● Infusion-related reactions ● Off-label paediatric use
● Prolonged activated partial

thromboplastin time
● Elevated liver enzymes

47
3.2.S.7.3 STABILITY (Stability Data)
Duration (term) Temperature Period
Long Term -20 ± 5°C 12 months

-20°C ± 5°C 36 months
Short Term 5 ± 3°C 3 month
Proposed shelf life : 24 months at -20°C ± 5°C
48
Executive Summary
Regulatory CMC
01 02 03
Drug Drug Product

Introduction
Substance
About Gaucher’s
Disease
About Elelyso Analytical Stability
49
Drug Product
50
3.2.P.1 DRUG PRODUCT
3.2.P.2.1 COMPONENTS OF THE DRUG PRODUCT (Drug Substance, Excipients)
3.2.P.2.2 DRUG PRODUCT (Formulation Development, Overages, Physicochemical and Biological

Properties)
3.2.P.2.3 MANUFACTURING PROCESS DEVELOPMENT
3.2.P.2.4 CONTAINER CLOSURE SYSTEM
3.2.P.2.5 MICROBIOLOGICAL ATTRIBUTES
51
3.2.P.2.1 COMPONENTS OF THE DRUG PRODUCT (Drug Substance, Excipients)
Quantity(mg)/
Ingredient 200mL Vial Function Quality
Drug Substance
Taliglucerase alfa 212 active ingredient Manufacturer's specification
Excipients
Mannitol 206.7 tonicity-adjusting agent USP
Polysorbate 80 0.56 stabilizer USP
Sodium citrate* 30.4 buffering agent USP
Citric acid** - pH adjustment USP
Water for Injection*** N/A diluent prior to lyophilization USP
Composition of Elelyso
*Introduce during manufacturing of Drug Substance
**To adjust pH to 6; amount depends on pH of ingredient 52
***Removed from finished product during lyophilization
3.2.P.2.2 DRUG PRODUCT (Formulation Development, Overages, Physicochemical
and Biological Properties)
Physicochemical and
Formulation Development: Overages: Biological Properties:
Taliglucerase alfa - Drug Substance - ● Sodium citrate and citric acid

● a hydrolytic lysosomal ● 12 units overfill (6%) of (pH adjustment to 6)
glucocerebroside-specific Taliglucerase alfa ● Mannitol is added to adjust the tonicity
enzyme ● Polysorbate 80 for stabilizing
● recombinant active form of
the lysosomal enzyme, β-
glucocerebrosidase
● expressed in genetically
modified carrot plant root
cells
● cultured in a disposable
bioreactor system
(ProCellEx®)
53
3.2.P.2.3 MANUFACTURING PROCESS DEVELOPMENT
Thawing of Drug 05
Fill and Finish
Substance
01 04
Sterile Filtration
Separation
02 03
Purification
54
3.2.P.2.4 CONTAINER CLOSURE SYSTEM
Protects from light Protect from solvent leakage protects sterile products
55
3.2.P.2.5 MICROBIOLOGICAL ATTRIBUTES
Purification Filling
56
3.2.P.2.6 COMPATIBILITY
3.2.P.3.1 MANUFACTURERS
3.2.P.3.2 BATCH FORMULA
3.2.P.3.3 DESCRIPTION OF MANUFACTURING PROCESS AND PROCESS CONTROLS
3.2.P.3.4 CONTROLS OF CRITICAL STEPS AND INTERMEDIATES
3.2.P.3.5 PROCESS VALIDATION AND-OR EVALUATION
57
3.2.P.2.6 COMPATIBILITY
As of March 12, 2020:
In the absence of compatibility studies, ELELYSO should not be mixed with other
medicinal products, except those mentioned in the DOSAGE AND ADMINISTRATION
section.
58
3.2.P.3.1 MANUFACTURERS
Pfizer Global Manufacturing, Pharmacia and Upjohn Company LLC,

7000 Portage Road, Kalamazoo, MI 49001, USA
59
3.2.P.3.2 BATCH FORMULA
Sterile Fluid Path Component Quantity per compounding Quality

Mixture of Taliglucerase alfa 200 U/vial; Mannitol
200mg/vial; Polysorbate 80 .56mg/vial; Sodium Nitrate Manufacturer's
30.4mg/vial 475 mg specification
Citric acid** - USP
Water for Injection*** 100mL USP
**To adjust pH to 6; amount depends on pH of ingredient

To take account of the overage and the addition of citric acid for
adjusting the pH
60
3.2.P.3.3 DESCRIPTION OF MANUFACTURING PROCESS AND PROCESS CONTROLS
61
3.2.P.3.4 CONTROLS OF CRITICAL STEPS AND INTERMEDIATES
2 x
Purification Process
62
3.2.P.3.5 PROCESS VALIDATION AND-OR EVALUATION
63
3.2.P.4.1 CONTROL OF EXCIPIENTS (Specifications)
3.2.P.4.2 CONTROL OF EXCIPIENTS (Analytical Procedures)
3.2.P.4.3 CONTROL OF EXCIPIENTS (Validation of Analytical Procedures)
3.2.P.4.4 CONTROL OF EXCIPIENTS (Justification of Specifications)
3.2.P.4.5 CONTROL OF EXCIPIENTS (Excipients of Human or Animal Origin)
3.2.P.4.6 CONTROL OF EXCIPIENTS (Novel Excipients)
3.2.P.5.1 SPECIFICATIONS
3.2.P.5.2 ANALYTICAL PROCEDURES
64
3.2.P.4.1 CONTROL OF EXCIPIENTS (Specifications)
Included within the 200 unit(s) vial of the finished product, there are four excipients:
Quantity(mg)/
Ingredient 200mL Vial Function Quality
Excipients
Mannitol 206.7 tonicity-adjusting agent USP
Polysorbate 80 0.56 stabilizer USP
Sodium citrate* 30.4 buffering agent USP
Citric acid** - pH adjustment USP
Water for Injection*** N/A diluent prior to lyophilization USP
65
3.2.P.4.2 CONTROL OF EXCIPIENTS (Analytical Procedures)
● Infrared spectroscopy
● Ultraviolet spectroscopy
● Thin-layer chromatography
66
3.2.P.4.3 CONTROL OF EXCIPIENTS (Validation of Analytical Procedures)
1. Gas Chromatography
2. Liquid Chromatography
67
3.2.P.4.4 CONTROL OF EXCIPIENTS (Justification of Specifications)
The excipient is close to 100% purity, it is often necessary to widen the acceptance criteria
to “NLT 98.0% and NMT 102.0%”, which is typical for chromatographic procedures.- USP-
NF
Both Infrared spectroscopy ultraviolet spectroscopy should yield that all four known
excipients fall into the 98- 102 % purity range which is acceptable as outline in the USP-
NF.
68
3.2.P.4.5 CONTROL OF EXCIPIENTS (Excipients of Human or Animal Origin)
69
3.2.P.4.6 CONTROL OF EXCIPIENTS (Novel Excipients)
No novel excipients are found in the manufacturing of ELELYSO drug product.
70
3.2.P.5.1 SPECIFICATIONS
Quantity(mg)/
Ingredient 200mL Vial Characterization Quality
Excipients
Mannitol 206.7 odorless white crystalline powder USP
Polysorbate 80 0.56 viscous, water-soluble yellow liquid USP
Sodium citrate* 30.4 white, crystalline powder USP
odorless and colorless crystals with an acidic
Citric acid** - taste USP
71
3.2.P.5.2 ANALYTICAL PROCEDURES
The specific assays in which Protalix used for its analytical procedures were:
1. Cellular uptake assay

2. Moisture content specification
3. iCE assay
4. Mass spectrometry
72
3.2.P.5.3 VALIDATION OF ANALYTICAL PROCEDURES
3.2.P.5.4 BATCH ANALYSES
3.2.P.5.5 CHARACTERIZATION OF IMPURITIES
3.2.P.5.6 JUSTIFICATION OF SPECIFICATIONS
3.2.P.6 REFERENCE STANDARDS OR MATERIALS
3.2.P.7 CONTAINER CLOSURE
3.2.P.8.1 STABILITY SUMMARY AND CONCLUSIONS
3.2.P.8.2 POST APPROVAL STABILITY PROTOCOL AND STABILITY COMMITMENT
3.2.P.8.3 STABILITY DATA
73
3.2.P.5.3 VALIDATION OF ANALYTICAL PROCEDURES
The applicant has provided acceptable data for the transfer of eight release and stability
assays.
For the cellular uptake assay limited data in relation to the transfer of this assay to the facility
at Pfizer Ireland Pharmaceuticals, Grange Castle was provided, however this was deemed
acceptable, A revised specification range should be introduced after release testing with the
improved assay of at least 30 batches of drug substance and 15 batches of the finished
product manufactured using the commercial process.
74
3.2.P.5.4 BATCH ANALYSES
Due to the nature of non-viral threat from mammalian cells with the plant-cell based
ProCellEx platform, all assessments of available information for ELELYSO batch analysis are
thought to be conducted on a periodic basis (Skip Testing) for the 128 batch analyzation as
outlined in ICH Q6A.
75
3.2.P.5.5 CHARACTERIZATION OF IMPURITIES
The purity of the active substance is estimated by a combination of methods. The choice of
analytical procedures is focused on the separation of the taliglucerase alfa product from product
related substances (variants and impurities) and from process related impurities.
The quantitative composition of the lyophilized drug product is as follows: a 200 unit vial is
composed of taliglucerase alfa (212 units, which allows for a withdrawal of 200 units), mannitol
(206.7 mg), sodium citrate (30.4 mg) and Polysorbate 80 (0.56 mg). Citric acid anhydrous to
adjust pH.
76
3.2.P.5.6 JUSTIFICATION OF SPECIFICATIONS
Process validation was performed at the commercial manufacturing site for the taliglucerase
alfa active substance process, Protalix (Carmiel, Israel), according to process validation
protocols.
Assays, including electrophoresis, RP-HPLC, capillary IEF, and Size Exclusion
Chromatography are performed to assure the purity of taliglucerase alfa and characterize the
product-related impurities. Process-related Impurities such as Host Cell Protein and Host Cell
DNA are controlled at release, while clearance of other potential process-related impurities has
been demonstrated.
77
3.2.P.6 REFERENCE STANDARDS OR MATERIALS
ICH guidances (e.g., Q3A(R) Impurities in New Drug Substances, Q3C Impurities: Residual
Solvents, and Q6A Specifications: Test Procedures and Acceptance Criteria for New Drug
Substances and New Drug Products: Chemical Substances)
78
3.2.P.7 CONTAINER CLOSURE
ELELYSO (taliglucerase alfa), is supplied as a sterile, non-pyrogenic, white to off-white

lyophilized product for intravenous infusion.
ELELYSO is supplied in Type 1 glass vials packaged in single vial cartons.
79
Lyophilized vial: Store ELELYSO at 2° - 8 °C and protect vials from light. Reconstituted and
diluted solution for infusion: After reconstitution, promptly dilute vial. The reconstituted solution
may be stored for up to 12 hours at room temperature OR for up to 24 hours at 2 to 8°C,
protected from light. The diluted solution may be stored for up to 24 hours at 2 to 8°C, protected
from light. The medicinal product should be used immediately. If not used immediately, in-use
storage times and conditions prior to use are the responsibility of the user.
adjust pH.
80
3.2.P.8.2 POST APPROVAL STABILITY PROTOCOL AND STABILITY COMMITMENT
No post approval stability protocol and stability commitment were performed.
81
If not used immediately, Taliglucerase alfa finished product powder for solution for injection is
stable when stored at 5 ± 3°C for the shelf life of 24 months. The applicant provided the results
of the primary stability studies after 24 months storage, including data for eleven batches of
which three were full scale validation batches.
Only up to 9 month of stability data was available at long term condition (5 ± 3°C) for the full
scale batches but the stability data of the primary stability batches (up to 36 months) were
considered to be supportive to the proposed shelf-life
82
Stability data which was available showcased that the finalized product supplied evidence that
ELELYSO would have a shelf life of 24 months when stored at 5 ± 3°C.
Based on the evaluation of two batches of pooled reconstituted finished product diluted with
saline solution in infusion bags, reconstituted taliglucerase alfa finished product diluted into
infusion bags is considered stable for at least 18 hours at 2-8°C.
83
From the stability studies, 18 of the 26 adults who completed the 9 month clinical trial
extension, as an open-label extension trial with an additional 27 months. Patients maintained
stability within the clinical parameters of:
● Spleen volume
● Liver Volume
● Platelet count
● Hemoglobin
10 of the 18 patients completed the 27 month extension of Elelyso with 7 of these patients
seeing a reduction in spleen and liver volume for a total of 36 months
84
Executive Summary
Regulatory CMC
01 02 03 04

Substance
Facilities and
Equipments

Disease Recipients
Post-Approval
About Elelyso Analytical Stability Change
85
Appendices
86
3.2.A APPENDICES
3.2.A.1 FACILITIES AND EQUIPMENT
3.2.A.2 ADVENTITIOUS AGENTS SAFETY EVALUATION
3.2.A.3 NOVEL EXCIPIENTS
3.2.R.1 REGIONAL INFORMATION
3.2.R.2 METHODS VALIDATION PACKAGE
87
3.2.A APPENDICES
● Protalix does all manufacturing within a 20,000 sq/ft facility.

○ Additional 48,000 sq/ft (labs, storage, & offices)
88
3.2.A APPENDICES
Step 1: Cell bank thawing and recovery
● Single use cryovials

● Industrial sized cryo-freezers (N2 cooled)
89
3.2.A APPENDICES
Step 2: Scale up to 800 L disposable bioreactors
● Estimate 20-30 800 L disposable bioreactors
90
3.2.A APPENDICES
Step 3: Protein expression vis ProCellEx system
Step 4: Cell harvest and separation

● Depth separation (via centrifuge)
● Filtration (via vacuum filtration)
91
3.2.A APPENDICES
Step 5: Cell mass re-suspension in extraction buffer
Step 6: Filter press and supernatant collection

● Plate filter in tandem with centrifuge
92
3.2.A APPENDICES
Step 7: Column chromatography
● No chemical consumption
● Low operating costs
● 24/7 operation
93
3.2.A APPENDICES
3.2.A.2 ADVENTITIOUS AGENTS SAFETY EVALUATION
With the use of a plant cell line, viral contamination is a non-factor when considering the safety
evaluation with respect to users of ELELYSO.
Also for consideration, plant viruses are again considered a non-factor as no plant virus has
been link to infect mammalian cells.
94
3.2.A APPENDICES
3.2.A.3 NOVEL EXCIPIENTS
No novel excipients are found in the manufacturing of ELELYSO drug product.
95
3.2.A APPENDICES
3.2.R Regional
All ELELYSO product is manufacturing within Protalix’s only plant which is located in
Carmeil, Israel.
96
3.2.A APPENDICES
3.2.R.1 REGIONAL INFORMATION
Being that Protalix has noted that all materials they recieve for the manufacturing process are
sourced from one supplier, which has been validated through the FDA. Regional information on
materials has not been provided.
97
3.2.A APPENDICES
3.2.R.2 METHODS VALIDATION PACKAGE

ELELYSO (taliglucerase alfa), is supplied as a sterile, non-pyrogenic, white to off-white
lyophilized product for intravenous infusion.
adjust pH. ELELYSO is supplied in Type 1 glass vials packaged in single vial cartons
98
Executive Summary
Regulatory CMC
01 02 03 04

Substance
Facilities and
Equipments

Disease Recipients
Information and
About Elelyso Analytical Stability References
POST- APPROVAL CHANGE 99

Post Approval Change
Proposed Change
100
https://onlinelibrary.wiley.com/doi/epdf/10.1002/biot.201300548
Data Justification
pH STAR AX Additional Purification Conclusion
Effectiveness of Beta- A salt-tolerant anion Additional Steps of Implementation of

glucocerebrosidase is exchange resin which is buffer exchange STAR AX resin will
maximized at pH 5.5 since compatible with plant followed by result in higher amount
native isoelectric point is pH extracts that have high Hydrophobic interaction of target protein in the
6.0 salt concentrations. chromatography and Downstream
Removes initial sulfopropyl Purification Process
conditioning steps
101
Economic Rationale
Ideal purification No need of Resulting in the host cell protein

process would be (HCP) - glucocerebrosidase in
able to capture buffer carrots - to fall into the flow-
through fraction
target proteins exchange
102
Change Risk Assessment
STAR AX STAR AX STAR AX STAR AX
Flow-through mode Capacity Acidic proteins Volume

Extremely effective Must be reevaluated like Having a large
in separating the glucocerebrosidase, column volume will
target protein from having a pH of 5.5, help with the
the HCP, in carrots; require a large differentiation
column volume in
order to capture it
103
Sections of CTD Affected
1. Section 3.2.P.3.3, titled DESCRIPTION OF MANUFACTURING

PROCESS AND PROCESS CONTROLS,
2. 3.2.S.2.2 MANUFACTURE (Description of Manufacturing and
Manufacturing Controls),
3. 3.2.S.2.4 MANUFACTURE (Control of Critical Steps and
Intermediates)
104
QUESTIONS?
105

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ALS 419: Chemistry, Manufacturing, Controls

Elelyso (Taliglucerase alfa)

Alex Acosta, Uchechukwu Anyaduba, Cathy Pham, Diti Shah

May 07, 2020

Alex Acosta Uchechukwu Cathy Pham Diti Shah

Introduction Drug Drug Product Appendices

About Gaucher’s Agents and

POST- APPROVAL CHANGE 3

Biopharmaceutical company Pipeline consists of

● Life long disease

● Diet and exercise combined with ERT,

About Protalix Characterization

About Elelyso Analytical

3.2.S.1.1 GENERAL INFORMATION (Nomenclature)

3.2.S.1.2 GENERAL INFORMATION (Structure)

3.2.S.1.3 GENERAL INFORMATION (General Properties)

3.2.S.2.1 MANUFACTURE (Manufacturers)

3.2.S.2.2 MANUFACTURE (Description of Manufacturing and Manufacturing Controls)

3.2.S.1.1 GENERAL INFORMATION (Nomenclature)

● Trade Name- “Elelyso”

3.2.S.1.2 GENERAL INFORMATION (Structure)

● Protein chemical formula (Structure)

● Purified taliglucerase alfa is a monomeric glycoprotein containing 4 N linked

● Taliglucerase alfa differs from native human glucocerebrosidase by two amino

Predicted Amino Acid Sequence for taliglucerase alfa:

3.2.S.1.3 GENERAL INFORMATION (General Properties)

● Taliglucerase alfa is supplied as a sterile, non-pyrogenic, lyophilized product

● Taliglucerase alfa is a white to off-white lyophilised powder that may form a

3.2.S.2.1 MANUFACTURE (Manufacturers)

3.2.S.2.2 MANUFACTURE (Description of Manufacturing and Manufacturing

Prior to final formulation, the

● This system was developed originally by Osmotek LTD

● More recently has been modified by Protalix to

● FDA-approved and successfully commercialized

Extraction, The standard

Filtered using a 0.2

Prior to final formulation, the

3.2.S.2.3 MANUFACTURE (Control of Materials)

3.2.S.2.4 MANUFACTURE (Control of Critical Steps and Intermediates)

3.2.S.2.5 MANUFACTURE (Process Validation and-or Evaluation)

3.2.S.3.1 CHARACTERIZATION (Elucidation of Structure and other Characteristics)

3.2.S.3.2 CHARACTERIZATION (Impurities)

3.2.S.4.1 CONTROL OF DRUG SUBSTANCE (Specifications)

3.2.S.2.3 MANUFACTURE (Control of Materials)

The corrective actions for

Large number of parameters aimed

3.2.S.2.4 MANUFACTURE (Control of Critical Steps and Intermediates)

3.2.S.2.5 MANUFACTURE (Process Validation and-or Evaluation)

● Improved validation studies for better control of the bioburden.

● The pool hold times of intermediates were validated.

3.2.S.3.1 CHARACTERIZATION (Elucidation of Structure and other Characteristics)

The structure had been evaluated using

3.2.S.3.2 CHARACTERIZATION (Impurities)

● Product related substances (variants and impurities) and process

● Electrophoresis, RP-HPLC, capillary IEF, and Size

● Process-related Impurities -Host Cell Protein and Host Cell

Appearance, pH European Pharmacopoeia Standards

Identification Peptide Map, SDS-PAGE – Western blot analysis

Taliglucerase alfa content RP-HPLC

Enzymatic Kinetics Michaelis-Menten kinetics

Glycan Structure Analysis NP-HPLC combined with glycosidase digestion

Cellular Uptake Cell based assay

Mannose Content Mass spectrometry