role of equipment in the manufacturing of pancreatic … · §standardization of standard operating...

ROLE OF EQUIPMENT IN THE MANUFACTURING OF PANCREATIC ISLETS

Elina Linetsky, M.Sc., MTUM cGMP Facility, Cell Transplant Center

University of Miami Miller School of Medicine

Produce insulin

Glucose sensors

Release insulin when

needed

Maintain Maintain normoglycemianormoglycemia

Why Islets?

InsulinGlucagonSomatostatin

Produce glucagon

Micro-organs

PANCREAS AND ISLETS OF LANGERHANSPANCREAS AND ISLETS OF LANGERHANS

FDA does not regulatetransplantation of whole

vascularized organs

Tx of islet cells meet criteria for regulation

as a biologic

ØBiologic Product according to Section 351 of PHSA

ØSomatic cell therapy according to FDA • Not a “practice of medicine”• Considered experimental in the U.S.• Clinical studies must be performed under IND (21

CFR Part 312) or BLA (21 CFR Part 600, 601 & 610)

ØDrug according to Federal Food, Drug and Cosmetic Act

Weber DJ, et al. Transplantation 2002; 74(12): 1816-1820.

Pancreatic Islet Cells

BLA RequirementsPrior to marketing the product as therapy for Type 1 diabetic patients the following need to be demonstrated by the manufacturer:

ØSafety (sterility)ØPurity (Endotoxin)ØPotency (viability, dose, stability,

composition & biological activity)ØEffectiveness (reproducibility, consistency)

Wonnacott K. American Journal of Therapeutics 2005; 12: 600-604.

BLA RequirementsComplex nature of pancreatic islets (final product)

Limited ability to characterize final product prior to administration into a patient

Control of the Source Material Control of the Manufacturing Process

Regulatory Requirements1. Current Good Manufacturing Practices

(cGMP’s, 21 CFR Part 210 & 211)Ø minimum requirements of any manufacturing processØ Equipment (qualification, validation, & calibration)Ø Control of components (qualification)§ Receipt and storage§ Testing, approval and use

Ø Process and product control (standardization, testing)

2. Current Good Tissue Practices(cGTPs, 21 CFR Part 1271)Ø Govern donor eligibility rules and other tissue practicesØ Prevention of introduction, transmission and spread of

communicable diseases§ HIV 1 & 2, HBV, HCV, TSE, Treponema pallidum, HTLV I/II§ Donor qualification (social, medical history & physical

examination)

Regulatory Requirements3. Biologic Products

(21 CFR Part 600, 601 & 610)Ø govern standards for biologicsØ Product testing for

ü Sterility, identity, purity & potency Ø Product licensure

4. Standards For Cellular Therapy Product Services, 2nd Ed.Ø Quality SystemsØ Safety of

§ Procurement§ Processing§ Storage§ Administration

5. FACT-JACIE International Standards for Cellular Therapy Product Collection, Processing and Administration, 3rd Ed.

Evolution of islet isolation process…

… is a story of imagination & innovation

Initial attempts to isolate islet cells from a donor pancreas involved crude & disruptive mechanical methods.

Can Glucose Stimulate Insulin Release “In Vitro”?

Islet Isolation and TransplantationIslet Isolation and Transplantation

1967 Lacy islet isolation using intraductal distention

1969 Lindall Islet purification by density gradients

1981 Horaguchi collagenase via duct perfusion

1984 Gray large scale isolation of human islets

Scientific Concerns

Distention, Digestion, Dilution & Purification Purified Islets (shipment)

Donor Pancreas

vConcern: control of final product

vConcern: control of source materialDonor selection & testingOrgan preservation

vConcern: process control

Required Equipment (general)Required Equipment (general)

•• BSCBSC’’ss•• COCO22 incubatorsincubators (37(37°° & 22& 22°° C)C)•• RefrigeratorsRefrigerators•• FreezersFreezers•• Refrigerated centrifuges Refrigerated centrifuges •• Inverted microscopeInverted microscope

(Light/fluorescent & camera)(Light/fluorescent & camera)•• TT°° Monitoring SystemMonitoring System•• Peristaltic PumpsPeristaltic Pumps

Required Equipment (general)Required Equipment (general)•• DensitometerDensitometer•• WaterbathsWaterbaths (37(37°° & 45& 45°°))

•• Automatic Pipette AidsAutomatic Pipette Aids•• BalancesBalances•• pH meterpH meter•• Computer & printerComputer & printer•• Heat sealerHeat sealer•• MicroplateMicroplate reader reader (fluorescent & (fluorescent &

spectrophotometricspectrophotometric))

•• Manual cell counterManual cell counter•• Glass washerGlass washer•• AutoclaveAutoclave

Islet Isolation Islet Isolation –– SpecificSpecificEquipmentEquipment

•• Organ transport set (oxygenated PFC/UW)Organ transport set (oxygenated PFC/UW)•• Continuous pancreas perfusion apparatus Continuous pancreas perfusion apparatus

(cold/warm) & perfusion tray(cold/warm) & perfusion tray•• Chamber, screen & hollow stainless steel beadsChamber, screen & hollow stainless steel beads•• Ricordi isolator with heating, cooling, pressure Ricordi isolator with heating, cooling, pressure

control, and flow & pH monitoring capabilitiescontrol, and flow & pH monitoring capabilities•• COBE 2991 Cell ProcessorCOBE 2991 Cell Processor•• Islet shipping containerIslet shipping container•• Ricordi infusion bagRicordi infusion bag

•Reduce variability•Simplify process

Manufacturing ControlsControl of Source Material

Ø Source material – pancreata, from deceased heart-beating donors

Ø Can not be controlled in a traditional sense Ø Establishment of acceptance criteria (both inclusion &

exclusion)§ Age: 15 – 65 years old§ Acceptable cause of death§ CIT: ≤ 12 hrs§ Cold storage: UW, UW/PFC, HTK, HTK/PFC§ Acceptable physical examination, social & medical history§ Negative for HIV 1 & 2, HBV, HCV, TSE, Treponema

pallidum, HTLV I/II, WNV

Ice

PFC

EC or UW

Outlet Inlet: O2 95%CO2 5%

Pancreas

Oxygenated PFC/UW TransportOxygenated PFC/UW Transport

Islet Isolation Islet Isolation –– SpecificSpecificEquipmentEquipment

Matsumoto I. World Journal of Surgery 1996; 20(8): 1030-1034

Transplantation 2003; 75(9): 1524-7

Improved human islet isolation outcome from marginal donors following addition of oxygenated perfluorocarbon to the cold-storage solution. Ricordi C, Fraker C, Szust J, Al-Abdullah I, Poggioli R, Kirlew T, Khan A & Alejandro R.

Group N AGE CITPFC+UW* 15 56.7±5.4 7.6±5.5UW 18 55.9±4.2 8.4±3.2

IEQ

* 8 preparations transplanted1 patient off insulin after receiving a single islet preparation

0

50,000

100,000

150,000

200,000

250,000

300,000

350,000

PFC+UW UW

Two-Layer Method

All Organs Count!!!All Organs Count!!!

Manufacturing ControlsProcess Controls

(goal: to optimize & standardize the manufacturing process)

Ø Control of raw materials through reagent qualificationØ Control of process, according to cGMP§ Critical process controls§ Standardization of standard operating procedures (SOP)

Ø Tracking of final product to donor organ / tissueØ Qualification and validation of equipmentØ Lot-to-lot reproducibility§ In-process specifications § Final product specifications (lot release criteria)

Continuous Pancreas Perfusion SystemContinuous Pancreas Perfusion System

Islet Isolation Islet Isolation –– Specific EquipmentSpecific Equipment

2007Prior to 2007•Touch-screen controls•Automatically pumps fluid at a constant pressure•Automatic adjustment of the flow rate to maintain constant pressure • Automatic T° control•Capacity to download data to PC with Perfusion Data Acquisition software

The Ricordi Chamber


Reusable Ricordi Chamber•Ultem Polyetherimide - tough, rigid biocompatible plastic of superior thermostatic strength• Resistant to high T°, can be repeatedly sterilized •Comes with stainless steel screen & O-ring

Disposable Ricordi Chamber•Durastar – clear, tough, chemically resistant plastic•Clamp replaces stainless steel screws•Sold individually packed & autoclaved•Comes with stainless steel screen & O-ring

After 2006

•New generation of shakers•Touch-screen user interface•Controlled heating & cooling•Capacity to monitor T°, flow rate, pH & shaking parameters (stroke length, frequency of vertical and rotational strokes)

•Data saved on a memory card

Ricordi IsolatorRicordi Isolator

Cell Transplantation 2004; 13(5): 497-502.

Multiparametric Monitoring of the Islet Isolation Procedure: A Potential Tool for In-process Corrections of Critical Physiological Factors. Fraker C, Montelongo J, Szust J, Khan A and Ricordi C.

HP 1478 5-20-03

Time (minutes)0 20 40 60 80

pH

6.046.086.126.166.246.286.326.366.446.486.526.566.646.686.726.766.846.886.926.967.047.087.127.167.247.287.327.367.447.487.527.567.647.687.727.76

6.00

6.20

6.40

6.60

6.80

7.00

7.20

7.40

7.60

7.80

X

XXXXX

XXXXXXXX

XX

XXXXXXXXXXXXXXXXXX

XXXXXX

XXXXX

XXXXXXXXXXXXXXXXXXXXXXXX

pCO

2 (m

m H

g)

0

20

40

60

pO2

(mm

Hg)

0

50

100

150

200

250

300

Tem

pera

ture

(C)

0

10

20

30

40

pHX

pCO2 pO2 Temp

Diabetes 1997; 46: 1120-1123

Improved Human Islet Isolation Using a New Enzyme Blend, Liberase TM.Linetsky E, Bottino R, Lehmann R, Alejandro R, Inverardi L, Ricordi C.

0

50,000

100,000150,000

200,000

250,000

300,000

350,000

400,000

450,000

500,000

IN IEQ

Liberase Collagenase

•Reduced Endotoxin•Improved islet yields

NewNew

OldOld

•Low Endotoxin•Improved islet quality•GMP grade

•Comparable islet yield

Transplantation 2006; 79: 91-97

Assessment of a Novel Two-Component Enzyme Preparation for Human Islet Isolation and Transplantation.Bucher P, Mathe Z, Morel P, Bosco D, Andres A, Kurfuest M, Friedrich O, Raemsch-Guenther N, Buhler LH, and Berney T

Liberase HICollagenase NB1

Profound Degradation of Serva’s Collagenase NB1 vs. Roche’s Liberase HI Revealed by Ion-Exchange HPLC on

ProteinPak DEAE 5PW Column in Imidazole.HCl Gradient Buffer System at pH 6.3

Liberase HI Collagenase NB1Collagenase II

38.5%

Collagenase Ia

37.1%

Collagenase Ib

8.8% Collagenase IIa

19.9%

Collagenase IIb

24.4%

Collagenase Ia

10.0%

Collagenase Ib

35.5%

Roche’s Thermolysin vs. Serva’s Neutral Protease: Homogeneityand Trio-teeth as a Result of Separation by Ion-Exchange

HPLC on ProteinPak DEAE 5PW Column in L-Histidine.HClGradient Buffering System at pH 5.5

Thermolysin Neutral Protease NPSingle Peak 90.2% 1st Peak 22.2%

2nd Peak 29.3%

3d Peak 20.2%


COBE 2991 Cell ProcessorCOBE 2991 Cell Processor•FDA approved Class II device (# BK840009, cleared Date: 08-JUN-1984 •Used as a large capacity centrifuge, @ 4°C•Operated in a semi-automated fashion with continuous Ficoll gradients

American Journal of Transplantation 2005; 5(1): 21-30

Rescue purification maximizes the use of human islet preparations for transplantation.Ichii H, Pileggi A, Molano RD, Baidal DA, Khan A, Kuroda Y, Inverardi L, Goss JA, Alejandro R and Ricordi C.

23

56789

10111213141516171819

2021

2223

2524

1

4

0 5,000 10,000 15,000 20,000

Recipient No.

CGP-isletsRGP-islets

Pre-purification

(IEQ/kg r.b.w.)

Specifications for In-Process TestingØIdentity (visual inspection using DTZ)ØPotencyüInsulin release assay: ≥ 1üIslet quantity: ≥ 5,000 IEQ/kg, üViability: ≥ 70%üCellular composition and β-cell fractional viability

ØPurity: ≥ 30%ØSafety (sterility testing for aerobic, anaerobic and

fungal organisms: no growth)

Manufacturing Controls

American Journal of Transplantation 2005; 5: 1635-1645

A novel method for the assessment of cellular composition and beta-cell viability in human islet preparations.Ichii H, Inverardi L, Pileggi A, Molano RD, Cabrera O, Caicedo A, Messinger S, Kuroda Y, Berggren P-O and Ricordi C.

Non-ReversalReversal

0.10 0.20 0.30 0.40 0.50 0.60ß-cell viability index

100%69%30%0%Reversal rate23163013No.

0.4 < x 0.3 < x ≤ 0.40.2 < x ≤ 0.3x ≤ 0.2Index

β-cell composition (%)NG

7AAD

β-cells viability (%)

dead cells (%)

TMRE

TMRE

Other cellsviability (%)

90%

Relationship between β-cell viability index and in vivo transplantation success rate (reversal rate).

x = ß-cell content in islet x ß-cell viability

prospectively assess and correlate β-cell mass and fractional viability of an islet product to its functional performance

Islet Shipping Device


•Insulated plastic coolers•Several room T° Sebra gel packs on the bottom / sides•One bag / metal shelf (x3) in the device•Cells are in gas permeable bags•T° monitoring devices enclosed•Shipping T° is room T°•Cooler sealed with tape & transported upright•Containers validated

American Journal of Transplantation 2007; 7(4): 1010-20

Shipment of human islets for transplantation. Ichii H., Sakuma Y., Pileggi A., Fraker C., Alvarez A., Montelongo J., Szust J., Khan A., Inverardi L., Naziruddin B., Levy M.F., Klintmalm G.B., Goss J.A., Alejandro R. and Ricordi C.

Centralized ICPC

-Islet isolation -Islet purification -Islet assessment-Islet shipment

RemoteCITP

-Pancreas recovery-Pancreas shipment-Islet assessment

-Islet transplantation

Control ConicalTube

Gas permeable

Bag

0

5

10

15

20 **

IEQ

(x10

3 )

0

20

40

60

β-ce

ll co

nten

t in

Isle

t (%

)

*

Control ConicalTube

Gas permeable

Bag

0

1.0

2.0

3.0 **

GSI

R (S

I)

Control ConicalTube

Gas permeable

Bag

Manufacturing Controls

Control of the Final Product

Ø Each preparation is considered to be a product lot

Ø Each product lot must be tested before release for transplant

Ø Testing determined by § Regulatory requirements§ Manufacturers

Ø Each test must contribute meaningful scientific information about the final product

Manufacturing ControlsSpecifications for Lot Release Testing

ØIdentity (visual inspection of product by DTZ & in its final labeled container)ØProduct Volume (< 200 ml/bag)ØPotency (insulin release assay: ≥ 1;

islet quantity: ≥ 5,000 IEQ/kg; viability: ≥ 70%)ØPurity (≥ 30%)ØSafety (Gram Stain: negative; Endotoxin: < 5

EU/kg)

Cell Transplantation 2003; 12:809-813

The Bag Method for Islet Cell Infusion.Baidal D. A., Tatiana Froud T, Ferreira J. V., Khan A., Alejandro R and Ricordi C.

Camillo Ricordi, ChairpersonJames Shapiro and Bernhard Hering, Co-Chairpersons

Phase II Pilot Clinical Trials & Phase III Licensure

Clinical Islet Transplantation ConsortiumiCClinical Islet Transplantation ConsortiumiC

Multi-center Single Arm Phase 3 Trial of Islet Transplantation for T1D

RandomizationRituximabPhase 2 Study(N=12)

RandomizationDSG Phase 2 Study(N=20)

RandomizationExenatide and LysophyllinePhase 2 Studies(N=12)

RandomizationLEA29Y Phase 2 Study(N=12)

•Subjects are screened for inclusion/exclusion criteria common to all trials•All sites have identical manufacturing procedures except for modifications specific to phase 2

trials

U. PennsylvaniaU. MinnesotaU. MiamiU. Alberta

Oxygenated PFC/UW Oxygenated PFC/UW TransportTransport

Islet Isolation: The FutureIslet Isolation: The Future

Oxygenated Tissue CultureOxygenated Tissue Culture

Transplant DevicesTransplant Devices

Stem Cells 2007; 25(12): 3155-64

Enhanced oxygenation promotes beta-cell differentiation in vitro. Fraker C.A., Alvarez S., Papadopoulos P., Giraldo J., Gu W., Ricordi C., Inverardi L., Domínguez-Bendala J.

B

C

05

101520253035404550

Insulin 1 Insulin 2

Fold increase

CHigh O2PFC/Si

D

Standard culture

“Oxygen sandwich”

O2

O2

O2PFC/Si membrane

Oxygen grad Size (d 3) Hypoxia (d 3) INS + GLU (d 3)

áO2 áO2 áO2 áO2

PFC/Si PFC/Si PFC/Si PFC/Si

C C C C

A

p

s

A B

Days after transplantation0 20 40 60 80 100 120 140 160 180 200N

on-fa

stin

g B

lood

Glu

cose

(mg/

dL)

0

100

200

300

400

500

600

700

LIVER DEVICE

Transplantation 2006; 81(9):1318-24Reversal of diabetes by pancreatic islet transplantation into a subcutaneous, neovascularized device.Pileggi A., Molano R. D., Ricordi C., Zahr E., Collins J., Valdes R. and Inverardi L.

role of equipment in the manufacturing of pancreatic … · §standardization of standard operating...

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