PDA Standard 02-2021

Cryopreservation of Cells for Use in Cell Therapies,

Gene Therapies, and Regenerative Medicine
Manufacturing: An Introduction and Best Practices
Approach on How to Prepare, Cryopreserve, and
Recover Cells, Cell Lines, and Cell-Based Tissue
Products

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 PDA Standard 02-2021 Cryopreservation of Cells for Use in Cell Therapies,
Gene Therapies, and Regenerative Medicine Manufacturing: An Introduction
and Best Practices Approach on How to Prepare, Cryopreserve, and Recover
Cells, Cell Lines, and Cell-Based Tissue Products

Authors

Brian Hawkins (Chair)

Pluristyx, Inc.

Kathy Loper (Co-Chair)

NMDP Be The Match

Alireza Abazari
Pluristyx, Inc.

Brenda Alder
Northside Hospital Cell Therapy Program

Judith A. Arcidiacono
U.S. Food and Drug Administration (FDA/CBER)

Rabia Ballica
U.S. Food and Drug Administration
Center for Biologics Evaluation and Research (FDA/CBER)

Stacey Brower
Cook MyoSite, Inc.

John T. Elliott
National Institute of Standards and Technology (NIST)

Bassem Gayed
Celgene

Choon Wee Goh

Roche Singapore Technical Operations Pte. Ltd.

Dawn Henke
The Standards Coordinating Body for Regenerative Medicine (SCB)

Pennie E. Hylton
U.S. Department of Health and Human Services (HHS/ASPR/BARDA)

Peter Kilbride
Cytiva

Seth Kreger
Cook MyoSite, Inc.

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Sanjibita Mishra
Kite Pharma

Shannon Pasley
Akron Biotech

Darius Pillsbury
ValSource

Samuel D. Stevens
Fred Hutchinson Cancer Research Center

Anthony Tavera
U.S. Department of Health and Human Services (HHS/ASPR/BARDA)

Melanie Tellers
Organon

Wen Bo Wang*
Fate Therapeutics, Inc.

Claudia Zylberberg
Akron Biotech

*In Memoriam of Wen Bo Wang, a contributor and a great member of this scientific community
who unfortunately passed away before this standard was published.

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 About PDA Standards
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Sanctioning Advisory Board: BioAB

Chair: Morten Munk Co-Chair: Andrew Chang Past-Chair: Michael DeFelippis

Ed Balkovic Michael Blackton Jose Caraballo

Dayue Chen Maxwell De Long John Geigert

Brian Hawkins Olivia Henderson Christopher Hwang
Margit Jeschke Stephan Krause Peter Makowenskyj

Nicola Rose Sebastian Teitz Stephanie Verbrugghe

Tami Wu Earl Zablackis

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PDA Officers and Directors

Officers:

Chair: Jette Christensen, Novo

Nordisk

Chair-Elect: Susan Schniepp,

RCA

Treasurer: Melissa Seymour,

Biogen, Inc.

Secretary: Emma Ramnarine,

Genentech/Roche

Immediate Past-Chair:
Rebecca Devine, Consultant

President: Richard Johnson

Directors:

Barbara Allen, Eli Lilly and

Company

Michael Blackton,
Adaptimmune

Bettine Boltres, West

Pharmaceutical Services

Tia Bush, Amgen

Javier Camposano, Celltrion

Ghada Haddad, Merck & Co.

Joyce Hansen, Johnson &

Johnson

Stephan Krause, AstraZeneca

Biologics

Mary Oates, Emergent

Bioscience

Mathias Romacker, Pfizer

(ret.)

Anil Sawant, Merck & Co.

Osamu Shirokizawa,
Lifescientia

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ANSI/PDA Standard 02-2021 Cryopreservation of Cells for Use in Cell Therapies, Gene Therapies, and
Regenerative Medicine Manufacturing: An Introduction and Best Practices Approach on How to
Prepare, Cryopreserve, and Recover Cells, Cell Lines, and Cell-Based Tissue Products

©2021 Parenteral Drug Association

All rights reserved.

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Contents
1 Introduction .............................................................................................................................................................. 3
2 Scope ........................................................................................................................................................................... 3
3 Normative references .......................................................................................................................................... 3
4 Terms and definitions ............................................................................................................................................. 4
5 Acronyms ................................................................................................................................................................... 5
6 Critical Quality Attributes of the Cell-based Product .................................................................................... 6
7 Cryopreservation Process Steps and Critical Process Parameters .............................................................. 6
7.1 Material qualification ............................................................................................................................................. 7
7.2 Process parameters.................................................................................................................................................. 8
8 Cell Characterization and Testing ....................................................................................................................... 9
8.1 Identity/Appearance............................................................................................................................................. 10
8.2 Purity ....................................................................................................................................................................... 10
8.3 Viability ................................................................................................................................................................... 10
8.3.1 Viability testing ............................................................................................................................................... 10
8.3.2 Viability timepoint considerations.............................................................................................................. 10
8.4 Biological function ................................................................................................................................................ 11
8.5 Sterility assurance and testing ........................................................................................................................... 11
8.6 Mycoplasma ........................................................................................................................................................... 11
8.7 Viral and other adventitious agent testing...................................................................................................... 11
8.8 Stability ................................................................................................................................................................... 11
9 Formulation Development .................................................................................................................................. 11
9.1 Cryopreservation medium .................................................................................................................................. 11
9.2 Cryoprotectant agent ........................................................................................................................................... 12
9.2.1 Qualification for cryoprotectant agents .................................................................................................... 12
9.2.2 Cryoprotective agent exposure.................................................................................................................... 12
10 Cryogenic Container Closure Systems ............................................................................................................ 12
10.1 Cell package selection, qualification, quality control, and integrity testing ........................................... 12
10.1.1 Container integrity/secondary container considerations ...................................................................... 13
11 Cryopreservation Procedures ............................................................................................................................ 13
11.1 Qualification of freezing device ......................................................................................................................... 13
11.2 Ice nucleation ........................................................................................................................................................ 13
11.3 Transfer................................................................................................................................................................... 14
11.4 Documentation....................................................................................................................................................... 14
12 Cryogenic Storage and Transport .................................................................................................................... 14
12.1 Cryogenic storage ................................................................................................................................................. 14
12.2 Alert limits and action ......................................................................................................................................... 14
12.3 In-process hold ...................................................................................................................................................... 14
12.4 Transport ................................................................................................................................................................ 15
12.5 Label integrity ....................................................................................................................................................... 15
12.6 Equipment and qualification.............................................................................................................................. 15
13 Thawing................................................................................................................................................................... 15
13.1 Duration of thaw ................................................................................................................................................... 15
13.2 Holding before freezing or after thawing process (post-thaw hold) ......................................................... 15
13.3 Washing................................................................................................................................................................... 16
14 Bibliography/References ..................................................................................................................................... 17

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