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Assuring the Safety of Cultivated Meat: HACCP plan development and application to a cultivated meat target-product

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Assuring the Safety of Cultivated Meat:
HACCP plan development and
application to a cultivated
meat target-product
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Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product 2
Authored by
Anderson S. Sant’Ana
Professor, University of Campinas (UNICAMP), Brazil
Amanda Leitolis
Science and Technology Specialist, The Good Food Institute, Brazil
Cristiana Ambiel
Science and Technology Manager, The Good Food Institute, Brazil
Kamila Habowski
Doctoral student, University of Campinas, (UNICAMP), Brazil
Aline Bruna da Silva
Chief Scientific Officer, MOONDO Biotech, Brazil
Bibiana Franzen Matte
Chief Scientific Officer, Cellva ingredients, Brazil
Denise Rosane Perdomo Azeredo
Professor, Federal Institute of Rio de Janeiro, Brazil
Maristela S. Nascimento
Professor, University of Campinas (UNICAMP), Brazil
Raíssa Canova
Researcher, Cellva ingredients, Brazil
Kamilla Swiech Antonietto
Consultant, BioImprove Pharmaceutical Biotechnology, Brazil
Reviewers
Isabela de Oliveira Pereira
Science and Technology Analyst, Brazil
Yeshi Liang
Science and Technology Specialist, GFI Consultancy, Singapore
Elliot Swartz
Lead Scientist, The Good Food Institute, United States of America
Katherine Helena Oliveira de Matos
Independent Consultant, Brazil
Vinícius Gallon de Aguiar
Communication Manager, The Good Food Institute, Brazil
Design
Fabio Cardoso
Communication Analyst, The Good Food Institute, Brazil
Correspondence
Credits
Sant’Ana, Anderson S. et al.
Assuring the safety of cultivated meat: HACCP plan development and application to a cultivated meat
target-product / Anderson S. Sant’Ana, Amanda Leitolis, Cristiana Ambiel, Kamila Habowski, Aline
Bruna da Silva, Bibiana Franzen Matte, Denise Rosane Perdomo Azeredo, Maristela S. Nascimento,
Raíssa Canova and Kamilla Swiech Antonietto. – São Paulo: The Good Food Institute Brazil, 2023.
E-Book: PDF, 72 p.; IL.
ISBN :978-65-87080-57-4 .
1. Food. 2. Food Production Chain. 3. Food Technology. 4. Food Safety. 5. Food Safety Plan. 6. HACCP. 7.
Critical Control Point. 8. Meat. 9. Cultivated Meat. I. Title. II. HACCP plan development and application to
a cultivated meat target-product. III. Methodology to develop HACCP Plan. IV. The results. V. Sant’Ana,
Anderson S. VI. Leitolis, Amanda. VII. Ambiel, Cristiana. VIII. Habowski, Kamila. IX. Silva, Aline Bruna
da. X. Matte, Bibiana Franzen. XI. Azeredo, Denise Rosane Perdomo. XII. Nascimento, Maristela S. XIII.
Canova, Raíssa. XIV. Antonietto, Kamilla Swiech.
CDU 664 CDD 664
S232
Cataloging prepared by Regina Simão Paulino – CRB 6/1154
International Cataloging in Publication Data – CIP
ciencia@gfi.org
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product 3
Table of contents
Figures and Tables 5
Acknowledgements 6
Abbreviations and Acronyms 7
Definitions 9
Executive Summary 12
Introduction 13
1. Bapckground . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2. Overview of Food Safety Aspects of Cultivated Meat . . . . . . . . . . . . . . . . . . . . . . . . 15
3. Scope of this study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Methodology to develop HACCP Plan 19
1. Approach to accomplish the preliminary tasks . . . . . . . . . . . . . . . . . . . . . . . . . . .20
2. Approach to Process Flow Diagram Construction . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3. Approach to HACCP plan development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.1. Conduct a hazard analysis (Principle 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.2. Determine critical control points (Principle 2) . . . . . . . . . . . . . . . . . . . . . . . . .23
3.3. Establish critical limits (Principle 3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.4. Establish monitoring procedures (Principle 4) . . . . . . . . . . . . . . . . . . . . . . . . .24
3.5. Establish corrective actions (Principle 5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.6. Establish verification procedures (Principle 6) . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.7. Documentation and record-keeping (Principle 7) . . . . . . . . . . . . . . . . . . . . . . . 24
4
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
The results 25
1. Flow Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
1.1. Process Flow Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
1.2. Description of steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28
2. HACCP Plan for Cultivated Meat Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.1. Assumptions and considerations for the HACCP plan . . . . . . . . . . . . . . . . . . . . . 33
2.2. Product Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34
2.3. Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.4. Hazard Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.4.1. Biological hazards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35
2.4.2. Chemical hazards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
2.4.3. Physical hazards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38
2.5 HACCP Worksheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Conclusions and Research priorities 49
References 52
Appendix 01 - Seed train expansion design 62
Appendix 02 - Decision tree to identify CCPs6 64
Appendix 03 - Ingredients, culture media and processing aids composition 65
Appendix 04 - dentification of critical material 68
The Good Food Institute Brasil 71
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product 5
Figures and Tables
Figures
Figure 1 - Flow diagram of cultivated meat burger . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Tables
Table 1 - Product Description Form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Table 2 - Product Composition Form (Raw Materials, Ingredients, Additives, etc.) . . . . . . . .35
Table 3 - Hazard Analysis Worksheet - (Principle 1) Physical,
Chemical and Biological Hazards related to process steps . . . . . . . . . . . . . . . . . . . . . .39
Table 4 - HACCP Worksheet (Principles 3 to 7) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Table A3.1 - Burger Patty - Ingredients composition . . . . . . . . . . . . . . . . . . . . . . . . . 65
Table A3.2 - Culture media - Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Table A3.3 - Processing Aids - Classification and Composition . . . . . . . . . . . . . . . . . . . . 66
Table A4 - Ingredient or Processing Aids and Packaging Critical Material . . . . . . . . . . . . .68
6
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
The Good Food Institute Brazil and the authors
of this study would like to express their deepest
gratitude to all those who generously provided
knowledge and expertise during its development.
International experts from cultivated meat
companies provided invaluable technical
contributions for the development of the process
flow diagram. To ensure information confidentiality,
we shall reference them anonymously here.
International GFI colleagues from Brazil, US
and GFI Consultancy provided technical reviews
throughout the text.
We thank the training offered by “HACCP application
in the pharmaceutical industry,” taught by Dr. Elezer
Monte Blanco Lemes (Fundação Oswaldo Cruz) and
the technical contributions of Dr. Katherine Oliveira
de Matos.
Acknowledgements
Researcher: UFMG (Federal University of Minas Gerais - Brazil)
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product 7
Abbreviations and Acronyms
Buffer-ACK
Ammonium-Chloride-Potassium Lysing Buffer.
aw
Water activity.
ADI
Acceptable Daily Intake.
ANVISA
Brazilian Health Regulatory Agency
ATP
Adenosine Triphosphate.
BSE
Bovine Spongiform Encephalopathy.
BSA
Bovine Serum Albumin.
CAC
Codex Alimentarius Commission.
CCP
Critical Control Point.
CDC
Center for Disease Control and Prevention.
CIP
Clean-in-place.
CRISPR-Cas 9
Clustered Regularly Interspaced Short Palindromic
Repeats/associated protein 9.
DMEM
Dulbecco’s Modied Eagle Medium.
DMSO
Dimethyl sulfoxide.
DNA
Deoxyribonucleic acid.
DO
Dissolved oxygen.
EDTA
Ethylenediaminetetraacetic acid.
EGF
Epidermal growth factor
EMA
European Medicines Agency.
FACS
Fluorescence-Activated Cell Sorting.
FAO
Food and Agriculture Organization.
FBO
Food Business Operator.
FBS
Fetal Bovine Serum.
U.S. FDA
The United States Food And Drug Administration.
FGF
Fibroblast Growth Factor-Basic.
FSIS
Food Safety and Inspection Service.
gRNA
Guide of ribonucleic acid.
GAP
Good Agricultural Practices.
GCCP
Good Cell Culture Practices.
GHP
Good Hygienic Practices.
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product 8
GMP
Good Manufacturing Practices.
GRAS
Generally recognized as safe.
HACCP
Hazard Analysis and Critical Control Point.
HSA
Human Serum Albumin.
IARC
International Agency for Research on Cancer.
ICMSF
International Commission on Microbiological
Specications for Foods.
IGF-1
Insulin-like Growth Factor 1.
JECFA
Joint FAO/WHO Expert Committee on Food Additives.
LLDPE
Linear Low-Density Polyethylene.
LOG
Letter of Guarantee.
mAbs
Monoclonal antibodies.
MAPA
Brazilian Ministry of Agriculture, Livestock, and Food
Supply.
MCB
Master Cell Bank.
MWCB
Manufacturer’s Working Cell Bank.
PBS
Phosphate buffered saline.
PGA
Polygalacturonic Acid.
pH
potential hydrogen.
PS
Penicillin-Streptomycin.
PTFE
polytetrafluoroethylene.
RASFF
Rapid Alert System for Food and Feed.
SFD
Staphylococcal food-borne disease.
SIP
Sterilized-In-Place.
STEC
Shiga-Toxigenic E. coli.
STR
Stirred Tank Reactor.
UNICAMP
University of Campinas
USA
United States of America.
USDA
United States Department of Agriculture.
USFDA
United States Food and Drug Administration.
WCB
Working Cell Bank.
WHO
World Health Organization.
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product 9
Definitions
Allergen cross-contact
Unintentional incorporation of an allergenic food or
ingredient into another food that is not intended to
contain that allergenic food or ingredient (CAC, 2020).
Batch
A specic quantity that is intended to have uniform
character and quality within specied limits and is
produced according to a single manufacturing order
during the same manufacture cycle. (FDA, 2023).
Bioreactor
A device where cells growth under closed and controlled
conditions. (Allan, De Bank and Ellis, 2019).
Cell banking
Cell bank system consists of two tiers: a master cell bank
(MCB); and a working cell bank (WCB), sometimes called
a manufacturer’s working cell bank (MWCB) (EMA, 1998).
CCP Decision Tree
A sequence of questions to assist in determining whether
a control point is a CCP. (USFDA, 1997).
Control Measure
Any action or activity that can be used to prevent,
eliminate or reduce a signicant hazard to an acceptable
level (CAC, 2020).
Corrective Action
Procedures followed when a deviation occurs to
re-establish control, segregate and determine the
disposition of the affected product, if any, and prevent or
minimize the reoccurrence of the deviation (CAC, 2020).
Critical Control Point (CCP)
A step at which a control measure or control measures,
essential to control a signicant hazard, is/are applied to
a HACCP system (CAC, 2020).
Critical Limit
A maximum and/or minimum value to which a biological,
chemical or physical parameter must be controlled at a
CCP to prevent, eliminate or reduce to an acceptable level
the occurrence of a food safety hazard (USFDA, 1997).
Deviation
Failure to meet a critical limit (CAC, 2020).
Downstream
Downstream processing refers to the transformation of
the active ingredient into the nal products. It includes
steps such as cell harvesting, concentration, and product
formulation. (Allan, De Bank and Ellis, 2019).
Food Business Operator (FBO)
Entity responsible for operating a business at any step in
the food chain. Includes primary producers, importers,
manufacturers/processors, food warehouse/logistics
operators, food service operators, retailers and traders
(CAC, 2020).
Food Additive
Any substance not normally consumed as a food by
itself and not normally used as a typical food ingredient,
whether or not it has nutritive value, the intentional
addition of which to food for a technological (including
organoleptic) purpose in the manufacture, processing,
preparation, treatment, packaging, transport or holding
of such food results, or may be reasonably expected
to result, (directly or indirectly) in it or its by-products
becoming a component of or otherwise affecting
the characteristics of such foods. The term does not
include “contaminants” or substances added to food
for maintaining or improving nutritional qualities (CAC,
2021).
Food safety
Assurance that food will not cause adverse health effects
to the consumers when it is prepared and/or eaten
according to its intended use. (CAC, 2020).
Food Safety Plan
A Food Safety Plan consists of the primary documents in
a preventive control food safety system that provides a
systematic approach to identifying food safety hazards
that must be controlled to prevent or minimize the
likelihood of foodborne illness or injury. It contains a
collection of written documents that describes activities
that ensure the food safety during manufacturing,
processing, packing, and storage (FDA, 2016).
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Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Good Agricultural Practices (GAP)
Basic environmental and operational conditions
necessary for producing safe and wholesome food. Good
Management Practices (GMP) or Good Handling Practices
(GHPs) are general practices to reduce microbial food
safety hazards. The term may include both “good
agricultural practices” used in growing, harvesting,
sorting, packing, and storage operations and “good
handling practices” used in sorting, packing, storage, and
transportation operations (USDA, 2009).
Good Cell Culture Practices (GCCP)
Guidance document that presents some principles
intended to support best practice in all aspects of using
cells and tissues in vitro. (OECD, 2018).
Good Hygiene Practices (GHPs)
Fundamental measures and conditions applied at any
step within the food chain to provide safe and suitable
food (CAC, 2020).
Growth factors
Comprise molecules that can stimulate cellular events
such as growth, differentiation, migration, morphological
changes during the development and healing of tissues.
(Seeger; Paller, 2015).
HACCP
Hazard Analysis and Critical Control Point is a science-
based and systematic approach that identies specic
hazards and measures for their control to ensure food
safety (CAC, 2020).
HACCP Plan
Document or set of documents prepared in accordance
with the HACCP principles to ensure control of signicant
hazards in the food business (CAC, 2020).
HACCP System
Development of a HACCP plan and the implementation of
procedures according to that plan (CAC, 2020).
HACCP Team
Group of people who are responsible for developing,
implementing and maintaining the HACCP system
(USFDA, 1997).
Hazard
A biological, chemical or physical agent in food with the
potential to cause an adverse health effect (CAC, 2020).
Hazard Analysis
Process of collecting and evaluating hazard information
identied in raw materials and other ingredients, the
environment, in the process or in the food, and conditions
leading to their presence to decide whether or not these
are signicant hazards (CAC, 2020).
Ingredient
Any substance, including a food additive, used in the
manufacture or preparation of a food and present in the
nal product, although possibly in a modied form (CAC,
2009).
Ionizing radiation sterilization
Low-temperature sterilization method used for medical
products and food.
Letters of Guarantee (LOG)
Legal documents that protect facilities from penalties
if a supplier provides adulterated or misbranded food
additives, raw materials, packages, etc.
Master cell bank (MCB)
Represents a collection of cells of uniform composition
derived from a single source prepared under specic
culture conditions. (EMA, 1998).
Monitor
The act of conducting a planned sequence of observations
or measurements of control parameters to assess
whether a control measure is operating as intended (CAC,
2020).
Prerequisite Programs
Programmes including good hygiene practices, good
agricultural practices and good manufacturing practices,
as well as other practices and procedures such as training
and traceability, that establish the basic environmental
and operating conditions that set the foundation for
implementation of a HACCP system (CAC, 2020).
Processing Aid
Substance or material, not including apparatus or
utensils, and not consumed as a food ingredient by itself,
intentionally used in the processing of raw materials,
foods or its ingredients to fulll a certain technological
purpose during treatment or processing and which may
result in the non-intentional but unavoidable presence of
residues or derivatives in the nal product. (CAC, 2018)
Raw material
All materials which are in the nal product (PAHO, 2005).
Significant hazard
A hazard identied by a hazard analysis, as reasonably
likely to occur at an unacceptable level in the absence
of control, and for which control is essential given the
intended use of the food (CAC, 2020).
Step
A point, procedure, operation or stage in the food system
from primary production to nal consumption (CAC,
2020).
11
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Serial subculture
The sequential transferring of some or all cells from
previous cell culture to a new cell culture containing a
fresh growth medium. (GIBCO, 2020).
Satellite cells
Multipotent cells found in mature muscle. Satellite cells
are precursors to skeletal muscle cells, able to give rise
to satellite cells or differentiated skeletal muscle cells.
(Asakura; Komaki; Rudnicki, 2001).
Upstream
Upstream processing refers to the rst part of
bioprocessing where the target product is produced,
i.e., the synthesis stage. It includes steps such as cell
isolation or cell line development, media preparation, cell
banks, seed train (inoculum production) and bioreactor
production. (Allan; De Bank; Ellis, 2019).
Validation of control measures
Obtaining evidence that a control measure or combination
of control measures, if properly implemented, is capable
of controlling the hazard to a specied outcome (CAC,
2020).
Verification
The application of methods, procedures, tests and other
evaluations, in addition to monitoring, to determine
whether a control measure is or has been operating as
intended (CAC, 2020).
Working cell bank (WCB)
A collection of cells derived from one or more vials of
cells from a master cell bank and expanded by serial
subculture. (EMA, 1998).
12
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Of rapid development in recent years, cellular
agriculture technology has emerged as an
alternative to solve many food system problems.
The successful combination of knowledge from
fields such as biotechnology, tissue engineering
and molecular biology enables the production of
food products using cell culture, like cultivated
meat. As the technology advances, so does the
challenge for regulatory authorities. Regulatory
agencies must obtain reliable technical-scientific
information to base their regulatory authorization
of different products in an environment whose
technology advancements are disruptive, varied
and most often protected within companies.
At the same time, entrepreneurs and scientists
require more precise guidelines to conduct the
development of their products properly and swiftly.
In this scenario, a key issue for regulation and
technology development concerns the food safety
of cultivated meat products.
Attesting food safety is a prerequisite for product
commercialization and the first concern of
regulatory agencies. Hence, after suggestion of
the Brazilian Health Regulatory Agency (ANVISA)
and the Brazilian Ministry of Agriculture, Livestock,
and Food Supply (MAPA), the Good Food Institute
(GFI) Brazil in collaboration with the University of
Campinas (UNICAMP) has developed the present
study.
This study aimed to establish a Food Safety Plan
for a cultivated meat target product and contribute
toward assessing the safety aspects of cultivated
meat production by employing the Hazard Analysis
and Critical Control Points (HACCP) approach.
Hazard analysis was performed according to FAO
(1998), FDA (2022), and Codex Alimentarius (CAC,
2020) guidelines. Through interactive meetings,
the study team developed a HACCP plan for the
cultivated meat burguer. Besides the process flow
diagram and worksheets used, we also present the
research priorities identified during the study.
Lastly, we hope the information presented here
may provide the basis for future food safety studies,
indicating some steps toward ensuring the food
safety of cell culture food products and assisting all
stakeholders interested in assuring safe cultivated
meat production.
Executive Summary
Cultivated chicken sandwich: UPSIDE Foods
13
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Cultivated burger: Ivy Farm
CHAPTER 1
Introduction
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Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Cultivated meat is an alternative protein produced
via animal cell culture under controlled conditions
that can potentially replicate the sensory and
nutritional profile of conventional meats (Porto;
Berti, 2022). This technology can expand the
frontiers and ways in which meat is produced and
consumed, reducing the environmental impacts
caused by human food systems, supporting the
increasing global demand for protein to human
consumption, and tackling global food insecurity.
Cultivated meat production can also become an
alternative to mitigate ethical and health concerns
associated with traditional livestock agriculture
based on raising and slaughtering animals.
In recent years, cultivated meat companies
announced the construction of pilot production
facilities, indicating an approaching commercial-
scale production (Swatz, 2023). Rapid advances in
cultivated meat technology have been overcoming
the bottlenecks preventing industrial production,
such as scaling cell production and developing an
animal-free and cost-effective culture medium. At
the same time, food safety competent authorities
are challenged to follow these advances and
create the basis for regulatory authorization
flexible enough to cover the innovative and varied
methods used in cultivated meat production while
considering future scientific advances.
The present study was elaborated in a context
where the cultivated meat industry is still in its
early stages of development. Although progress in
cultivated meat technologies occurs fast, policy and
regulatory landscapes are still under elaboration.
Only Singapore and the USA have approved
cultivated meat products for commercialization,
for example.
While governments worldwide are working to
develop regulatory standards to ensure cultivated
meat safety, multilateral organizations such as The
Food and Agriculture Organization of the United
Nations (FAO) and the World Health Organization
(WHO) are initiating discussions around the use of
cell culture for food production aiming to identify
ways to ensure its safety.
Working to advance the fundamental scientific
knowledge in alternative proteins, the Good Food
Institute (GFI) presents this technical report
to contribute to future food safety analyses for
cultivated meat products.
1. Background
Bioreactors: UPSIDE Foods
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Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Despite the cultivated meat industry’s growth
and the pace at which technologies are moving
from research and development settings to
commercialization, cellular agriculture as a
research field has been slower to develop due to
the lack of dedicated funding from government
funding agencies (Ong et al., 2021). This field of
research is just beginning to be recognized as an
academic research activity, and it has a long way to
go regarding basic science generation.
Similarly, open-access scientific studies involving
the food safety of cultivated meat products are
still scarce. Most comprise review/opinion articles
proposing safety and regulatory aspects for
producing cultivated meat, indicating research
priorities and contributing to compile information
on the first steps toward building the fundamental
scientific basis for ensuring the safety of cultivated
meat (Ketelins; Kremers; De Boer, 2021; Ong et al.,
2021; FAO, 2022). Recently, numerous relevant data
for the safety of these products has been generated
from reports issued by regulatory agencies1 and
from a series of FAO/WHO technical documents,
including hazard analyses, country case studies
and terminology issues, putting us one step ahead
in terms of ensuring food safety for cultivated meat
(FAO; WHO, 2023a). However, as knowledge gaps
on this topic abound, answering safety questions to
gain insights and further develop effective control
measures to ensure cultivated meat products’
safety and safeguard public health.
Broadly speaking, microorganisms such as
bacteria, viruses, and yeast are the primary cause
of biological hazards in food safety. Transmission
sources can range from poor hygienic practices,
contamination of raw materials, processing failures,
or environmental contamination. Some bacteria,
such as Staphylococcus aureus and pathogenic
types of Escherichia coli, may produce exotoxins
that can suppress the consumer’s immune response
and cause illness. Presence of any chemical residue
(e.g., veterinary drugs, antibiotics, reagent residue,
sanitizers, and growth factors) in food products
can lead to chemical hazards, and should either
be found in acceptable levels set by the regulatory
authorities or not be found entirely in the food
2. Overview of Food Safety
Aspects of Cultivated Meat
1 FDA Completes First Pre-Market Consultation for Human Food Made Using Animal Cell Culture Technology; FDA Completes Second
Pre-Market Consultation for Human Food Made Using Animal Cell Culture Technology.
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Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
product. Physical hazards include the discovery
of foreign materials such as broken glass, plastic,
stones, wood, or metal (Malik; Krishnaswamy;
Mustapha, 2021). Below we describe some of the
safety aspects raised for cultivated meat from a
centralized perspective.
Risk of microbial contamination constitutes one
of the main safety concerns regarding cultivated
meat. While meat contamination by pathogenic
microorganisms in traditional livestock agriculture
is frequent due to the use of feedlots, animal
features, and failures across the meat processing
chain (Cassin et al., 1998, Møller et al., 2016, Han
et al., 2022, Brashears; Chaves, 2017), cultivated
meat is produced under well-controlled sanitary
conditions and using cells that were previously
subjected to rigid screening procedures for
microbial contamination (FAO, 2022, Ong et al.,
2021). Moreover, the entire cultivation process
must be conducted in bioreactors, equipment and
processing premises under rigid quality control
practices and proper monitoring for microbial
contaminants. By controlling cell origin and
implementing strict hygienic measures during
processing one can ensure that cultivated meat
is less prone to contamination by pathogenic
microorganisms commonly associated with
foodborne disease outbreaks linked to meat
consumption such as Salmonella spp., pathogenic
Escherichia coli, Listeria monocytogenes, among
others (FAO, 2022).
Another core safety concern of cultivated meat
is potential allergenicity. As animal cells are
employed to produce cultivated meat, it is expected
that any allergies to meat obtained by traditional
animal agriculture could also be observed when
cultivated meat is consumed. Hence, cultivated
meat producers will need to assess the product’s
potential allergenicity and properly inform
consumers through labeling, for instance.
On the other hand, cultivated meat technology can
help mitigate risks related to traditional livestock
agriculture, such as reducing the use of antibiotics
in animal husbandry. This practice is a significant
contributor to the rise of antibiotic-resistant
microorganisms, which is detrimental to treating
human infections (Wallinga et al., 2022, Xu et al.,
2022, Wu et al., 2023). Cultivated meat production
is expected to require much lower use of antibiotics
or none at all, potentially contributing to reduce the
occurrence of antibiotic-resistant microorganisms
in the food systems.
17
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Another beneficial contribution is a potential
decrease in the risk of zoonotic diseases, i.e.,
diseases transmitted from animals to humans,
as using properly screened animal cells grown
under controlled conditions to produce cultivated
meat will require reduced or no slaughtering.
Lastly, due to the strict safety and quality control
measures assigned to cultivated meat production,
the occurrence of physical hazards associated with
meat obtained from traditional livestock agriculture
should be mitigated (Cavalheiro et al., 2020, Iko
Afé et al., 2021, Smulders; Rietjens; Rose, 2019,
FAO, 1998).
Considering all these aspects, a crucial step to
ensure food safety for a new product and one of
the requirements to acquire regulatory approval
is identifying potential hazards in food production.
Given the potential presence of chemical,
biological, and physical hazards in cultivated meat
(FAO, 2022), the Hazard Analysis and Critical
Control Point (HACCP) approach can be used to
manage food safety.
HACCP is a systematic preventive tool that can
be used to identify and control hazards in food
production chain, in addition it is also adopted
by the Codex Alimentarius Commission to assess
hazards and establish control systems focusing
on prevention rather than relying on end-product
testing. Developing a HACCP plan may require
changes in raw materials, processing parameters,
manufacturing technology, end-product
characteristics, and distribution method employed
in the intended use or in the prerequisite program
applied. Any HACCP system should be capable
of accommodating change, such as advances in
equipment design, processing procedures, or new
technology (CAC, 2020).
Cultivated meat production’s potential to become a
safer and more sustainable option than traditional
meat production will likely be achieved if these
and other safety concerns are properly assessed.
Addressing them will require bridging research
gaps and cooperation between producers,
regulatory agencies, consumers, researchers,
and other stakeholders in designing, validating,
implementing, and monitoring proper and strict
measures to ensure the production of high-quality
and safe cultivated meat.
18
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
• This study presents a Food Safety Plan for a
hypothetical product, the cultivated meat burger. A
process flow diagram of the process for obtaining
the target product was modeled and cross-checked
by international experts in the cultivated meat field,
thus enabling hazard analysis;
• The process modeled includes 24 steps, from
selection of the animal donor to product storage
and distribution. Upstream processing includes
immortalization and expansion of bovine satellite
cells using 500L Stirred Tank Reactor (STR)
bioreactors in parallel. Downstream processing
covers unit operations to harvest cell biomass, which
is added with plant-based ingredients, shaped into
the target product, stored and distributed;
• The multidisciplinary study team, consisting of
ten scientists, held discussion sessions to elucidate
questions and decisions regarding every study
stage and to complete the hazard assessment.
To ensure as much as possible complete hazard
identification, safety assessment considered
all ingredients, processing aids, raw materials,
equipment, and packaging used in the process;
• Despite the study’s relevance in contributing to
ensure cultivated meat food safety, limitations
should be highlighted;
• This study was proposed in the context of the few
regulatory processes on cultivated meat publicly
available. Moreover, cultivated meat products
can be manufactured using a wide variety of
ingredients, raw materials and bioprocess designs,
most of which are currently developed under the
intellectual property protection of companies;
• Hence, the modeled process proposed here is only
representative of how a cell culture-based food
production can be carried out, as well as how its
inputs are applied and steps conducted. Our hazard
analysis, therefore, does not intend to exhaust
all potential hazards and appropriate control
measures for all cultivated meat products, nor to
meet regulatory requirements of any particular
country or region. Food safety assessments should
always be product-specific.
3. Scope of this study
19
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
CHAPTER 2
Methodology
to develop
HACCP Plan
Cell expansion in scaffolds: UFMG (Federal University of Minas Gerais - Brazil)
20
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Given the technical complexity of producing
cultivated meat and the need to conduct a robust
hazard analysis, we assembled a multidisciplinary
study team of ten scientists from five different
institutions with specific knowledge and expertise
in the fields of food safety, food microbiology, quality
control, processing technologies for conventional
meat, cultivated meat, tissue engineering, cellular
and molecular biology, material engineering,
biotechnology and bioprocessing.
Due to this broad and multidisciplinary background,
we deemed it necessary to conducted an initial
training with all experts involved to standardize
the basic knowledge on the core topics required
to develop the food safety plan, namely: HACCP
system, the microbiology of foods and meat, food
business hazards, good manufacturing practices,
cell culture techniques, large-scale bioprocessing,
HACCP application in the pharmaceutical industry,
and HACCP application in the meat industry.
The target product of the study is the cultivated
meat burger. This product was chosen because
it can be representative of the first generation
of cultivated meat products developed by the
industry. In other words, processed foods (e.g.,
hamburgers, sausages, ham, and nuggets) made of
a mix of animal cells and plant-based ingredients
and intended to be sold in restaurants. Likewise,
during process conception we considered using
some inputs applied by the industry at is early stage
(such as FBS and other animal-derived inputs).
However, those inputs have been progressively
replaced by more suitable versions that meet
industry demands for ingredients at lower costs
and environmental impacts. When describing the
product’s intended use, we considered features
of similar conventional products and the current
status of the cultivated meat industry (Table 1), The
team also included an intermediate product used
as an ingredient for burger production—bovine
muscle cell biomass.
1. Approach to accomplish
the preliminary tasks
21
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
The flow diagram is the systematic representation
of the sequence of steps used in food production
(CAC, 2020). Its purpose is to provide a detailed
outline of the steps involved in the process in order
to work as the basis for hazard analysis and the
application of the other HACCP principles.
Typically, a HACCP team would develop the flow
diagram and perform an on-site review of the
operation to validate its accuracy and completeness,
but as this is a case study based on a theoretical
product, the study team built the flow diagram using
available scientific data describing methodologies
and steps used in cultivated meat production
(Bomkamp et al., 2022; Bodiu et al., 2020; Bradley,
1978; Brasil, 2004; Brasil, 2015b; USHHS, 2022;
Ding et al., 2018; Rodríguez Escobar et al., 2021;
Geraghty et al., 2014; USHHS, 2010; Hanga et al.,
2020; Hanga et al., 2021; USHHS, 2009; Humbird,
2021; Ianovici et al., 2022; Joo et al., 2022; Kang
et al., 2021; Kern et al., 2016; Inamdar et al., 2012;
Flecknell, 2009; Letti et al., 2021; Li et al., 2021;
Pereira; Oliveira, 2020; Post et al., 2020; Marga et
al., 2015; Melzener et al., 2021; Melzener et al.,
2022; Messmer et al., 2022; Ramezani et al., 2019;
Sart; Agathos, 2016; Shit; Shah, 2014; Skrivergaard
et al., 2021; Ben-Arye et al., 2020; Verbruggen et
al., 2018; Genovese, 2017). The experts’ previous
experience establishing bioprocesses was also
valuable to this outlining.
Once finalized, we submitted the flow diagram and
its process description for extensive reviews by
international experts from seven companies based
in different countries operating in cultivated meat
and one academic researcher working in the field,
all performed anonymously. Pertinent suggestions
were considered to ensure that the methodologies
used as a model complied with the current practices
and challenges of cultivated meat production.
2. Approach to Process
Flow Diagram Construction
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Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Work dynamics for developing the Food Safety Plan
included synchronous and asynchronous work,
including offline activities. During the discussion
sessions held, the study team deliberated on
queries and decisions regarding every stage until
reaching an agreement. Due to the limited scientific
data on several aspects of cultivated meat safety
the team used a conservative approach to decide
and reach a consensus during the HACCP plan
development. Below we describe details of how
the team addressed each HACCP principle.
3.1. Conduct a hazard analysis
(Principle 1)
Hazard analysis was performed according to FAO
(1998), U.S.FDA (2022), and Codex Alimentarius
(CAC, 2020) guidelines. Qualitative risk
assessment of each hazard or set of hazards should
be conducted according to the severity of the
adverse health effect caused by the hazard and the
3. Approach to HACCP
plan development
likelihood of it occurring. According to FAO (1998),
low-probability and low-severity hazards should
not be tackled by the HACCP system, but rather
through Good Manufacturing Practices (GMPs) and
Good Hygiene Practices (GHPs).
Despite knowledge of the severity around each
biological hazard, probability of occurrence and
the behavior of potential foodborne pathogens in
cultivated meat products are unknown and can
vary according to the process adopted by each food
business operators (FBOs). Thus, in the present
HACCP plan, hazard analysis was performed based
on epidemiological scientific data on foodborne
illness linked to conventional bovine burgers, and
experts’ opinions.
In our quest to adequately address possible
concerns and provide the most accurate information
to readers, we posed several questions during the
HACCP plan development, such as:
Cultivated burger: Mosa Meat
23
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Once the hazard analysis concluded, the study team
listed some control measures, i.e., any actions and
activities that can prevent, eliminate, or reduce a
food hazard to an acceptable level, for each hazard.
According to Codex Alimentarius (CAC, 2020),
more than one measure may be required to control
a specific hazard, and a specific measure may
control more than one hazard.
3.2. Determine critical control
points (Principle 2)
CCPs determination was performed according to
Codex Alimentarius (CAC, 2020) and U.S.FDA (2022)
guidelines, an aided by the Codex Alimentarius
decision tree (CAC, 2020) (Appendix 02). CCPS
were established at steps where significant
hazards were identified during the hazard analysis
(Table 3) and were essential to produce safe food.
CCPs are shown in the HACCP worksheet (Table 4)
and highlighted at the appropriate step on the flow
diagram (Figure 1).
It is essential to emphasize whether a CCP step is
multifactorial and varies between FBOs and from
process to process. As more than one CCP may be
applied to control a specific hazard, or a CCP may
control more than one hazard (CAC, 2020), the
CCPs presented are specific to the processes and
formulation conditions described in this document.
3.3. Establish critical limits
(Principle 3)
Critical limits should be measurable or observable,
and separate acceptable from unacceptable
products. We established critical limits for each
step classified as CCP based on regulations or
literature data. Given the technological novelty,
a real process will require some of the critical
limits as well as control measures to be previously
validated to obtain evidence that they are capable
of controlling the hazard and ensuring safe food.
• Is there a chance for product contamination with hazardous substances?
• Is there a chance of using ingredients above the critical limit allowed by local or international regulations?
• What hazards may result if the food composition is not controlled?
• Does the cultivated meat permit the survival or growth of pathogens in the product during processing?
• Is there a chance for biological or chemical cross-contamination during processing?
• Does the package include instructions for safe handling, storage, and food preparation by the end-product
consumer?
• Is the packaging material resistant to damage, thereby preventing microbial contamination?
• Are there any potential allergens in the ingredients which should be included in the list of ingredients on
the label?
• Can cleaning and disinfection affect the safety of the product?
• Would improper storage lead to microbiologically unsafe food?
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Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
3.4. Establish monitoring
procedures (Principle 4)
According to FAO (1998), monitoring procedures
for CCPs should be capable of timely detection of a
deviation from an established critical limit to allow
isolation of the affected products. The suggested
monitoring procedures aim simply to illustrate
the numerous possibilities available. As such,
each FBO should consider routine procedures
and particularities to establish the most suitable
procedure and its frequency for CCP monitoring.
3.5. Establish corrective actions
(Principle 5)
A corrective action plan must be enforced
immediately after monitoring indicates a critical
limit deviation. According to USDA (2021), a
corrective action plan comprises four actions:
1. Product destination: Segregate all products
processed after the last acceptable check until
appropriate disposition is taken. Analyze processing
parameters and products to determine whether the
product must be discarded, reprocessed, or sent
for by-product processing. Product destination
depends on the sensory characteristics of the
product, the magnitude and type of deviation,
among others;
2. Deviation cause: Determine and eliminate the
root cause of the deviation;
3. Re-establishment of CCP control: Take actions to
bring the CCP under control;
4. Prevention of recurrence: Take measures to
prevent future occurrences.
Since the corrective actions are deviation-specific,
the present HACCP plan suggested no corrective
actions as each company should perform a case-
by-case analysis to establish the best corrective
action to control CCP and prevent recurrence.
3.6. Establish verification
procedures (Principle 6)
According to Codex Alimentarius (CAC, 2020),
verification procedures ensure that the control
measures effectively control the hazards as
intended. The verification procedures suggested
here are examples of numerous possibilities that
companies can adopt.
Each FBO should consider routine and particularities
to establish the best method and its frequency for
each CCP, which can include observations, auditing
(internal and external), calibration, sampling and
testing, and record review. Verification should
be conducted by a third person responsible for
monitoring and implementing corrective actions
(FAO, 1998), and also tackle the HACCP plan to
ensure the whole system is controlled.
3.7. Documentation and record-
keeping (Principle 7)
Documentation and record-keeping should be
appropriate to the nature of the operation and
sufficient to assist the FBO in verifying whether the
CCPs and the HACCP plan are under control (FAO,
1998). As for principles 4, 5 and 6, the record-keeping
developed here comprises an example among
numerous possibilities. Each FBO should consider its
reality to establish the best record keeping system.
Table 4 summarizes the HACCP plan developed for
the target product, covering all the above principles.
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Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
CHAPTER 3
The results
Cultivated burger: Mosa Meat
26
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
1.1. Process Flow Diagram
Figure 1 illustrates the process flow diagram for
producing cultivated meat burger and describes
each step to complete the 24-step process, from
the donor animal selection to product storage and
distribution, including cell isolation, cell banking,
upstream, downstream, and product processing.
Broadly speaking, the bioprocess includes a
sequential set of unit operations separated into
‘upstream’ and ‘downstream’ steps. Upstream
processing include cell expansion, inoculum
production and bovine muscle cell biomass
production in batches in 500L stirred-tank (STR)
bioreactors in parallel. Appendix 01 details the
seed train expansion designed in this modeled
process. Downstream processing covers unit
operations such as harvesting and centrifugation,
involved in the obtention of the concentrate cell
intermediary product (bovine muscle cell biomass
used to formulate the target-product.
CCP steps were labeled in the flow diagram with a
number followed by the type of hazard—biological
(B), chemical (C) and physical (P). Moreover,
we analyzed all the inputs to determine critical
materials among raw materials, ingredients,
processing aids and packaging (Table A4, Appendix
04).
1. Flow Diagram
Bioreactors: UPSIDE Foods
27
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
28
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
1.2. Description of steps
This section offers a detailed description of each
step of the cultivated meat burger production
process, including all inputs, equipment and
activities used.
1 - Donor animal selection
A two-year old, male bovine (Bos taurus) live
animal was chosen as donor animal for the muscle
sample. Veterinary inspection, which considers
clinical parameters, vaccines, and disease history,
including administration history of veterinary drugs
such as antibiotics, was performed to attest to
health conditions in the field.
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Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
2 - Muscle sampling
After the semimembranosus muscle region is
washed, shaved, and cleaned with ethanol, the
animal receives local lidocaine-based anesthesia
and muscle sampling is performed via incision using
a sterile metallic tube. Muscle sampling is performed
in the field (open environment) at room temperature.
3 - Muscle sample transport
The sample (muscle tissue) is then transferred to a
sterile tube with Dulbecco’s Modified Eagle Medium
(DMEM) and Penicillin-Streptomycin (PS) and
stored at 4°C inside a thermal box. Muscle samples
are transported to the laboratory within two hours.
3.A - Preparation of culture medium for
sample transport
Under aseptic conditions (biological safety
cabinet), previously sterile-filtered liquid DMEM
culture medium are mixed with sterile Penicillin-
Streptomycin (PS) to a final concentration of 1%.
The media is placed in sterile flasks and stored in a
cold chamber (2-8°C) until use.
4 - Muscle sample reception
Upon reception at the industry, the thermal box
and plastic bag protecting the muscle sample are
externally decontaminated with 70% Ethanol.
Temperature and preservation conditions of the
sample are verified (visual inspection of the sample
and internal temperature control of the box).
5 - Ingredients and processing aids
reception and storage
All ingredients and processing aids (described
in Table A3.1 and A3.3) are purchased from
suppliers and received by the FBO. All materials are
checked to ensure adequate shipping conditions
(temperature), packing integrity, expiration date,
and sterility (when required). Processing aids
and ingredients are kept under the supplier’s
recommended storage conditions until use.
Information regarding batch, brand, and expiration
date of the ingredients and processing aids received
are recorded.
6 - Media preparation/sterilization and
storage and processing aids preparation
Media ingredients (from Culture Medium 1, 2, and 3,
described in Table A3.2) are dosed and mixed with
ultrapure water in a single-use mixing vessel. The
desired pH (7.2-7.4) is adjusted using phosphoric
acid (H3PO4; 1M) or sodium hydroxide (NaOH; 1M)
basic solutions, previously weighed and diluted in
water. DMEM is purchased fractionated and added
as a powder directly into the mixing vessel. A piping
connected to filtration units removes the media
from the mixer, where the media is then sterilized
through a filtration process (0.2 or 0.1 µm). The
sterilized media is placed in sterile bags and stored
in a cold chamber (2-8°C) until use.
Processing aids that are sterile and that will have
direct contact with the product must be handled
(mixed, diluted, aliquoted, etc.) in a clean room, using
sterile materials inside the biological safety cabinet.
7 - Cell isolation
Before opening, the thermal box and sample tube
are cleaned with 70% ethanol. The tube is then
placed under sterile conditions, and the muscle
sample is transferred to an appropriate new
sterile tube. Next, the tissue is washed with 70%
30
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
resuspended in Culture medium 2. The cells are then
transfected using CRISPR-Cas9, guide of ribonucleic
acid (gRNA), and selected by first isolating individual
clonal populations from the parental pool of cells
subjected to one transfection cycle using 0.1%
gelatin-coated wells. Plasmid DNA construct
delivery is mediated by non-liposomal DNA complex
forming transfection reagent (Fugene HD). gRNAs
targeting the 5’region of the Bos taurus CDKN2A
gene encoding p16 are designed for expression from
a transfected plasmid. Plasmids are diluted in sterile
deionized water before use. After immortalization,
immortalized satellite cells are placed in Culture
medium 2 and cultivated in a CO₂ incubator.
10 - Cell Expansion
The immortalized satellite cells are submitted to
serial subculture until a sufficient number of cells
is reached. For the subculture procedure, the
immortalized satellite cells are washed (with PBS),
detached from flasks (Accutase), washed with new
Culture medium 2, centrifuged, replated in culture
medium 2 and cultivated in T-flasks (CO₂ incubator).
11 - Cell banking and storage
When a sufficient cell number is reached, the
subculturing steps are repeated to prepare a master
cell bank (MCB) and a working cell bank (WCB).
The cells are resuspended in a cryopreservation
medium (Serum-Free Cell Freezing Medium),
dispensed into individual cryovials, and frozen for
at least 4 hours at -80ºC (Ultra-freezer) and then
permanently at -150ºC. The WCB is derived from
one or more cell vials from the MCB. MCB and WCB
must be stored in ultra-freezers at -150ºC (which
are placed in a room with controlled temperature
Ethanol and Phosphate buffered saline (PBS)-PS,
dissociated (scalpel), digested with collagenase
in DMEM-PS at 37°C (CO₂ incubator) for 1.5 h, and
mixed (vortex) for 10 minutes. After digestion,
20% Fetal Bovine Serum (FBS) in DMEM is added
to the sample and mixed with a pipette. Muscle
fragments are centrifuged at 80 xg for 3 minutes,
and the supernatant collected. The precipitated
debris is again triturated with a 20-gauge needle
in PBS and centrifuged at 80 xg for 3 minutes. The
supernatant is collected, mixed, and centrifuged
at 1,000 xg for 5 minutes. The cells are washed
twice with PBS, followed by DMEM with 20% FBS.
Next, the cells are filtered (100 μm and 40 μm cell
strainers), centrifuged at 1,000 g for 5 minutes
at 4°C and incubated with Ammonium-Chloride-
Potassium Lysing Buffer (ACK Buffer) for 5 minutes
on ice. Finally, the cells are washed twice with PBS.
8 - Cell selection
Cells are centrifuged in a sterile tube, and the
pellet is reconstituted with buffer (1% BSA
2
in PBS)
plus antibodies (conjugated mAbs). Then, the cell
suspension is incubated in ice. Subsequently, labeled
cells are washed with PBS, reconstituted in Culture
medium 1 and selected by cell sorting in a flow
cytometry equipment located under a laminar flow
module. The selected cell suspension of satellite
cells is transferred to culture flasks and cultivated in a
CO₂ incubator for two passages in Culture medium 1.
9 - Immortalization
After removal of the culture flasks containing the
satellite cells from the CO₂ incubator and placement
under sterile conditions, the cells are washed with
PBS, detached from flasks with Accutase, and
2 Bovine Serum Albumin
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Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
15 - Gas filtration
Gases—N2, O2, CO2, and synthetic air, which is a
mixture of Nitrogen (80%) and oxygen (20%)—
must undergo a filtration process (0.2 μm
polytetrafluoroethylene - PTFE - hydrophobic
membranes, single-use or sterilized in place) before
entering into the bioreactors at a determined rate.
The bioreactors are fed a mixture of the four gases
(proportions will vary throughout the culture) to
maintain the dissolved oxygen (DO) concentration
during the proliferation and differentiation stages.
16 - Cell inoculation and biomass
production
Before inoculation in the bioreactor (previously
sterilized), cells are washed (with PBS), detached
from flasks (Accutase), rewashed (Culture medium
2), centrifuged, resuspended in Culture medium
2 and placed inside sterile bags. After overnight
equilibration, the cell suspension is inoculated
in the bioreactor using sterile connections. Cells
adhered to the PGA microcarriers are kept inside
the stirred-tank bioreactor with Culture medium 2
under controlled temperature (37ºC), pH (7.2-7.4),
and dissolved oxygen (DO) concentration. The pH
is controlled by adding CO2/basic solution (Sodium
bicarbonate buffered medium) or, alternatively, by
adding an acid and base solution. DO concentration
is maintained at the desired setpoint by controlling
the impeller speed (agitation) and the gas flow
using the 4-gas mixture (N2, O2, CO2, and synthetic
air). During biomass production, partial medium
exchange (50%) can be conducted by adding fresh
Culture medium 2. This biomass production step is
held until the desired number of cells is reached.
Then, a full medium exchange (100%) to Culture
medium 3 is performed to start skeletal muscle
differentiation.
between 20 and -25ºC), under the same conditions
in two or more separate locations.
12 - Cell thawing
One or more WCB vials are removed from the ultra-
freezers and thawed rapidly by immersion in a
bead bath at 37ºC. Vials are placed under sterile
conditions (biological safety cabinet), the cells are
washed with PBS, centrifuged using a laboratory
centrifuge, and resuspended in a Culture medium 2.
13 - Cell inoculum production
Cells are cultured and expanded in Culture medium
2 at 37ºC using monolayer culture systems (T-flasks,
Cell Factories, or Cell Stacks) until the number of
cells required to inoculate the bioreactor is reached.
14 - Preparation of microcarriers
Under sterile conditions (biological safety
cabinet), dry sterile Polygalacturonic Acid (PGA)
microcarriers are placed inside a sterile glass
bottle siliconized with a food-grade silicone spray.
After adding distilled water and gently swirling the
suspension, it is transferred to sterile plastic bags
and stored for up to 1 week at 4°C before use.
The microcarrier suspension is placed in a sterile
Schott bottle to settle and the water is aspirated
with a pipette. A small volume of Culture medium
2 is then added, and the liquid is aspirated again
to remove all water from the suspension. Lasty,
the desired volume of Culture medium 2 is added,
and the suspension is transferred to a sterile
bag. Culture mediums 2 and 3 and microcarriers
suspension stored in bags are transported to the
bioreactor area and transferred to the bioreactor
using aseptic connections and peristaltic pumps.
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Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
step is conducted under non-aseptic conditions at
a room temperature ≤10°C.
21 - Forming
The mass obtained is sent to automatic burger
formatters for proper size and thickness.
22 - Freezing
Quick freezing is performed by feeding the burger
patties through a liquid nitrogen tunnel, reaching
a temperature of ≤–18°C. The burger patties are
then separated individually by paraffin paper.
23 - Packaging and labeling
Frozen cultivated meat burgers are packed in a
paper box, interleaved paraffin paper in a room at
≤10°C temperature. The paper boxes pass through
a metal, magnetic, or x-ray detector.
24 - Storage and distribution
The product must be stored and distributed at
≤-18°C.
17 - Harvesting
Harvesting requires the agitation/aeration to be
turned off so that the cells settle and spent Culture
medium 3 is removed for the washing procedure
with sterile 0.9% Saline Solution. Agitation is then
turned on, pH of the saline solution is reduced to
pH 5.5-5.8 using phosphoric acid solution (H3PO4),
and the bioreactor temperature is reduced to 15°C.
The solution is mixed until equilibrium of pH and
temperature. Subsequently, the cell biomass is de-
watered by centrifugation (Disk-stack centrifuge).
Spent saline solution is a waste product removed
during centrifugation.
18 - Biomass chilling and holding
The de-watered cell biomass is transferred to a tank
and chilled to ≤5°C. Once chilled, the cell biomass
is kept in the holding tank, automatically weighted,
and transferred to the mixing tank.
19 - Weighing of ingredients
The ingredients for the cultivated meat burger (as
described in Table A3.1) are placed in a clean room
for weighing on a calibrated scale. Food handlers
must be well-trained and follow procedures for
correctly weighing ingredients.
20 - Mixing
The de-watered cell biomass is mixed with
textured pea protein, coconut fat, ascorbic acid,
transglutaminase, methylcellulose, beetroot
colorant, salt, and ice in an appropriate mixing tank
for 10 to 15 minutes until a homogeneous mass with
fine binding is obtained. Mass temperature must be
kept ≤5°C to allow proper forming while avoiding
fat separation and microbiological growth. This Cultivated salmon: Wildtype
33
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
and processing differences can directly affect
hazard analysis. Thus, the potential hazards
identified in this HACCP plan may not be the only
hazards associated with all cultivated meat and
cultivated meat-based products. For any food
product, the safety assessment should always be
product-specific.
Nonetheless, the information presented here might
contribute to developing safe cultivated meat-
based products and support FBOs in meeting future
regulatory requirements related to pre-requisite
programs and HACCP.
2.1. Assumptions and
considerations for the HACCP plan
As technology for cultivated meat production
is still under development, we adopted some
assumptions to guide the HACCP plan execution and
interpretation, as detailed below. Each assumption
may be directly linked to one or more steps of the
cultivated bovine biomass and cultivated meat
burger production processes.
This section presents the HACCP plan for
producing cultivated meat burger from cultivated
bovine muscle cell biomass (an intermediary
bioproduct used as an ingredient), as an example
of a cultivated meat-based product intended to be
commercialized in restaurants.
Here we detail the hazards more likely to be
associated with raw materials, ingredients
and processing steps, as well as their control
measures. We developed it by gathering data
from the scientific literature on the safety of
conventional meat products and conditions
employed in pharmaceuticals, bioprocesses, cell
culture, and biological products. Tables 1 and 2
give a full description of the target product and its
composition. Table 3 summarizes the results of the
Hazard Analysis (principle 1), and Table 4 presents
the HACCP worksheet to meet principles 2 to 7.
Since we used a batch process as reference,
differences may exist in terms of hazards and
control measures if a FBO employs a continuous
process. Similarly, facility, equipment, formulation,
2. HACCP Plan for
Cultivated Meat Production
34
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
2.2. Product Description
• If unlike the modeled process, which considers the use of cells collected directly from a donor animal,
the cells are purchased from a third-party cell bank, a letter of guarantee (LOG) and good manufacturing
practices (GMP) certificate should be provided;
• The modeled process considers the use of ingredients and processing aids purchased from qualified
external suppliers;
• The modeled process considers that all equipment, such as bioreactors and filters (for gas and media
sterilization), were cleaned-in-place (CIP)/sterilized-in-place (SIP) using food grade cleaning products,
except for the single-use mixer (for media preparation);
• For the approach used here, labeling was deemed a key control measure for specific hazards identified.
But since labeling regulations may vary among countries, in concrete cases, FBOs should comply with the
country’s regulations/guidelines, and if needed, rework the hazard analysis.
• Different FBOs could employ many different processes to produce cultivated meat. If any element or
aspect of a concrete process differ from those considered here, significant hazards may be modified
leading to changes in the process flow diagram and the hazard analysis.
• Since the conditions, parameters, specifications, standards, and critical limits employed were based on
the available scientific data, use of updated references and validated critical limits may imply the need to
revise the plan.
Table 1. Product Description Form
1. Product name Cultivated bovine muscle cell biomass Cultivated meat burger
2. Product definition An aggregate of microcarriers and bovine
skeletal muscle cells produced in bioreactors
A patty consisting of cultivated bovine muscle cells
biomass and added ingredients, shaped and subjected to
an adequate technological process
3. Important characteristics of the
final products, such as, pH, water
activity (aw), etc.
pH=5.5-5.8 (desirable)
aw=0.98-0.99
pH=5.5-5.8 (desirable based on beef meat).
aw=0.98-0.99.
4. Instruction for use and/or
consumption
Ingredients for the process of obtaining
cultivated meat products Heat treatment (grilled, roasted, fried, cooked, etc.)
5. Packaging characteristics Single-use or reusable (by sanitizing) plastic
buckets/bowls Plastic packaging for frozen products
6. Expiration date To be defined (additional research is needed) At least 90 days under freezing (based on conventional
meat)
7. Where the product will be sold Business-to-business Restaurants
8. Storage Between 0 to 4ºC Frozen at -18ºC
9. Information included in the label
• Product name;
• Ingredient list;
• Batch;
• Expiration date;
• Storage instruction.
• Product name;
• Ingredient list;
• Batch;
• Expiration date;
• Storage instruction;
• Allergen declaration;
• Preparation/instruction for use.
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Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
2.3. Composition
Table 2. Product Composition Form (Raw Materials, Ingredients, Additives, etc.)
Product name Cultivated bovine muscle cell biomass Cultivated meat burger
Raw material Bovine semimembranosus muscle Cultivated bovine muscle cell biomass
Ingredients
Cultivated bovine muscle cells
Textured pea protein
Coconut fat
Ascorbic acid
Transglutaminase
Methylcellulose
Beetroots colorant
Salt
Water/ice/deionized water
Polygalacturonic Acid Sodium Salt (PGA) microcarrier
Facility Gases O2, CO2, N2, synthetic air (mixture of nitrogen (80%) and oxygen (20%)
Packaging material
Single-use bioprocess bags Paraffin paper
Cell culture flasks (Polystyrene) Carton packs
Plastic buckets/drums (LLDPE) Plastic material (LLDPE)
2.4. Hazard Analysis
Below we present an overview and considerations
about the physical, chemical, and biological hazards
analysis conducted. Details on the steps in which
these hazards were identified and their respective
control measures are shown in the Hazard Analysis
Worksheet (Table 3).
2.4.1. Biological hazards
Our analysis identified the following biological
hazards: foodborne pathogens that could be
introduced from the donor animal (bovine),
ingredients, and processing aids or entered by cross-
contamination due to improper storage, handling,
or sanitation. Given the current lack of public
scientific data on whether and what foodborne
pathogens can grow or survive in cultivated meat,
the experts agreed to use a conservative approach
contemplating the analysis of groups of the major
pathogens recognized as meat contaminants.
On that basis, this HACCP plan addressed the
following potential biological hazards: Brucella
abortus, Mycobacterium sp., prion - Bovine
Spongiform Encephalopathy (BSE), Toxoplasma
gondii; Shiga-toxigenic Escherichia coli (STEC),
Salmonella, Cryptosporidium parvum, Listeria
monocytogenes, Staphylococcus aureus, Bacillus
cereus. In conventional ways of meat processing,
most of these hazards can be inactivated by heat
treatments (cooking, frying, grilling).
In the cultivated meat burguer production, the
initial phases of the process, which includes a
sampling procedure conducted in an open field can
36
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
be problematic in terms of biological contamination
of the sampled tissue. Thus, it is essential to follow
GAPs and conduct sampling using sterile material
and adding antibiotics at the proper concentration
during sample transportation. Once the cells
undergo cultivation under controlled conditions,
pathogens are not expected to propagate in the cell
culture and go undetected until the end process
(FAO; WHO, 2023a; Treich, 2021).
Those hazards can also be avoided or controlled by
aseptic handling of cells and inputs, monitoring the
cultures to verify signs of contamination, ensuring
the materials’ quality by adopting a supplier
qualification program and implementing programs
such as GHP and GCCP. For the process evaluated
here, ensuring the adequate application of the
method to sterilize media and inspect cultures
for signs of contamination before inoculation in
the bioreactors are specific control measures to
prevent contamination by foodborne pathogens.
Additional testing to detect microbiological
contaminants in cells and sterility testing of the
culture medium are also recommended to avoid
biological contamination in cell cultures (Geraghty
et al., 2014).
Salmonella, Shiga-toxigenic E. coli (STEC), and
L. monocytogenes can be found in the farm
environment (feces, manure, soil, feeding silage,
etc.), and can also colonize meat processing
facilities and equipment (Roberts, 2005; ICMSF,
2011; Singh; Thippareddi, 2019; Matle; Mbatha;
Madoroba, 2020). These pathogens have been
isolated from raw meat or linked to a foodborne
outbreak caused by meat and meat products
(RASFF, 2022a; 2022b; 2022c; FSIS, 2022a;
2022b; 2022c, 2022d, CDC, 2019a, 2019b, 2022).
Staphylococcus aureus can be found in cattle’s fur,
hide, or skin as part of the microbiota (Roberts,
2005), or introduced into the manufacturing plant
by cross-contamination or handling, reaching the
final product (Roberts, 2005; Singh; Thippareddi,
2019; Fang; Chen; Kuo, 1999). Staphylococcal
foodborne disease (SFD) is one of the most common
worldwide, resulting from food contamination by
preformed S. aureus enterotoxins (Kadariya; Smith;
Thapaliya, 2014).
Bacillus cereus was considered a potential
biological hazard here due to some plant-based
ingredients used across the process and the
likelihood of biofilm formation (Ellouze et al., 2021;
Akamatsu et al., 2019; Majed et al., 2016; Lin;
Briandet; Kóvacs, 2022). Moreover, this foodborne
pathogen has already been isolated from meat and
meat products (Tewari; Singh; Singh, 2015).
Cultivated meat burger processing applies
recombinant proteins as processing aids at different
stages of production. Recombinant proteins are
produced using microorganisms as host systems;
in the present study, all recombinant proteins
used are produced using Escherichia coli, which
has a history of safe use in food production and
approval for food additives production in Europe
(Kallscheuer, 2018) and GRAS approval in the US
(FDA, GRN 000897). Except for strains known
to be pathogenic, E. coli is considered a Class 1
Agent under the National Institutes of Health (NHI)
guidelines, which covers all non-human organisms
or animal pathogens. Its use as a cell factory is well-
established and has been commercially exploited
by various industries. The strain E. coli K-12, for
example, is non-pathogenic, non-toxigenic, and
not likely to pose a risk to human or animal health,
plants, or other microorganisms, and has been
utilized for decades, often in industrial settings with
high volumes and cell densities. In cases where
37
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
consumed and on the amount of contaminant
present in the animal production chain as residues
or in the culture media and reagents used in the
cultivated meat production.
Accordingly, the potential chemical hazards
identified were veterinary drugs, antibiotics
(PS), growth factors (IGF-1, FGF, EGF), chemical
substances from the packaging material and
substances capable of eliciting allergic responses,
albumins (HSA and BSA) and pea protein isolate
(Taylor et al., 2021). In the present plan, pea protein
is the only input intended to be present in the final
product. Veterinary drugs could be unintentionally
introduced during production, while all other
chemicals are intentionally introduced but are not
intended to be present in the final product. Control
measures for these chemical hazards include
source control (supplier qualification program),
adopting GMP, and labeling control (allergens). For
our proposed process, controlling the antibiotic
formulation and applying a validated washing
procedure before harvesting the cell biomass are
specific controls to reduce the concentration of
chemical hazards in the end product.
Moreover, the gene editing technique (CRISPR-
Cas 9) to which satellite cells are subjected
for immortalization could result in knock-out
expressions of protein p16, extending the cell’s
replicative capacity to allow pilot production of
the biomass. Previous publications have shown
that similar methods could affect the levels of
endogenous bioactive substances produced
by cells, resulting in the expression of novel
substances with potential allergenic or hazardous
properties for consumers (FAO; WHO, 2023a, Ong
recombinant proteins are produced using recipient
strains that lack a history of safe use, their safety
must be first established.
2.4.2. Chemical hazards
Chemical hazards in conventional food production
generally include natural toxins (mycotoxins, animal
toxins, and phytotoxins), pesticides, veterinary
drugs, environmental pollutants, heavy metals,
allergens and additives in inadequate levels. These
toxic chemicals may contaminate food at different
stages of the production chain (Arisseto-Bragotto;
Feltes; Block, 2017), from production (including
operations carried out in crop, livestock, and
veterinary medicine) to manufacture, processing,
preparation, treatment, packaging, transport, or
holding of such food, or as a result of environmental
contamination (CAC, 2016).
Analysis of chemical hazards consulted
publications from recognized entities, like the
Codex Alimentarius Commission (CAC), the United
Nation’s Joint Food and Agriculture Organization
(FAO), the World Health Organization (WHO) Expert
Committee on Food Additives (JECFA) and the
International Agency for Research on Cancer (IARC/
WHO). Carcinogenicity of each listed chemical was
verified by consulting the IARC database (IARC
Monographs34)(IARC, [2023]). For allergens, we
consulted the US FDA database which reinforces
the need to control cross-contact and labeling of
nine major food allergens (soybeans, sesame, milk,
eggs, fish, crustacean shellfish, tree nuts, peanuts,
and wheat)5. Severity of the chemical hazard
identified was scored using Acceptable Daily Intake
(ADI), and its probability depended on the amount
3 https://monographs.iarc.who.int/wp-content/uploads/2019/07/Classifications_by_cancer_site.pdf
4 https://monographs.iarc.who.int/list-of-classifications
5 https://www.fda.gov/food/food-labeling-nutrition/food-allergies
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Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
reagents added in low concentration and applied
up to the cell banking step are unlikely to remain
in the end product after multiple washes, medium
fluid exchanges and increases in cell volume (see
seed train details in Appendix 01).
On the other hand, residues from inputs added in
the final steps, notably prior to harvesting, such
as HSA, growth factors, p38 inhibitor and MEM,
could reach the final product. For the proposed
process, formulation control and application of a
validated washing procedure before cell biomass
harvesting are specific measures needed to
eliminate chemical hazards in the final product. In
a concrete production process potential adverse
effects (allergenic, mutagenic, carcinogenic, toxic)
from processing aids and their maximum residue
limits must be determined. If this information is
unknown, alternatives must be sought for their use
in production. Regarding growth factors and other
recombinant proteins, we must determine their
equivalence with the native protein, its presence and
acceptable levels in the conventional counterparts,
and the presence of the microorganisms used to
express the recombinant protein in the end product
(WHO, 2003; Swartz et al., 2023).
2.4.3. Physical hazards
Although the physical hazards considered were
mostly those known for conventional ground
meat and burger processing, we considered some
particular characteristics of the cultivated meat
processing and its ingredients and processing aids.
According to FAO (1998), the main physical hazards
to be considered in a HACCP plan are metal, stones,
glass, and plastics, as foreign materials have
been responsible for the recall of meat and meat
products (FSIS, 2019, 2022e). Examples of control
et al., 2021, Soice ; Johnston, 2021). We thus
considered them as chemical hazards in the hazard
analysis (Table 3). Expression of novel substances
could be controlled by inspecting the cultures to
identify signs of culture disruption, such as altered
growth and viability (FAO; WHO, 2023a). Moreover,
testing control measures could be applied to verify
that the editing technique resulted in no further
changes to the genome (e.g., off-target effects)
and to directly identify new protein expression.
Besides foods derived from cell culture, gene
editing techniques have been applied to plants,
animals and microorganisms for agrifood use
already commercialized. Thus, the same potential
unintended effects may also occur and have been
managed in these products (FAO, 2023).
Of the 49 inputs used to produce the cultivated
meat burger, 19 contain hazardous substances
in their composition and were considered critical
materials (Table A4). Of these, most are inputs
commonly used for biomedical research purposes
and, in general, have not been used in conventional
food production. Although these inputs may be
hazards in the cultivated meat burger production,
due to the lack of information supporting an
evidence-based safety assessment, some of them
were not listed in Table 3 (see an indication in Table
A3.3) but commented detailed in the following text.
All the processing aids intentionally introduced in
the processing are not intended to be present in
the final product. Nonetheless, those identified
as critical materials contain in their composition
substances that could lead to adverse events (e.g.,
sodium azide, phenylalanine, putrescine, etc.) or
directly modulate cell function (e.g., growth factors
and small molecules). Considering the cultivated
meat burger production process, critical materials
such as antibiotics, antibodies and immortalization
39
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
measures for physical hazards include source control (supplier qualification program), processing control
(magnets, metal detectors, sifter screens, de-stoners), and environmental control (GMP, including employee
training program and equipment maintenance program).
Table 3. Hazard Analysis Worksheet – (Principle 1) Physical,
Chemical and Biological Hazards related to process steps
(1) Step (2) Identify potential hazards
introduced, controlled or
enhanced at this step
P= Physical
C= Chemical
B= Biological
(3) Does this
potential hazard
need to be
addressed in the
HACCP plan?
Yes* No**
(4) Justify your decision for
column 3
(5) What measure(s) can
beapplied to prevent or
eliminate the hazard or reduce it
to an acceptable level?
1. Donor animal
selection
PNone
CVeterinary drug x
Residues from veterinary drugs
used for herd treatment can
potentially reach the final product
if the withdrawal period and/or
the critical limits are not properly
respected. Those residues can
be a health hazard or elicit an
allergic reaction when handled or
consumed.
Follow the withdrawal period
established for each drug.
Veterinary inspection (require vet
drug residue analysis reports).
Follow relevant good practices¹.
B
Foodborne pathogens
(Brucella abortus,
Mycobacterium sp.,
prion – Bovine Spongiform
Encephalopathy (BSE),
Toxoplasma gondii; Shiga-
toxigenic Escherichia
coli (STEC), Salmonella,
Cryptosporidium parvum)
x
Foodborne pathogens can be
present in tissues such as the
muscle and/or blood of the donor
animal and can survive and/or
grow, potentially reaching the final
product and causing illnesses in
consumers.
Animal health report from the
donor.
Health inspection of the animal
prior to sampling.
Follow relevant good practices¹.
2. Muscle
sampling
PNone
CNone
B
Foodborne pathogens
(STEC, Salmonella,
L. monocytogenes;
Staphylococcus aureus)
x
Food borne pathogens can be
present in the environment, animal
skin or hair and contaminate the
tissue during sampling, potentially
reaching the final product causing
illnesses in consumers
Trichotomy, cleaning and
antisepsis of the animal’s
semimembranosus muscle region
prior to sampling.
Health inspection of the sampled
tissue for signs of infection.
Use of sterile material for tissue
sampling.
3. Muscle
sample
transport
PNone
CNone
B
Foodborne pathogens
(STEC, Salmonella, L.
monocytogenes; S.
aureus)
x
Failure to properly cool the
material can result in foodborne
pathogen growth and toxin
formation, potentially reaching the
final product causing illnesses in
consumers
Control sample storage
temperature (4ºC) and time of
transportation (2h).
Use of sterile culture medium to
maintain sample viability during
transport.
Add antibiotic in proper
concentration.
Follow relevant good practices¹.
¹ Good practices may include good agricultural practices (GAP); good manufacturing practices (GMPs); good hygiene practices (GHPs); and
good cell culture practice (GCCP).
40
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Table 3. Hazard Analysis Worksheet – (Principle 1) Physical,
Chemical and Biological Hazards related to process steps
(1) Step (2) Identify potential hazards
introduced, controlled or
enhanced at this step
P= Physical
C= Chemical
B= Biological
(3) Does this
potential hazard
need to be
addressed in the
HACCP plan?
Yes* No**
(4) Justify your decision for
column 3
(5) What measure(s) can
beapplied to prevent or
eliminate the hazard or reduce it
to an acceptable level?
3.A –
Preparation of
culture medium
for sample
transport
PNone
CPenicillin-Streptomycin-
(PS) x
Failure in preparing antibiotic
solutions can potentially result in
concentrations above the critical
limit set for these substances in the
final product and become a health
hazard or elicit an allergic reaction
when handled or consumed
Apply a validated washing
procedure to remove PS or reduce
their concentration.
Food handling training.
Follow other relevant good
practices¹.
B
Foodborne pathogens
(STEC, Salmonella, L.
monocytogenes; S.
aureus)
x
Failure in preparing and/or
sterilizing the culture medium can
result in contamination, survival or
growth of foodborne pathogens,
potentially reaching the final
product causing illnesses in the
consumers
Aseptic handling of cell culture
inputs.
Follow other relevant good
practices¹.
4. Muscle
Sample
Reception
PNone
CNone
B
Foodborne pathogens
(STEC, Salmonella, L.
monocytogenes; S.
aureus)
x
Failure in sample reception can
lead to failure in identifying
samples previously contaminated
by foodborne pathogens,
potentially reaching the final
product causing illnesses in the
consumers
External decontamination of the
thermal box and the plastic bag.
Checking sample temperature
and preservation conditions.
Visual inspection of the material
(turbidity, color, viscosity.
Follow relevant good practices¹.
5. Ingredients
and processing
aids reception
and storage
P
Foreign materials (plastic,
metal, insect fragments,
stones) x
Fragments of foreign materials
can come from ingredients or
processing aids and potentially
reach the final product, causing
health issues/injury to consumers
Adoption of a supplier
qualification program.
Follow other relevant good
practices¹.
C
Allergens – Pea protein
Packaging material
x
x
Pea protein will be present in
the final product and could elicit
allergic reaction when handled or
consumed.
Toxic substances can potentially
migrate from the packaging
material to the final product,
causing health issues to
consumers.
Food allergen labeling.
Adoption of an allergen control
program.
Adoption of a supplier
qualification program.
B
Fetal Bovine Serum (FBS)
(Príons, L. monocytogenes,
S. aureus, Bacillus cereus,
STEC, Salmonella, B.
abortus)x
Failures in good practices can
result in foodborne pathogen
growth and toxin formation,
potentially reaching the final
product causing illnesses in the
consumers.
Control storage temperature
(≤-10°C).
Adoption of a supplier
qualification program.
Follow other relevant good
practices¹.
¹ Good practices may include good agricultural practices (GAP); good manufacturing practices (GMPs); good hygiene practices (GHPs); and
good cell culture practice (GCCP).
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Assuring the Safety of Cultivated Meat: HACCP plan development
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Table 3. Hazard Analysis Worksheet – (Principle 1) Physical,
Chemical and Biological Hazards related to process steps
(1) Step (2) Identify potential hazards
introduced, controlled or
enhanced at this step
P= Physical
C= Chemical
B= Biological
(3) Does this
potential hazard
need to be
addressed in the
HACCP plan?
Yes* No**
(4) Justify your decision for
column 3
(5) What measure(s) can
beapplied to prevent or
eliminate the hazard or reduce it
to an acceptable level?
6. Media
preparation/
sterilization
and storage and
Processing aids
preparation
PForeign materials (plastic
and metal) x
Fragments of foreign materials can
potentially reach the final product,
causing health issues/injury in the
consumers.
Adoption of an equipment
maintenance program.
Follow other relevant good
practices¹.
C None
B
Foodborne pathogens
(Príons, L. monocytogenes,
S. aureus, Bacillus cereus,
STEC, Salmonella, B.
abortus)
x
Failure in preparing and/or
sterilizing the culture medium can
result in contamination, survival or
growth of foodborne pathogens,
potentially reaching the final
productcausing illnesses in the
consumers.
Apply a validated sterilization
procedure.
Control media flow rate and
pressure of filters during
sterilization.
Aseptic handling of cells and cell
culture inputs.
Follow other relevant good
practices¹.
7. Cell isolation
PNone
CNone
B
Foodborne pathogens
(Príons, S. aureus, L.
monocytogenes, STEC,
Salmonella, B. abortus,)
x
Failures in good practices can
result in contamination and
growth of foodborne pathogens,
potentially reaching the final
product causing illnesses in the
consumers.
Aseptic handling of cells and cell
culture inputs.
Follow other relevant good
practices¹.
8. Cell selection
PNone
CBovine Serum Albumin
(BSA) x
Residues of albumin can be carried
to the final product and elicit
allergic reaction when handled or
consumed
Food allergen labeling.
Apply a validated washing
procedure to remove albumin
residues or reduce their
concentration.
Adoption of an allergen control
program.
B
Foodborne pathogens
(S. aureus, L.
monocytogenes, STEC,
Salmonella)
x
Failures in good practices or
during flow cytometry can result
in contamination and growth of
foodborne pathogens, causing
illnesses in the consumers of the
final product.
Cell sorter cleaning.
Aseptic handling of cells and cell
culture inputs.
Follow relevant good practices¹.
¹ Good practices may include good agricultural practices (GAP); good manufacturing practices (GMPs); good hygiene practices (GHPs); and
good cell culture practice (GCCP).
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Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Table 3. Hazard Analysis Worksheet – (Principle 1) Physical,
Chemical and Biological Hazards related to process steps
(1) Step (2) Identify potential hazards
introduced, controlled or
enhanced at this step
P= Physical
C= Chemical
B= Biological
(3) Does this
potential hazard
need to be
addressed in the
HACCP plan?
Yes* No**
(4) Justify your decision for
column 3
(5) What measure(s) can
beapplied to prevent or
eliminate the hazard or reduce it
to an acceptable level?
9.
Immortalization
PNone
C
Novel allergenic or
hazardous substances
generated by unintended
effects of immortalization
x
Failures in the immortalization
process can potentially lead to
the expression of hazardous or
allergenic substances. These
substances can persist in the cell
culture and reach the final product,
becoming a health hazard or
eliciting an allergic reaction when
handled or consumed.
Regular inspection of the cultures
examining cell morphology and
signs of culture disruption (e.g.,
altered growth and viability).
B
Foodborne pathogens
(S. aureus, L.
monocytogenes, STEC,
Salmonella)
x
Failures in good practices can
result in contamination and growth
of foodborne pathogens, causing
illnesses in the consumers of the
final product
Aseptic handling of cells and cell
culture inputs.
Follow other relevant good
practices¹.
10. Cell
expansion
PNone
CNone
B
Foodborne pathogens
(S. aureus, L.
monocytogenes, STEC,
Salmonella)
x
Failures in good practices can
result in contamination and growth
of foodborne pathogens, causing
illnesses in the consumers of the
final product
Regular visual inspection of the
cultures using microscope. Cell
morphology examination: signs
of deterioration (e.g., granularity,
detachment and vacuolation)
and signs of contamination (e.g.,
medium turbidity, color, viscosity).
Aseptic handling of cells and cell
culture inputs.
Follow other relevant good
practices¹.
11. Cell banking
and storage
PNone
CNone
B
Foodborne pathogens
(S. aureus, L.
monocytogenes, STEC,
Salmonella)
x
Failure to properly cool the
material can result in foodborne
pathogen growth and toxin
formation, causing illnesses in the
consumers of the final product
Control storage temperature
(≤-80ºC).
Correctly label cryovials and
check for leakage.
Quarantine the cells until their
origin has been authenticated
and are shown to be free of
microorganisms.
Aseptic handling of cells and cell
culture inputs.
Follow other relevant good
practices¹.
¹ Good practices may include good agricultural practices (GAP); good manufacturing practices (GMPs); good hygiene practices (GHPs); and
good cell culture practice (GCCP).
43
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Table 3. Hazard Analysis Worksheet – (Principle 1) Physical,
Chemical and Biological Hazards related to process steps
(1) Step (2) Identify potential hazards
introduced, controlled or
enhanced at this step
P= Physical
C= Chemical
B= Biological
(3) Does this
potential hazard
need to be
addressed in the
HACCP plan?
Yes* No**
(4) Justify your decision for
column 3
(5) What measure(s) can
beapplied to prevent or
eliminate the hazard or reduce it
to an acceptable level?
12. Cell thawing
PNone
CNone
B
Foodborne pathogens
(S. aureus, L.
monocytogenes, STEC,
Salmonella)
x
Failures in good practices can
result in contamination and growth
of foodborne pathogens, causing
illnesses in the consumers of the
final product
Aseptic handling of cells and cell
culture inputs.
Follow other relevant good
practices¹.
13. Cell
inoculum
production
PNone
CNone
B
Foodborne pathogens
(S. aureus, L.
monocytogenes, STEC,
Salmonella)
x
Failures in good practices can
result in contamination and growth
of foodborne pathogens, causing
illnesses in the consumers of the
final product
Regular visual inspection of the
cultures using microscope. Cell
morphology examination: signs
of deterioration (e.g., granularity,
detachment and vacuolation)
and signs of contamination (e.g.,
medium turbidity, color, viscosity).
Aseptic handling of cells and cell
culture inputs.
Follow other relevant good
practices¹.
14. Preparation
of microcarriers
PNone
CNone
B
Foodborne pathogens
(S. aureus, L.
monocytogenes, STEC,
Salmonella)
x
Failures in good practices can
result in contamination and growth
of foodborne pathogens, causing
illnesses in the consumers of the
final product
Control storage temperature
(4°C).
Aseptic handling of microcarriers
and inputs.
Follow other relevant good
practices¹.
15. Gas
filtration
PNone
CNone
B
Foodborne pathogens
(STEC, Salmonella, L.
monocytogenes, B.
cereus)
x
Failures in gas filtration can result
in survival or growth of foodborne
pathogens, causing illnesses in the
consumers of the final product
Adoption of a filter maintenance
program.
¹ Good practices may include good agricultural practices (GAP); good manufacturing practices (GMPs); good hygiene practices (GHPs); and
good cell culture practice (GCCP).
44
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Table 3. Hazard Analysis Worksheet – (Principle 1) Physical,
Chemical and Biological Hazards related to process steps
(1) Step (2) Identify potential hazards
introduced, controlled or
enhanced at this step
P= Physical
C= Chemical
B= Biological
(3) Does this
potential hazard
need to be
addressed in the
HACCP plan?
Yes* No**
(4) Justify your decision for
column 3
(5) What measure(s) can
beapplied to prevent or
eliminate the hazard or reduce it
to an acceptable level?
16. Cell
inoculation
and Biomass
production
PForeign materials (plastic
and metal) x
Fragments of foreign materials
originating from equipment,
cell culture plastics, packaging
materials, can reach the final
product, causing health issues or
injuries to consumers
Adoption of an equipment
maintenance program.
Follow other relevant good
practices¹.
C
Human Serum Albumin
(HSA) – allergen x
Residues of albumin can be carried
to the final product, becoming
a health hazard or eliciting an
allergic reaction when handled or
consumed.
Food allergen labelling.
Adoption of an allergen control
program.
Fibroblast Growth Factor-
Basic (FGF),
Insulin-like Growth Factor-1
(IGF-1), EGF (Epidermal
Growth Factor)
xResidues of these substances can
potentially reach the final product,
becoming a health hazard or
eliciting an allergic reaction when
handled or consumed.
Formulation control – ensure
the use of the minimal levels for
effective action.
B
Foodborne pathogens
(STEC, Salmonella,
L. monocytogenes)
x
Failures in good practices can
result in contamination and growth
of foodborne pathogens, causing
illnesses in the consumers of the
final product
Monitor signs of contamination
(e.g., pH, high consumption
of alkaline solution for Ph
maintenance, turbidity, color).
Aseptic handling of cells and
inputs.
Follow other relevant good
practices¹.
17. Harvesting
PForeign materials (plastic
and metal) xFragments of foreign materials can
reach the final product, causing
health issues/injury to consumers
Adoption of an equipment
maintenance program.
Follow other relevant good
practices¹.
C
Fibroblast Growth Factor-
Basic (FGF),
Insulin-like Growth Factor-1
(IGF-1), EGF (Epidermal
Growth Factor)
x
Residues of these substances can
potentially reach the final product,
becoming a health hazard or
eliciting an allergic reaction when
handled or consumed
Apply a validated washing
procedure to remove residues of
chemical compounds.
Adoption of a residual testing
program.
B
Foodborne pathogens
(STEC, Salmonella, L.
monocytogenes)
x
Failures in good practices can
result in contamination and growth
of foodborne pathogens, causing
illnesses in the consumers of the
final product
Control biomass temperature and
Ph prior to harvesting.
Aseptic handling of cells and cell
culture inputs.
Follow other relevant good
practices¹.
¹ Good practices may include good agricultural practices (GAP); good manufacturing practices (GMPs); good hygiene practices (GHPs); and
good cell culture practice (GCCP).
45
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Table 3. Hazard Analysis Worksheet – (Principle 1) Physical,
Chemical and Biological Hazards related to process steps
(1) Step (2) Identify potential hazards
introduced, controlled or
enhanced at this step
P= Physical
C= Chemical
B= Biological
(3) Does this
potential hazard
need to be
addressed in the
HACCP plan?
Yes* No**
(4) Justify your decision for
column 3
(5) What measure(s) can
beapplied to prevent or
eliminate the hazard or reduce it
to an acceptable level?
18. Biomass
chilling and
holding
PForeign materials (plastic
and metal) xFragments of foreign materials can
reach the final product, causing
health issues/injury to consumers
Adoption of an equipment
maintenance program.
Follow other relevant good
practices¹.
CNone
B
Foodborne pathogens
(STEC, Salmonella,
L. monocytogenes)
x
Failures in GHP can result in
contamination and growth of
foodborne pathogens, causing
illnesses in the consumers of the
final product
Follow relevant good practices¹
19. Weighing
of ingredients
(burger
formulation)
P
Foreign materials (insect
fragments, stones, plastic,
metal) xFragments of foreign materials can
reach the final product, causing
health issues/injury to consumers
Salt sifting.
Adoption of a supplier
qualification program.
Follow relevant good practices¹.
C Allergens – Pea protein
cross contact x
Residues of pea protein can be
carried to the final product and
could elicit allergic reaction when
handled or consumed
Food allergen labeling.
Adoption of an allergen control
program.
Follow other relevant good
practices¹.
B None
20. Mixing
PForeign materials (plastic
and metal) xFragments of foreign materials can
reach the final product, causing
health issues/injury to consumers
Adaptation of an equipment
maintenance program.
Follow other relevant good
practices¹
CNone
B
Foodborne pathogens
(STEC, Salmonella, L.
monocytogenes, S.
aureus)
x
Failures in GHP can result in
contamination and growth of
foodborne pathogens, causing
illnesses in the consumers of the
final product
Follow relevant good practices¹
21. Forming
PForeign materials (plastic
and metal) xFragments of foreign materials can
reach the final product, causing
health issues/injury to consumers
Adoption of an equipment
maintenance program.
Follow other relevant good
practices¹.
CNone
B
Foodborne pathogens
(STEC, Salmonella, L.
monocytogenes, S.
aureus)
x
Failures in GHP and in room
temperature control can result in
contamination and/or growth of
foodborne pathogens and toxin
production, causing illnesses in the
consumers of the final product
Temperature control of the
processing room (≤10ºC).
Follow relevant good practices¹.
¹ Good practices may include good agricultural practices (GAP); good manufacturing practices (GMPs); good hygiene practices (GHPs); and
good cell culture practice (GCCP).
46
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Table 3. Hazard Analysis Worksheet – (Principle 1) Physical,
Chemical and Biological Hazards related to process steps
(1) Step (2) Identify potential hazards
introduced, controlled or
enhanced at this step
P= Physical
C= Chemical
B= Biological
(3) Does this
potential hazard
need to be
addressed in the
HACCP plan?
Yes* No**
(4) Justify your decision for
column 3
(5) What measure(s) can
beapplied to prevent or
eliminate the hazard or reduce it
to an acceptable level?
22. Freezing
PForeign material (plastic,
metal) xFragments of foreign materials can
reach the final product, causing
health issues/injury to consumers
Adoption of an equipment
maintenance program.
Follow relevant good practices¹.
CNone
B
Foodborne pathogens
(STEC, Salmonella, L.
monocytogenes, S.
aureus)
x
Failure to properly cool the product
can result in foodborne pathogen
growth and toxin formation,
causing illnesses in the consumers
Temperature control of the
products (≤-18ºC)
23. Packaging
and labeling
PForeign material (plastic,
metal) xFragments of foreign materials can
reach the final product, causing
health issues/injury to consumers
Inspect the packaged product
(x-ray, metal detector, magnetics)
CAllergens x
Failure to declare the presence
of allergens on the label of the
final product can cause an allergic
reaction in consumers
Food allergen labeling
B
Foodborne pathogens
(STEC, Salmonella, L.
monocytogenes, S.
aureus)
x
Failures in package integrity and
GHP can result in contamination
and growth of foodborne
pathogens and toxin production,
causing illnesses in the consumers
of the final product
Temperature control of the
processing room (≤10ºC).
Visual inspection of package
integrity.
Food labeling (Consumer
instructions – preparation/use
instructions, storage conditions)
24. Storage and
distribution
PNone
CNone
B
Foodborne pathogens
(STEC, Salmonella, L.
monocytogenes, S.
aureus)
x
Failures in GHP and storage
temperature can result in
contamination and/or growth of
foodborne pathogens and toxin
production, causing illnesses in the
consumers of the final product
Temperature control (≤-18ºC) of
the product.
Follow relevant good practices¹.
To answer the question at column 3 as:
* Yes: The study team determined that if the potential hazard is not adequately controlled, consumption is
likely to result in an unacceptable health risk. Hence, the potential hazard was addressed in the HACCP plan.
**No: The study team determined that the potential hazard risk is low and good practices can adequately
control it. Hence, the potential hazard was not addressed in the HACCP plan.
¹ Good practices may include good agricultural practices (GAP); good manufacturing practices (GMPs); good hygiene practices (GHPs); and
good cell culture practice (GCCP).
47
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
2.5 HACCP Worksheet
Table 4. HACCP Worksheet (Principles 2 to 7)
Critical
Control
Points
Significant Hazards Critical limits
Monitoring
Corrective
actions
Verification
activities Records
What How When
(frequency) Who
CCP 1B
Step 3.
Muscle
sample
transport
B
Foodborne
pathogens (STEC,
Salmonella, L.
monocytogenes;
S. aureus)
Stored at 4°C
for up to 2h
Transport
time and
temperature
Data collection
using data
loggers
Continuous
(real time) Designee
Determine
and eliminate
the cause of
deviation.
Muscle
sample
segregation
for evaluation.
Retrain
employee.
Instrument
calibration
(data loggers).
Record review.
Intermediate
checks of
sensors and
measurement
devices
Microbiological
analysis of
the muscle
sample.
Data loggers’
calibration
certificate.
Time and
temperature
profile
sheet(s).
Microbiological
analysis
report(s).
CCP 1C
Step 3A
Preparation
of culture
medium
for sample
transport
C
Penicillin-
Streptomycin
(PS) (overdose
inappropriate
concentration of
antibiotic
Concentration
in the final
product:
Penicillin < 50*
µg/kg.
Streptomycin <
600 µg/kg.
Weighing of
antibiotics
and volume
added to
the culture
medium
Observe the
employee
preparing
the antibiotic
solution.
Record in the
proper form.
At each
medium
batch
preparation
Designee
Determine
and eliminate
the cause of
deviation.
Segregate for
evaluation.
Retrain the
employee.
Record review.
Observation of
the weighing
procedure.
Analysis of the
final product
to verify the
antibiotic
concentrations
Weighing/
volume sheet.
Employee
training
certificate.
Reports of
antibiotic
analysis in the
final product.
CCP 2B
Step 3A
Preparation
of culture
medium
for sample
transport
B
Growth of
Foodborne
pathogens (STEC,
Salmonella, L.
monocytogenes;
S. aureus)
Concentration
in the culture
medium (per
liter):
Penicillin.
100U/Ml.
Streptomycin
100µg/Ml.
Weighing of
antibiotics
and volume
added to
the culture
medium
Observe the
employee
measure the
components
and mix the
solution.
Record in the
proper form
At each
medium
batch
preparation
Designee
Determine
and eliminate
the cause of
deviation.
Segregate for
evaluation.
Retrain the
employee.
Record review.
Observation
of the
preparation
procedure.
Intermediate
checks of
sensors and
measurement
devices.
Microbiological
analysis of
the culture
medium.
Pipette
calibration
certificate.
Weighing/
volume sheet.
Employee
training
certificate.
Culture
medium
microbiological
analysis
reports.
CCP 3B
Step 6.
Media
preparation/
sterilization
and storage
and
Processing
aids
preparation
B
Foodborne
pathogens (S.
aureus, B.
cereus, STEC,
Salmonella,
B. abortus, L.
monocytogenes)
Filter integrity
Flow rate and
pressure limits
established
after validation
by FBO
Filter
integrity
Flow
rate and
pressure
of the filter
system
Visual
inspection of
filteremometer/
Manometer
At each
batch
preparation
Continuous
(real time)
Designee
Determine
and eliminate
the cause of
deviation.
Segregate for
evaluation.
Retrain the
employee
Microbiological
analysis of
the culture
medium and
processing
aids.
Intermediate
checks of
flow rate
and pressure
sensors and
measurement
devices.
Supervision of
filter integrity
systems.
Instrument
calibration
(flow rate
and pressure
measurement
devices).
Culture
medium and
processing
aids
microbiological
analysis
reports.
Flow rate
and pressure
measurement
devices
calibration
certificate.
Filter integrity
systems sheet.
Employee
training
certificate.
48
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Table 4. HACCP Worksheet (Principles 2 to 7)
Critical
Control
Points
Significant Hazards Critical limits
Monitoring
Corrective
actions
Verification
activities Records
What How When
(frequency) Who
CCP 4B
Step 13.
Cell
inoculum
production
B
Foodborne
pathogens
(S. aureus, L.
monocytogenes,
STEC,
Salmonella)
Absence of
microorganisms
No visual
changes
(turbidity, color,
viscosity)
Microbial
growth
Visual
aspect of
the cell
culture
Rapid sterility
test
Visual
inspection
Each batch Designee
Determine
and eliminate
the cause of
deviation
Segregate for
evaluation
Retrain the
employee
Microbiological
analysis/
sterility test
of the cell
culture.
Record review.
Supervision
of inspection/
sterility test.
Intermediate
checks of
equipment
Microbiological
analysis/
sterility test
reports.
Employee
training
certificate.
Equipment
calibration/
maintenance
certificate.
Inspection/
sterility test
sheet.
CCP 2C
Step 17.
Harvesting
C
Fibroblast Growth
Factor-Basic
(FGF),
Insuline-like
Growth Factor
1(IGF-1),
Epidermal growth
factor (EGF)
Minimum
washing cycles
to ensure
absence of
the chemical
hazard
Number of
washing
cycles and
conditions
Record in the
proper form Each batch Designee
Determine
and eliminate
the cause of
deviation.
Segregate the
product for
evaluation.
Retrain the
employee.
Intermediate
checks of
washing
procedure.
Residual
testing
program (e.g.,
ELISA test).
Recording
review.
Washing
procedures
form.
Employee
training
certificate.
Residual
testing
program
reports/
intermediate
checks.
CCP 1P
Step 23.
Packaging
and labeling
PForeign material
(plastic, metal) 2mm** Foreign
material
Inspection of
the packaged
product (e.g.,
x-ray, visual
inspection,
metal detector,
magnet)
Continuous
(real time) Designee
Determine
and eliminate
the cause of
the deviation.
Segregate the
product for
evaluation.
Retrain the
employee.
Equipment
calibration.
Record review.
Supervision of
the inspection.
Intermediate
checks of
inspection
(e.g., use of
specimens).
Equipment
calibration
certificate.
Inspection
sheet.
Employee
training
certificate
*Critical limit suggested based on FAO & WHO (2023b).
**Critical limit suggested based on ANVISA – RDC 623/2022 (Brasil, 2022).
49
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
CHAPTER 4
Conclusions
and research
priorities
Cultivated meat steak-tartare: Mosa Meat
50
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Alternative proteins such as cultivated meat might
offer solutions for food security challenges by
providing more sustainable products and methods
of production, but to effectively deliver cultivated
meat to consumers, we must ensure its safety
for human consumption. Although food safety
authorities worldwide have been confronted with
many innovative solutions to cultivated meat
production, only two regulatory frameworks
(Singapore in late 2020, and the USA in mid-
2023) formally processed complete applications
for these products. The present study conducted
a scientifically-based safety assessment of a
hypothetical cultivated meat product to serve as
an initial guide for developers working on their
products and food safety studies, and contribute
to regulators in building and providing clear food
safety guidelines for cultivated meat across
different regions.
As demonstrated in the present report, HACCP
system identified hazards in the cultivated meat
burger manufacturing process supporting their
safe production. The hazard analysis has indicated
most of the hazards identified in the cultivated
meat burger are already known and familiar to
conventional FBOs. Many potential hazards can
occur in producing conventional counterparts of the
cultivated meat burger (e.g., foodborne pathogens
and veterinary drugs) and other conventional food
products (e.g., pea protein). Other potential hazards
are unfamiliar to traditional meat processing but
may occur in biotechnology-derived foods, such as
fermented food and ingredients and foods produced
by conventional breeding or genetic engineering
(e.g., novel allergenic or hazardous substances). In
agreement with previous publications (FAO, 2023;
Ong et al., 2021), most of the identified hazards
can be controlled by existing control measures and
relevant good practices.
Due to the nature of the process and the lack of
a well-established supply chain, many of the cell
culture materials were not previously optimized for
food application. Using non-food-grade ingredients
and additives is a challenge for both industries
and regulators. Establishing appropriate inputs
for cultivated meat application is paramount for
meeting the lower costs and environmental impacts
needs of the final product. However, we highlighted
this requirement from the food safety perspective.
Besides costs and environmental impact, careful
selection of well-characterized and safe inputs, as
a safety-by-design strategy, from the early stages
of product development contributes to ensure
product and process safety while avoiding rework
by developers after significant investments in
terms of time and resources.
Despite limitations arising from using a modeled
flow diagram without in-loco validation, we were
able to accurately detail all the manufacturing
steps necessary to produce a cultivated meat
product and allow the safety assessment to map
potential hazards. Conventional meat processing
is well-known and relatively well-understood by
the general public. Conversely, cultivated meat
production is a new and disruptive food operation
about which relevant information must be provided
to consumers. Thus, the flow diagram developed
provides a detailed view of how a cell culture-based
production process could work from sampling
to final processing, providing transparency to
consumers and contributing to an informed
decision-making.
In short, this study serves as a starting guide for
developers interested in establishing a safety
assessment for their products and regulators
interested in designing legislation that stimulates
innovation and enables the safe development of
51
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Finally, we encourage all stakeholders in the
cultivated meat field to fill the research gaps
raised here using an evidence-based approach
and proactive work to advance the technology as
a whole.
cultivated foodstuffs. Discussion sessions held
by the team allowed to identify some knowledge
gaps. The research priorities described below may
serve to support hazard control, control measures,
corrective actions, monitoring, and verification
procedures, and be essential to implement the
HACCP plan and, consequently, to ensure the
safety of cultivated meat products:
• Given specific storage conditions,
characterize what foodborne pathogens can
grow or survive in the final product during
storage and distribution;
• Assess the behavior of spoilage
microorganisms in the product;
• Understand the role of microbiota in the
ingredients used in food processing (e.g., pea
protein, coconut fat, beetroot colorant, etc.)
and in cultivated meat shelf-life and safety;
• Verify potential cross-contamination
during packaging and the growth potential
of foodborne pathogens throughout the
product’s shelf-life;
• Determine the shelf-life of cultivated meat
products considering formulation, processing,
storage, distribution, and commercialization
conditions;
• Identify the limiting factor of shelf-life for
cultivated meat products;
• Establish strategies or validate procedures
needed to reduce or remove chemical
residues from the final product;
• Assess whether cell culture inputs not
commonly used in conventional food
production bear allergenic/mutagenic/
carcinogenic/toxicity potential and identify its
acceptable levels in the end product.
• Assess whether Staphylococcus aureus
toxin can be carried to the final product.
Despite the use of sterile microcarriers
during its preparation, contamination by this
toxin can occur due to inappropriate human
manipulation.
• Investigate differences and similarities in
transforming muscle into meat between
cultivated meat and conventional meat and
possible implications to safety and quality.
Cultivated chicken: GOOD Meat
52
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
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Appendix 01 - Seed
train expansion design
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and application to a cultivated meat target-product
After immortalization, cells need to be expanded
into monolayer cultures to produce the master
(MCB) and working cell banks (WCB). In this process,
conventional culture systems like well plates and
T-flasks can be employed in the first passages.
When a larger surface area is required, culture
systems such as HyperFlasks and HyperStacks are
preferable due to smaller footprint, when compared
to conventional monolayer systems, and possible
operation under closed conditions, being therefore
more GMP-compliant. Additional monolayer
passages should be employed to obtain MCB/WCB
with a higher number of vials/cell density.
Batch production begins with cell thawing
from WCB. After monolayer cell expansion in
HyperFlasks and HyperStacks (step 10), the cells
are inoculated in increasing volume bioreactors
until reaching the final bioreactor for biomass
production and differentiation. The flow diagram
developed considered a 5 times scale-up in the
bioreactor seed train, as is usual for microcarriers-
based processes. Growth area was estimated by
considering a microcarrier concentration of 5g/L
(5,000 cm2/g), as in Vebbrugen et al., 2018.
Operating multiple production bioreactors in
parallel requires multiple thawing vials and seed
train expansion processes. A process with a higher
volume biomass production bioreactor (1,000-
2,000L) might require an additional seed train step
in bioreactor.
64
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Appendix 02 - Decision
tree to identify CCPs
Reference: CAC, 2023
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Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Appendix 03 - Ingredients,
culture media and processing
aids composition
Table A3.1. Burger Patty - Ingredients composition
Ingredient Composition
Ultrapure Water Potable water
Deionized water Potable water
Ice Ice from potable water
Cell biomass Cultivated bovine muscle cells
Polygalacturonic Acid Sodium Salt (PGA) microcarrier
Pea protein Pea extract
Coconut fat Crude Coconut Oil
L-ascorbic acid 2-phosphate Vitamin C, Antiscorbutic factor
L-Threo Ascorbic acid: C6H8O6
Transglutaminase Glutaminase from E. coli
Methylcellulose Sodium carboxymethyl cellulose
Beetroot Red colorant Beet plant
Salt NaCl, Iodine
Table A3.2. Culture media - Formulation
Culture medium Composition
Culture medium 1
(Cell selection medium)
Dulbecco’s Modified Eagle Medium-F12
Penicillin-Streptomycin
Chemically-defined FBS replacement
Water
Phosphoric acid and sodium hydroxide (pH adjustment)
Culture medium 2
(Proliferation medium)
Dulbecco’s Modified Eagle Medium-F12
FGF (Fibroblast Growth Factor-Basic)
p38 MAP Kinase Inhibitor SB203580
Water
Phosphoric acid and sodium hydroxide (pH adjustment)
Culture medium 3
(Differentiation medium)
Dulbecc’'s Modified Eagle Medium-F12
EGF
IGF-1
Human Serum Albumin
L-ascorbic acid 2-phosphate (Vitamin C)
MEM amino acids solution
NaHCO3
Water
Phosphoric acid and sodium hydroxide (pH adjustment)
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Assuring the Safety of Cultivated Meat: HACCP plan development
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Table A3.3. Processing Aids - Classification and Composition
Processing aids Classification Composition
Ethanol Alcohol 70% ethyl alcohol
Penicillin-Streptomycin (PS) Antibiotic Stock solution contains 10,000 units/mL of penicillin 10,000µg/mL of
streptomycin in a 10 mM citrate buffer (for pH stability)
Phosphate-buffered saline (PBS) Buffer solution 1X working concentration contains 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4,
and 2 mM KH2PO4
Collagenase Enzyme Protease from Clostridium histolyticum
Fetal Bovine Serum (FBS) Serum Proteins, attachment factors, growth factors, amino acids, trace elements,
vitamins, lipids, and hormones.
Ammonium-Chloride-Potassium Lysing
(Buffer-ACK) Buffer solution Ammonium Chloride; Potassium Bicarbonate;
ethylenediaminetetraacetic acid (EDTA)
Bovine Serum Albumin (BSA) Protein Cohn Fraction V
Fibroblast Growth Factor-Basic (FGF) Growth factor Recombinant protein; 20mM potassium phosphate with 750mM NaCl
Insulin-like Growth Factor-1 (IGF-1) Growth factor Recombinant protein; 20mM potassium phosphate with 750mM NaCl
Epidermal growth factor(EGF) Growth factor Recombinant protein; 20mM potassium phosphate with 750mM NaCl
NCAM1-PE-Cy7* Monoclonal antibody Recombinant protein, sodium azide, BSA,
fluorochrome R-phycoerythrin (PE) coupled to the cyanine dye (Cy7)
CD29-APC* Monoclonal antibody Recombinant protein, sodium azide, BSA, Allophycocyanin (APC)
CD31-FITC* Monoclonal antibody Recombinant protein, sodium azide, BSA, Fluorescein isothiocyanate (FITC)
CD45-FITC* Monoclonal antibody Recombinant protein, sodium azide, BSA, Fluorescein isothiocyanate (FITC)
Collagen type 1 Protein Bovine collagen
Accutase Enzyme Invertebrate (crab)-derived enzyme; EDTA (ethylenediaminetetraacetic acid);
phenol red.
Serum-Free Cell Freezing Medium* Freezing medium 10% DMSO and methylcellulose
Agar Hydrocolloid Gum agar, Agar-agar
Calcium chloride Chemical compost CaCl2
Chemically-defined FBS replacement* Chemical compost Proteins, attachment factors, growth factors, amino acids, trace elements,
vitamins, lipids, and hormones
p38 MAP Kinase Inhibitor SB203580* ATP-competitive inhibitor 4-(4’-Fluorophenyl)-2-(4’-methylsulfinylphenyl)-5- (4’-pyridyl)-imidazole
Human Serum Albumin (HSA) Protein Recombinant protein
MEM amino acids solution* Culture media supplement Amino Acids: L-Arginine hydrochloride, L-Cystine, L-Histidine hydrochloride-
H2O, L-Isoleucine, L-Leucine, L-Lysine hydrochloride, L-Methionine,
L-Phenylalanine, L-Threonine, L-Tryptophan, L-Tyrosine, L-Valine.
Phosphoric acid Chemical compost H3PO4
Sodium bicarbonate Chemical compost NaHCO3
Gelatin Gelling agent Gelatin from bovine skin
Silicone spray Lubricating spray Dimethyldichlorosilane
Fugene HD* Transfection reagent Mixture of lipids; 80% ethanol
Cas9 protein*Enzyme Recombinant protein; glycerol
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Assuring the Safety of Cultivated Meat: HACCP plan development
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Table A3.3. Processing Aids - Classification and Composition
Processing aids Classification Composition
gRNAs* RNA Synthetic ribonucleic acid
Synthetic air Gases Nitrogen (80%) and Oxygen (20%)
Dulbecco’s Modified Eagle Medium High
glucose (DMEM)* Basal medium
Amino Acids: Glycine, L-Arginine hydrochloride, L-Cystine 2HCl, L-Glutamine,
L-Histidine hydrochloride-H2O, L-Isoleucine, L-Leucine, L-Lysine hydrochloride,
L-Methionine, L-Phenylalanine, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine
disodium salt dihydrate, L-Valine. Vitamins: Choline chloride, D-Calcium
pantothenate, Folic Acid, Niacinamide, Pyridoxine hydrochloride, Riboflavin,
Thiamine hydrochloride, I-Inositol. Inorganic salts: Calcium Chloride (CaCl2)
(anhyd.), Ferric Nitrate (Fe (NO3)3"9H2O), Magnesium Sulfate (MgSO4) (anhyd.),
Potassium Chloride (KCl), Sodium Chloride (NaCl), Sodium Phosphate
monobasic (NaH2PO4-H2O). Other Components: D-Glucose (Dextrose)
Dulbecco’s Modified Eagle
Medium-F12* Basal medium
Amino Acids: Glycine, L-Alanine, L-Arginine hydrochloride, L-Asparagine-H2O,
L-Aspartic acid, L-Cysteine hydrochloride-H2O, L-Cystine 2HCl, L-Glutamic Acid,
L-Glutamine, L-Histidine hydrochloride-H2O, L-Isoleucine, L-Leucine, L-Lysine
hydrochloride, L-Methionine, L-Phenylalanine*, L-Proline, L-Serine, L-Threonine,
L-Tryptophan, L-Tyrosine disodium salt dihydrate, L-Valine. Vitamins: Biotin,
Choline chloride, D-Calcium pantothenate, Folic Acid, Niacinamide, Pyridoxine
hydrochloride, Riboflavin, Thiamine hydrochloride, Vitamin B12, I-Inositol.
Inorganic Salts: Calcium Chloride (CaCl2) (anhyd.), Cupric sulfate (CuSO4-
5H2O), Ferric Nitrate (Fe(NO3)3"9H2O), Ferric sulfate (FeSO4-7H2O), Magnesium
Chloride (anhydrous), Magnesium Sulfate (MgSO4) (anhyd.), Potassium
Chloride (KCl), Sodium Bicarbonate (NaHCO3), Sodium Chloride (NaCl), Sodium
Phosphate dibasic (Na2HPO4) anhydrous, Sodium Phosphate monobasic
(NaH2PO4-H2O), Zinc sulfate (ZnSO4-7H2O) Other Components: D-Glucose
(Dextrose), Hypoxanthine Na, Linoleic Acid, Lipoic Acid, Putrescine 2HCl*,
Sodium Pyruvate, Thymidine.
* Inputs not addressed in Table 3. See comments in section 2.4.2 and table A.4 in Appendix 04.
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Assuring the Safety of Cultivated Meat: HACCP plan development
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Appendix 04 - Identification
of critical material
Table A4. Ingredient or Processing Aids and Packaging Critical Material*
Raw material ingredient
or processing aids and
packaging material
Hazard identified
P=Physical
C=Chemical
B=Biological
Question 1
Could the identified
hazard occur at levels
greater than acceptable
or could it increase to
undesirable levels?
If no. The raw material/
ingredient is not critical.
If yes, answer question
02.
Question 2
Will the process or consumer eliminate or
reduce the hazard to an acceptable level?
If no. The raw material/ingredient must
be considered critical, that is, the process
does not guarantee the safety of the
product. Modify the product or process.
If yes. It's not critical.
Repeat question 1 for another raw
material/ingredient.
Critical or
Non-critical**
Ultrapure water None Non-critical
Deionized water None Non-critical
Ice None Non-critical
Cell biomass (Bovine muscle
cells) None Non-critical
PGA Microcarriers None Non-critical
Textured pea protein C - Allergen Yes No Critical
Coconut fat None Non-critical
L-ascorbic acid 2-phosphate None Non-critical
Transglutaminase None Non-critical
Methylcellulose None Non-critical
Beetroots colorant None Non-critical
Salt None Non-critical
Ethanol None Non-critical
Penicillin-Streptomycin (PS) C - Antibiotic residue Yes No Critical
Phosphate buffered saline (PBS) None Non-critical
Collagenase None Non-critical
Dulbecco’s Modified Eagle
Medium High glucose (DMEM)
C - Presence of
L-Phenylalanine and
Putrescine 2HCl in the
composition
Yes No Critical
*Source: Mortimore and Wallace (2001).
** For all cases in which a hazardous component was identified in the processing aid, Question 2 was answered as ‘No’ due to the impossibil-
ity of experimentally evaluate the absence of residues in the final product.
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Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Table A4. Ingredient or Processing Aids and Packaging Critical Material*
Raw material ingredient
or processing aids and
packaging material
Hazard identified
P=Physical
C=Chemical
B=Biological
Question 1
Could the identified
hazard occur at levels
greater than acceptable
or could it increase to
undesirable levels?
If no. The raw material/
ingredient is not critical.
If yes, answer question
02.
Question 2
Will the process or consumer eliminate or
reduce the hazard to an acceptable level?
If no. The raw material/ingredient must
be considered critical, that is, the process
does not guarantee the safety of the
product. Modify the product or process.
If yes. It's not critical.
Repeat question 1 for another raw
material/ingredient.
Critical or
Non-critical**
Fetal Bovine Serum (FBS)
B - Foodborne
pathogens
(Prions, S. aureus,
L. monocytogenes,
STEC, Salmonella, B.
abortus,)
Yes No Critical
Ammonium-Chloride-Potassium
Lysing (Buffer-ACK) None Non-critical
Bovine Serum Albumin (BSA) C - Allergen Yes No Critical
Fibroblast Growth Factor-Basic
(FGF) C - Hormones Yes No Critical
Insulin-like Growth Factor-1
(IGF-1) C - Hormones Yes No Critical
Epidermal growth factor (EGF) C - Hormones Yes No Critical
NCAM1-PE-Cy7 (Conjugated
monoclonal Antibodies)
C - Presence of
sodium azide (NaN3)
in the composition Yes No Critical
CD29-APC (Conjugated
monoclonal Antibodies)
C - Presence of
sodium azide (NaN3)
in the composition Yes No Critical
CD31-FITC (Conjugated
monoclonal Antibodies)
C - Presence of
sodium azide (NaN3)
and Fluorescein
Isothiocyanate
Isomer 1 (FITC)
Yes No Critical
CD45-FITC (Conjugated
monoclonal Antibodies)
C - Presence of
sodium azide (NaN3)
and Fluorescein
Isothiocyanate
Isomer 1 (FITC)
Yes No Critical
Collagen type 1 None Non-critical
Accutase C - Presence of
Phenol red in the
composition Yes No Critical
Serum-Free Cell Freezing
Medium C - Presence of DMSO
in the composition Yes No Critical
Ágar None Non-critical
*Source: Mortimore and Wallace (2001).
** For all cases in which a hazardous component was identified in the processing aid, Question 2 was answered as ‘No’ due to the impossibil-
ity of experimentally evaluate the absence of residues in the final product.
70
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Table A4. Ingredient or Processing Aids and Packaging Critical Material*
Raw material ingredient
or processing aids and
packaging material
Hazard identified
P=Physical
C=Chemical
B=Biological
Question 1
Could the identified
hazard occur at levels
greater than acceptable
or could it increase to
undesirable levels?
If no. The raw material/
ingredient is not critical.
If yes, answer question
02.
Question 2
Will the process or consumer eliminate or
reduce the hazard to an acceptable level?
If no. The raw material/ingredient must
be considered critical, that is, the process
does not guarantee the safety of the
product. Modify the product or process.
If yes. It's not critical.
Repeat question 1 for another raw
material/ingredient.
Critical or
Non-critical**
Calcium chloride None Non-critical
Dulbecco's Modified Eagle
Medium-F12
C - Presence of
L-Phenylalanine and
putrescine 2HCl in the
composition
Yes No Critical
Chemically-defined FBS
replacement C - Hormones and
growth factors Yes No Critical
p38 MAP Kinase Inhibitor
SB203580
C - Presence of
imidazole in the
composition Yes No Critical
Human Serum Albumin (HSA) C - Allergen Yes No Critical
MEM amino acids solution C - Presence of
L-Phenylalanine in the
composition Yes No Critical
Phosphoric acid None Non-critical
Sodium bicarbonate None Non-critical
Gelatin None Non-critical
Silicone spray None Non-critical
Fugene HD None Non-critical
Cas9 None Non-critical
gRNAs None Non-critical
Synthetic air None Non-critical
Paraffin paper None Non-critical
Plastic Bags single use None Non-critical
Plastic drum (LLDPE - Linear
Low Density Polyethylene) None Non-critical
Grow bottle: Polystyrene None Non-critical
*Source: Mortimore and Wallace (2001).
** For all cases in which a hazardous component was identified in the processing aid, Question 2 was answered as ‘No’ due to the impossibil-
ity of experimentally evaluate the absence of residues in the final product.
71
Assuring the Safety of Cultivated Meat: HACCP plan development
and application to a cultivated meat target-product
Alexandre Cabral . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Policy VP
Alysson Soares . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Policy Specialist
Amanda Leitolis, Ph.D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Science & Technology Specialist
Ana Carolina Rossettini . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Development Manager
Ana Paula Rossettini . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human Resources Analyst
Bruno Filgueira . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Corporate Engagement Analyst
Camila do Nascimento . . . . . . . . . . . . . . . . . . . . . . . . . . . . Financial and Operational Analyst
Camila Lupetti . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Corporate Engagement Data Specialist
Cristiana Ambiel, MS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Science & Technology Manager
Fabio Cardoso . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Communication Analyst
Gabriela Garcia, MS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Policy Analyst
Gabriel Mesquita . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ESG Analyst
Graziele Grossi Bovi Karatay, Ph.D. . . . . . . . . . . . . . . . . . . Science & Technology Specialist
Guilherme de Oliveira Vilela . . . . . . . . . . . . . . . . . . . . . . . . Corporate Engagement Specialist
Gustavo Guadagnini . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . President
Isabela Pereira . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Science & Technology Analyst
Julia Cadete . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Operations Analyst
Karine Seibel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Operation’s and HR Manager
Lorena Pinho, Ph.D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Science & Technology Analyst
Luciana Fontinelle, Ph.D. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Science & Technology Specialist
Lívia Brito, MS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Communication Analyst
Manuel Netto . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Policy Analyst