CIRAD - Biological Systems Department

AnimalS, health, Territories, Risks, Ecosystems (ASTRE) Unit

OIE, FAO and UE Reference Laboratory for Peste des Petits
Ruminants

RESULTS OF ANALYSIS
ASSAY REPORT Quality Control of the homologous PPR Nigeria 75-1 vaccine
RE « 15-025 »

Date of issue 06/08/2018

CIRAD Reference PPR Nigeria 75/1 vaccine seed lot LK6
Vero 75, batch 130314: 2 vials from
lyophilisation of 14/03/2013.
Samples reception date 07/11/2012
End of tests 18/03/2015
Name and address of customer

Copy to: : CIRAD, A A-117/A Campus International de Baillarguet,

34398 Montpellier

CONCLUSION
The thorough purity control of the master seed, batch 130314 of the PPRV vaccine strain Nigeria 75/1
was based on conventional tests and high throughput sequencing (NGS). Our analyses confirmed that the PPR
vaccine seed held at the CIRAD PPR reference laboratory (Montpellier, France) is free from mycoplasma, viral,
bacterial and fungal contamination. Small quantities of pestivirus NGS reads were detected but
cell-based infectivity tests demonstrated that they are not from viable viral contamination.
The full genome obtained for the PPR vaccine batch showed 13 differences in comparison to the initial
sequence. This version is accessible on GenBank for the same strain.
This new genome should be used as a reference for the PPR Nigeria 75/1 vaccine strain.

Technical Manager Biologist: G. LIBEAU

Signature

In charge of analysis: Olivier KWIATEK

This report only concerns the objects submitted to analysis. This document belongs to CIRAD and shall neither be read
nor duplicated without permission.
CIRAD-BIOS-UMR117, TA A-117/E, Campus international de Baillarguet, 34398 MONTPELLIER CEDEX 5, France Tel : 33 (0)4 67 59 37 24
1/7
E-TD-09 Rapport d’essai V03 du 20/06/2018
 CIRAD - Biological Systems Department
AnimalS, health, Territories, Risks, Ecosystems (ASTRE) Unit
OIE, FAO and UE Reference Laboratory for Peste des Petits
Ruminants

RESULTS OF ANALYSIS
ASSAY REPORT Quality Control of the homologous PPR Nigeria 75-1 vaccine
RE « 15-025 »

PPR vaccine is a live attenuated vaccine. It has been obtained by attenuation through 75 successive
passages on Vero (African green Monkey Kidney) cell culture (Diallo et al., 1989) of the PPR virus
isolated in Nigeria in 1975 (Taylor and Abegunde, 1979).

The original master PPR 75-1 LK6 Vero 74 batch 1-960823 was tested on 14/01/1997 for quality
control. The titre obtained by the method of Spearman-Karber was 104.7 TCID50 according to results
of the 23.01.1997.

The seed prepared from the original master seed on the 14/02/2012 by subculture is the master seed
PPR 75-1 LK6 Vero 75, batch 130314 and submitted to quality control from the 07/11/2012. It is
recommended that the final production cultures obtained from the master seeds should not be more
than five passages.

This last seed is available from CIRAD - Biological Systems Department

AnimalS, health, Territories, Risks, Ecosystems (ASTRE)
TA 117/E Campus Int. Baillarguet
34398 Montpellier Cedex 5
FRANCE

The quality control is based on conventional tests, including targeted nucleic acid amplification;
cell-based infectivity assays as well as next-generation sequencing (NGS) that is a powerful tool for
ensuring vaccines are free of harmful contaminants, thus providing outstanding additional
guarantees on the vaccine purity and identity. Furthermore, it gives the opportunity to verify the full
sequence of the strain using advanced sequencing technology.

The following laboratory tests have been conducted on the above mentioned samples as summarised
here below:

 Determination of virus titre on microplate method (CPE reading)

 Determination of the identity of PPRV in the product by conventional and “Next

Generation Sequencing” (NGS)

 Detection and identification of adventitious viruses by conventional and NGS

methods
- Presence of Mycoplasma by specific PCR Amplification

- Presence of BVDV and PCV, Porcine circovirus (trypsin contaminant)

by specific Real-time RT-PCR Amplification

- Bacterial contamination by liquid culture

This report only concerns the objects submitted to analysis. This document belongs to CIRAD and shall neither be read
nor duplicated without permission.
CIRAD-BIOS-UMR117, TA A-117/E, Campus international de Baillarguet, 34398 MONTPELLIER CEDEX 5, France Tel : 33 (0)4 67 59 37 24
2/7
E-TD-09 Rapport d’essai V03 du 20/06/2018
 CIRAD - Biological Systems Department
AnimalS, health, Territories, Risks, Ecosystems (ASTRE) Unit
OIE, FAO and UE Reference Laboratory for Peste des Petits
Ruminants

RESULTS OF ANALYSIS
ASSAY REPORT Quality Control of the homologous PPR Nigeria 75-1 vaccine
RE « 15-025 »

- Fungal contamination by liquid culture

- Detection and identification of adventitious viruses by “Next Generation

Sequencing”

RESULTS

Virus titre of master seed:

The master seed batch 130314 have been titrated by the method of Spearman-Karber. Ten replicates
of serial dilutions of the vaccine were put in contact with sensitive cells (Vero). After 10 incubation
days, cytopathic effect was observed in all the wells. The titre obtain was 105.4 TCID50/ml or 103.4
TCID50 doses per vial.

Identity test:

This test allows confirming the presence or the absence of PPR virus in the product.

 Conventional test:

Peste des Petits Ruminants virus has been detected by PCR amplification using a one-step RT-PCR
kit from Qiagen and PPR specific primers targeting the nucleoprotein gene (Kwiatek & al., 2010)
A single band at 350pb was obtained, at the expected size.

 NGS test

Nucleic acids (both RNA and DNA) from 150 µl of the vaccine batch 130314 produced in Vero
cells were extracted, retro-transcribed and amplified by Multiple Displacement Amplification
(MDA) method using random primers. No enrichment step (viral concentration) has been
implemented in the protocol. MDA is a nonspecific and low-bias amplification method (Cheval J et
al., 2011). Sequencing was achieved on Illumina HiSeq 2000 with 100 Million clusters minimum
per sample (100Mio read pairs) and a read length of 101 bp, followed by Bioinformatics and data
analysis (Quality filtering, Assembly and Taxonomic assignment of contigs and single reads). A
total of 246,381,176 reads were generated of which, 63,556,517 were analysed after quality filtering.
8,647,010 reads (13.6%) matched the PPR genome, the rest being in majority reads of Vero cell
genome and unassigned reads. Coverage of the PPR genome was 7785x, thus giving a full
consolidated PPRV genome (submitted to GenBank Accession n° KY628761).

This report only concerns the objects submitted to analysis. This document belongs to CIRAD and shall neither be read
nor duplicated without permission.
CIRAD-BIOS-UMR117, TA A-117/E, Campus international de Baillarguet, 34398 MONTPELLIER CEDEX 5, France Tel : 33 (0)4 67 59 37 24
3/7
E-TD-09 Rapport d’essai V03 du 20/06/2018
 CIRAD - Biological Systems Department
AnimalS, health, Territories, Risks, Ecosystems (ASTRE) Unit
OIE, FAO and UE Reference Laboratory for Peste des Petits
Ruminants

RESULTS OF ANALYSIS
ASSAY REPORT Quality Control of the homologous PPR Nigeria 75-1 vaccine
RE « 15-025 »

Sterility control

The sterility control was undertaken on the master cell line, the sera and the master seed of the
vaccine to demonstrate that these are free of bacterial fungal and viral contaminants particularly
BVDV and mycoplasma. Absence of cytopathic effect was also verified.

Serum and master cell line

- Mycoplasma has been carried out using PCR amplification based on the 16S Ribosomal
mycoplasma universal primers (van Kuppeveld et al. 1994). Negative result obtained on the
10/12/2012.
- Pestiviruses contamination has been investigated by Real-time RT-PCR using the Adiavet BVDV
Real-time kit. This commercial kit (ADI105-50) uses two primers and a probe specific for BVDV,
BDV and part of CSF virus. Negative result obtained on the 26/02/2014.
- Bluetongue virus contamination has been investigated by Real-time RT-PCR based on the
publication of Toussaint et al., (2007). Negative result obtained on the 29/08/2014.
- Porcine circovirus contamination (trypsin contaminant) has been investigated by Real-time RT-
PCR using the TaqVet Porcine Circovirus 2 kit (PCVP-50). Negative result obtained on the
26/06/2014.

Vaccine: master seed

 Conventional tests

- Mycoplasma contamination has been investigated by PCR amplification using the 16S
Ribosomal mycoplasma universal primers for the generic detection of Mollicutes (van
Kuppeveld et al. 1994). No amplification was detected for the samples tested.

- Pestiviruses contamination has been investigated by Real-time RT-PCR using the Adiavet
BVDV Real-time kit. This commercial kit (ADI105-50) uses two primers and a probe specific
for BVDV, BDV and part of CSF virus. No amplification was detected for the samples tested.

- Bacterial contamination has been investigated by culture in SOC media and incubated 2 days at
37°C under shacking condition. No bacteria growth was detected in the media in contact with
the samples tested.

- Fungal contamination has been investigated by culture in liquid Sabouraud media and incubated
7 days at 25°C. No fungal growth was detected in the media in contact with the samples tested.

This report only concerns the objects submitted to analysis. This document belongs to CIRAD and shall neither be read
nor duplicated without permission.
CIRAD-BIOS-UMR117, TA A-117/E, Campus international de Baillarguet, 34398 MONTPELLIER CEDEX 5, France Tel : 33 (0)4 67 59 37 24
4/7
E-TD-09 Rapport d’essai V03 du 20/06/2018
 CIRAD - Biological Systems Department
AnimalS, health, Territories, Risks, Ecosystems (ASTRE) Unit
OIE, FAO and UE Reference Laboratory for Peste des Petits
Ruminants

RESULTS OF ANALYSIS
ASSAY REPORT Quality Control of the homologous PPR Nigeria 75-1 vaccine
RE « 15-025 »

 NGS test

Detection and Identification of adventitious viruses in vaccine batches by “Next Generation

Sequencing” is very efficient, thus providing outstanding additional guarantees on the vaccine
purity (Cabannes et al, 2014). This control has been made by a French company localised in the
Pasteur Institute (PathoQuest).

After assembly of resulting reads and taxonomic assignment, the main conclusion is:

The PPR vaccine contains sequences of BVDV3: a total of 141 reads (0.00022% of the total
number of quality filtered reads) matched the BVDV3 genome. The most likely explanation is
that BVDV3 reads correspond to virus particles contained within a culture reagent such as the
foetal calf serum. To assess or refute the presence of infectious particles of live BVDV, the
vaccine was further tested using a MDBK cell-based infectivity assay followed by a Real-time
RT-PCR targeting BVDV (see next section).

Sequences from already-known endogenous retroviruses of Vero cells are also present.

No circovirus reads are detected. The contamination could have come from the protease trypsin,
which is derived from pig and used in the production of the vaccines.

A single short sequence derived from a human papillomavirus is found. The source of the viral
fragment in the vaccine is unknown but it is believed to have come from a possible
contamination during sample handling.

Infectivity assay

To confirm or refute a BVDV3 contamination, CIRAD PPR vaccine was inoculated on BVDV
sensitive cells (MDBK) and three successive passages were realised followed by RNA
extraction on each cell culture and BVDV Real-time RT-PCR analysis using the Adiavet kit.
To be sure that this commercial kit.detects BVDV type 3, a corresponding RNA positive control
was included.

BVDV type 3 RNA positive control was well detected (Ct value of 29.8), evidencing the
efficacy of the commercial kit for this type of pestivirus.

RRT-PCR analysis of all extracted RNA after successive passages on MDBK did not exhibit
the presence of viral contamination, while the housekeeping gene was still detected (Ct value
from 23.3 to 23.71).

This report only concerns the objects submitted to analysis. This document belongs to CIRAD and shall neither be read
nor duplicated without permission.
CIRAD-BIOS-UMR117, TA A-117/E, Campus international de Baillarguet, 34398 MONTPELLIER CEDEX 5, France Tel : 33 (0)4 67 59 37 24
5/7
E-TD-09 Rapport d’essai V03 du 20/06/2018
 CIRAD - Biological Systems Department
AnimalS, health, Territories, Risks, Ecosystems (ASTRE) Unit
OIE, FAO and UE Reference Laboratory for Peste des Petits
Ruminants

RESULTS OF ANALYSIS
ASSAY REPORT Quality Control of the homologous PPR Nigeria 75-1 vaccine
RE « 15-025 »

In conclusion no Pestivirus replication was evidenced in the vaccine samples.

Pestiviruses detection Housekeeping gene

Well Name Dye Ct (dRn) Dye Ct (dRn)
RNA extraction Negative
FAM No Ct HEX No Ct
control
Water used for vaccine
FAM No Ct HEX No Ct
resuspension
Starting MDBK cells FAM No Ct HEX 23.81
Starting PPR vaccine FAM No Ct HEX 38.53
PPR vaccine 1st passage on
FAM No Ct HEX 23.71
MDBK
PPR vaccine 2nd passage on
FAM No Ct HEX 23.53
MDBK
PPR vaccine 3rd passage on
FAM No Ct HEX 23.30
MDBK
BVDV type3 RNA Positive
FAM 29.81 HEX No Ct
control (Sweden)
NTC FAM No Ct HEX No Ct
FAM positive control FAM 26.77 HEX 36.86

CONCLUSION
CIRAD PPR vaccine batch 130314 is free from Mycoplasma, viral, bacterial and fungal
contamination.

BVDV3 genome detected by NGS analysis is only RNA contamination (no infectious viral
particles), probably an outcome from irradiated foetal calf serum used in the cell culture for
vaccine production. This strengthens the importance to set up a continuous specific control to
confirm absence of pestiviruses in the vaccine production process: such a permanent control
has been in place at CIRAD since more than 15 years.

Retrovirus endogenous of Vero cells, identified also by NGS technology should be present in
all vaccines produced on these cells.

About the Papillomavirus, the rarity of specific reads in the sample (one out of 63,556,517
millions reads) is not representative of a vaccine contamination, but probably occurred during
RNA and NGS sample preparation.

This report only concerns the objects submitted to analysis. This document belongs to CIRAD and shall neither be read
nor duplicated without permission.
CIRAD-BIOS-UMR117, TA A-117/E, Campus international de Baillarguet, 34398 MONTPELLIER CEDEX 5, France Tel : 33 (0)4 67 59 37 24
6/7
E-TD-09 Rapport d’essai V03 du 20/06/2018
 CIRAD - Biological Systems Department
AnimalS, health, Territories, Risks, Ecosystems (ASTRE) Unit
OIE, FAO and UE Reference Laboratory for Peste des Petits
Ruminants

RESULTS OF ANALYSIS
ASSAY REPORT Quality Control of the homologous PPR Nigeria 75-1 vaccine
RE « 15-025 »

References

Cabannes E, Hébert C, Eloit M. Whole genome: next-generation sequencing as a virus safety test
for biotechnological products. PDA J Pharm Sci Technol. 2014, 68(6): 631-8. doi:
10.5731/pdajpst.2014.01015.

Cheval, J.; Sauvage, V.; Frangeul, L.; Dacheux, L.; Guigon, G.; Dumey, N.; Pariente, K.;
Rousseaux, C.; Dorange, F.; Berthet, N.; Brisse, S.; Moszer, I.; Bourhy, H.; Manuguerra, C. J.;
Lecuit, M.; Burguiere, A.; Caro, V.; Eloit, M. Evaluation of high-throughput sequencing for
identifying known and unknown viruses in biological samples. J. Clin. Microbiol. 2011, 49 (9),
3268 –3275.

Diallo, A, Taylor, W.P., Lefèvre, P.C. et Provost, A. Atténuation d’une souche de virus de la peste
des petits ruminants : candidat pour un vaccin homologue vivant. Rev. Elev. Méd. Vét. Pays trop.
1989. 42: 311-319.

Taylor W. P. and Abegunde A. Research in Veterinary Science, 1979, 26, 94-96 12.

Toussaint J.F., Sailleau C., Breard E., Zientara A. and De Clercq K. 2007. Bluetongue virus
detection by two real-time RT-qPCRs targeting two different genomic segments. Journal of
Virological Methods 140 (2007) 115–123

van Kuppeveld FJ, Johansson KE, Galama JM, Kissing J, Bölske G, van der Logt JT, Melchers WJ.
Detection of mycoplasma contamination in cell cultures by a mycoplasma group-specific PCR. Appl
Environ Microbiol. 1994 Jan;60(1):149-52.

This report only concerns the objects submitted to analysis. This document belongs to CIRAD and shall neither be read
nor duplicated without permission.
CIRAD-BIOS-UMR117, TA A-117/E, Campus international de Baillarguet, 34398 MONTPELLIER CEDEX 5, France Tel : 33 (0)4 67 59 37 24
7/7
E-TD-09 Rapport d’essai V03 du 20/06/2018
 UMR 117 Animals, health, Territories, Risks, Ecosystems
OIE, FAO, EU Reference Laboratory for Peste des Petits Ruminants

Montpellier, 09/06/2021

SANITARY CERTIFICATE

To whom it may concern,

By the present, we certify that the shipment to be sent to

HESTER BIOSCIENCES AFRICA LIMITED

Plot No. 647 & 648, NDC-Tamco Industrial Estate, Kibaha – Coast Region,
P.O. Box 30216, Kibaha,
Tanzania
TIN Number: 131-198-329
VAT Number: 40-030066-M

contains:

- Two flasks of Vero cells (African green Monkey Kidney) in MEM (volume of each flask:
25cm3)

- One bottle of Fetal Calf Serum (volume 500ml)

certified to be free of Mycoplasma, pestiviruses, Bluetongue virus, FMD virus, Circovirus, and
any bacterial or fungal contaminants.

Yours Sincerely,

CIRAD-BIOS-UMR117
TA A-117/E
Campus
international
de Baillarguet
34398 MONTPELLIER
Geneviève Libeau Arnaud Bataille
CEDEX 5, France

Co-heads of the OIE, FAO, EU Reference Laboratory for Peste des Petits Ruminants
Téléphone :
33 (0)4 67 59 37 24
Télécopie :
.
33 (0)4 67 59 37 98
Hester PPVR vaccine sequences bioinformatics analysis, by Jean Nepomuscene
Hakizimana, 06 February 2022
The fastq_pass file is used in the analysis. The fastq_pass contains data for 12 barcodes
(barcodes 01 to 12). After visualization of the content of each barcode file, I decided to continue
working with the barcode02 because it is the one the many sequencing reads as compared to
others.
1. Data concatenation
The “Cat” command was used to concatenate all the reads belonging to barcode02 as follow:
#cat *.fastq >mergedbarcode02.fastq
“gzip” was used to compress the concatenated file to be able to upload it in Kaiju for
metagenomics classification.
#gzip mergedbarcode02.fastq

2. Metagenomics classification of the sequencing reads

The GZ compressed file was uploaded to Kaiju for metagenomics classification, the results can
be assessed on the following links:
https://kaiju.binf.ku.dk/output/138400-
1398681584/krona.html?dataset=0&node=91&collapse=true&color=false&depth=13&f
ont=11&key=true

Figure 1: Metagenomics overview of the classified reads

 3. Remove of the sequencing adapter using PoreChop
The following command was used to remove sequencing adapters:
# porechop –i mergedbarcode02.fastq > mergedbarcode02trimmed.fastq --discard_middle
The defaults values of porechop were used to search for and trim adapters.

Figure 2: Screen shot of the adapter sequences trimming using porechop

4. Quality assessment of the reads

The quality of the trimmed reads was assessed using nanoplot version 1.35.1.
The following command line was used:
# NanoPlot –fastq mergedbarcode02trimmed.fastq –o nanoplotresults/
Below are the results of the quality assessment:

Figure 3: Basic statistics of the quality assessment of the sequencing reads

Figure 4: Some graphs obtained after quality assessment
 5. Mapping to the reference genome
The PPRV vaccine strain “Nigeria75/1” complete genome sequence (accession number:
KY628761.1) was downloaded from the NCBI database and used as reference genome. After
creating an index for the reference using the command:
# bwa index Nigeria751.fasta
The alignment of the sequencing reads to the reference genome was done as follow:
#bwa mem Nigeria751.fasta mergedbarcode02trimmed.fastq >
mergedbarcode02trimmedmapped.sam
Then the generated SAM file was converted into BAM file using the following command line:
#samtools view -S –b mergedbarcode02trimmedmapped.sam >
mergedbarcode02trimmedmapped.bam
The generated BAM file was sorted by coordinates as follow:
#samtools sort mergedbarcode02trimmedmapped.bam >
mergedbarcode02trimmedmappedsorted.bam
The sorted BAM file was indexed using the following command:
#samtools index mergedbarcode02trimmedmappedsorted.bam

The indexed BAM file was viewed in Integrative Genomics Viewer (IGV) version 2.8.9, the
results are shown below:
Figure 5: Distribution of the sequencing reads mapped to the PPRV vaccine strain
“Nigeria75/1” (accession number: KY628761.1) reference genome. A) General overview, B)
Zoom at the GC rich region.

6. Export of the reads mapped to the reference genome

To export only the sequencing reads mapped to the reference genome, the following command
line was used:
#samtools view -b -F 4 mergedbarcode02trimmedmappedsorted.bam >
mergedbarcode02mappedreadsonly.bam
Bedtools is used to convert the BAM file into fastq file. bamtofastq Convert BAM records to
FASTQ records.
The following command is used in our case:
#bedtools bamtofastq –i mergedbarcode02mappedreadsonly.bam -fq
mergedbarcode02mappedreadsonly.fastq

7. De novo genome assembly using Canu

The mapped reads were used for PPRV genome assembly using canu.
 Certificate of Analysis
Kerry Inc.
158 State Highway 320
Item: 5X59039 N-Z-AMINE AS (100# DR)
Norwich, NY
Customer ID: 023140260100000001 13815
HESTER BIOSCIENCES AFRICA LIMITED T: (315) 802-5900
PLOT NO 647 648 F: (607) 334-5022
Customer Order: 02-4612941
Customer PO: HBAL/004/19-20
Kerry Item: 5X59039
KIBAHA, TZ
Scheduled Ship Date: 01-Jul-2020

Lot: 1320031102 Date of Manufacture: 13-Apr-2020 LIMS ID: 15157051

Lot Qty: 1 DR Expiration Date: 12-Apr-2024

Analysis Test Method Specification Result Units

Enzyme, Porcine Origin USA
Bovine, Casein Origin New Zealand
Porcine Origin USA
Residue on Ignition Furnace, 650°C <=7.5 5.9 %
Loss On Drying Moisture Balance <=5.0 3.5 to 3.9 %
Degree of Digestion Amino Nitrogen/Total Nitrogen 45.3
Amino Nitrogen Formol Titration >=6 6 %
pH 2% Autoclaved Solution 6.4 - 7.0 6.8 to 6.8
Total Nitrogen Kjeldahl Nitrogen >=11.0 13.2 %
Total Aerobic Microbial Count USP <61> <=1000 <10 cfu/g
Enterobacteriaceae USP <62> <=10 <10 cfu/g
Salmonella AOAC 2003.09 Negative Negative
Clarity HACH 2100 AN <=1.1 0.1 to 0.4 NTU
Color UV <=0.18 0.11 to 0.13
Powder Test Visual Exam Fine-No Fine-No
Extraneous Matter Extraneous
Matter
Odor Sensory Conforms Conforms

Comments

Manufacturing Location: Kerry Inc.

158 State Highway 320, Norwich, NY , 13815

Generated on 6/30/2020 11:01:38 AM CST Page 1/2

 Certificate of Analysis
Kerry Inc.
158 State Highway 320
Item: 5X59039 N-Z-AMINE AS (100# DR)
Norwich, NY
Customer ID: 023140260100000001 13815
HESTER BIOSCIENCES AFRICA LIMITED T: (315) 802-5900
PLOT NO 647 648 F: (607) 334-5022
Customer Order: 02-4612941
Customer PO: HBAL/004/19-20
Kerry Item: 5X59039
KIBAHA, TZ
Scheduled Ship Date: 01-Jul-2020

Lot: 1320031102 Date of Manufacture: 13-Apr-2020 LIMS ID: 15157051

Lot Qty: 1 DR Expiration Date: 12-Apr-2024

Analysis Test Method Specification Result Units

Certified By: Rhea French, Quality Assurance
Supervisor

Generated on 6/30/2020 11:01:38 AM CST Page 2/2

 Kerry North America
3400 Millington Road
Beloit, WI 53511
T +1 608 363 1200
[email protected]
www.kerry.com

Regulatory Information Sheet

N-Z-Amine AS

Animal Origin

Not manufactured using animal origin material. Ingredients used to manufacture this
product are of non-animal origin, including any upstream materials (i.e., enzymes, etc.)
and their ancillary materials. Cleaning procedures mitigate risk for cross contamination
from other products / processes.

Manufactured by Kerry using animal origin material(s) sourced as follows:

Bovine Dairy: New Zealand
Bovine (Non-Dairy): NA
Equine: NA
Porcine: Canada, United States

BSE/TSE & Specified Risk Material

Non-Animal Origin
Not applicable as this product does not contain raw material of animal origin.

Non-Bovine Animal Origin

Not applicable as this product does not contain raw material of bovine origin.

Bovine-Dairy
RE: Statement Response to The Committee For Proprietary Medicinal Products’ (CPMP) Note For
Guidance On Minimising The Risk Of Transmitting Animal Spongiform Encephalopathy Agents
Via Human And Veterinary Medicinal Products (EMEA/410/01 Rev.3).

The following statement is a response to the document listed above and is provided in support of
users of Kerry Brand Products, which use bovine milk-derived raw materials in their
manufacture.

Specific to these bovine milk-derived products is Section 6.6 Milk And Milk Derivatives of the
Note For Guidance On Minimising The Risk Of Transmitting Animal Spongiform Encephalopathy
Agents Via Human And Veterinary Medicinal Products (EMEA/410/01Rev.3) adopted by the
Committee for Proprietary Medicinal Products (CPMP) and by the Committee for Veterinary
Medicinal Products (CVMP).

Please note that there are two conditions required for milk derivatives to be in compliance with
this note for guidance:

1. 'The milk is sourced from healthy animals in the same conditions as milk collected for human
consumption' and

Classified as General Business

 Kerry North America
3400 Millington Road
Beloit, WI 53511
T +1 608 363 1200
[email protected]
www.kerry.com

2.'No other ruminant materials, with the exception of calf rennet, are used in the preparation of
such derivatives (e.g. pancreatic enzyme digests of casein).'

This product contains only the bovine/ruminant material sources listed on the Certificate of
Animal Origin and does not use calf rennet. Any other animal sources used in the manufacture
of this product are also listed on the Certificate of Animal Origin and do not qualify as bovine/
ruminant origin. This product meets both requirements listed above, and therefore, does not
require a Certificate of Suitability.

Bovine-Non Dairy
Re: It is certified to the best of our knowledge that the above-named product does not contain
and is not derived from:
(a) as regards to bovine animals:
i. the skull excluding the mandible and including the brain and eyes, and the spinal cord of
animals aged over 12 months;
ii. the vertebral column excluding the vertebrae of the tail, the spinous and the transverse
processes of the cervical, thoracic and lumbar vertebrae and the median sacral crest and wings
of the sacrum, but including the dorsal root ganglia of animals aged over 30 months; and
iii. the tonsils, the intestines form the duodenum to the rectum and the mesentery of animals of
all ages.
(b) as regards ovine and caprine animals:
i. the skull including the brain and eyes, the tonsils, and the spinal cord of animals aged over 12
months or which have a permanent incisor erupted through the gum, and
ii. the spleen and ileum of animals of all ages.
(c) or mechanically separated meat obtained from bones of bovine, ovine or caprine animals;
(d) the animals from which this animal by-product is derived, have not been slaughtered after
stunning by means of gas injected into the cranial cavity or killed by the same method or
slaughtered by laceration of the central nervous tissue by means of an elongated rod-shaped
instrument introduced into the cranial cavity.

Country of Manufacture

This product was manufactured in the United States of America.

Genetically Modified Organisms

Non-GMO
This term is used to describe products that contain no ingredients, additives or processing
aids derived from commodities that have commercially grown GMO varieties in the supply
chain.

Non-GMO (validated)
This term is used to describe products that contain ingredients, additives or processing aids
derived from commodities that have commercially grown GMO varieties in the supply chain;

Classified as General Business

 Kerry North America
3400 Millington Road
Beloit, WI 53511
T +1 608 363 1200
[email protected]
www.kerry.com

however, they do not contain GMOs because 1) raw material records are maintained for
“Identity Preserved”, and/or 2) raw materials are tested for “PCR Negative” by our suppliers.

GMO
This term is used to describe products that contain ingredients, additives or processing aids
derived from commodities that have commercially grown GMO varieties in the supply chain.
Bioengineered

Contains detectable modified genetic material

The above product contains ingredients derived from a bioengineered source, and it
contains detectable modified genetic material.
BE ingredients: Soy

Contains ingredients derived from BE sources, but does not contain detectable modified
genetic material

Does not contain ingredients derived from BE sources

Melamine

IS NOT an at risk component, as it does not contain dairy derived ingredients.

IS an at risk component, as it does contain dairy derived ingredients. To mitigate risk, Kerry
has taken several steps to ensure the safety of the material. Kerry has controlled sourcing
programs in place. All suppliers are approved based on our Quality System standards,
ensuring product safety, regulatory compliance, and absence of Melamine. Melamine is not
used anywhere in the manufacturing facility. Products are shipped from our facilities in
sealed, tamper-evident packaging in order to prevent adulteration of this product during
distribution.

Residual Solvents

Does not contain residual solvents as noted in CPMP/ICH/283/95

Contains Class 1 solvents

Contains Class 2 solvents

Contains Class 3 solvents

Classified as General Business

 Kerry North America
3400 Millington Road
Beloit, WI 53511
T +1 608 363 1200
[email protected]
www.kerry.com

Nitrosamines

European Medicines Agency (EMA/369136/2020 and EMA/CHMP/409815/2020), the European

Directorate for the Quality of Medicines & HealthCare (Announcement to all CEP holders for
synthesized APIs regarding presence of nitrosamines of October 29, 2019), Health Canada (letter
19-119258-557) and the FDA's guidance on the Control of Nitrosamine Impurities in Human
Drugs.

This product:
Does not contain Sodium Nitrite or other nitrosating agents

Does not use Recycled or recovered solvents

Does not contain primary, secondary or tertiary amines used as a raw material,
intermediate, reagent catalyst or processing aid

Does not contain an ammonium salt used as a raw material, intermediate, catalyst or
processing aid

Does not use water as part of the manufacturing process

Proposition 65
With regard to California’s Safe Drinking Water and Toxic Enforcement Act of 1986 (Proposition
65), we have not carried out any specific analysis to confirm the absence of Proposition 65
chemicals. However, we hereby certify that, to the best of our knowledge, the above mentioned
product(s):

Does not contain any intentionally added chemicals on the Proposition 65 list.

Contains the following chemical(s) on the Proposition 65 list: __________________

Latex

Latex is not used as a raw material component or as a material of construction of any

equipment utilized to manufacture the Kerry products. Latex gloves are not utilized on site.

Revision Date: March 12, 2021

Supersedes: New

The information given and the recommendations made herein are based on our research and are believed to be accurate, but no guarantee of their accuracy is made. Data
presented herein are typical values; slight variations may occur. We guarantee that products shipped by us to you, are not, as of the date of shipment or delivery, adulterated or
misbranded within the meaning of the Federal Food, Drug and Cosmetic Act. Except as expressly set forth in the preceding sentence, the PRODUCTS DISCUSSED HEREIN ARE
SOLD WITHOUT ANY WARRANTY AS TO MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE OR ANY OTHER WARRANTY, EXPRESSED OR IMPLIED. Nothing contained
herein shall be construed to imply the nonexistence of any relevant patents or to constitute a permission, inducement or recommendation to practice any invention covered by
any patent, without authority from the owner of the patent.

Classified as General Business