The NorthBio Certificate of Analysis Explained

How is the C of A determined ?

During the bottle fill process, sample bottles are taken at regular intervals and used for testing and analysis, or held as retention samples for any future testing should it be required.  Once the sterile filtered FBS Lot is completely filled into sterile bottles, the serum is flash-frozen.  The Lot sample bottles are immediately sent out for testing.  Over 46 serum characteristics are measured and reported to Quality Control before theLotcan be released for sale.  If any characteristic does not meet Specification, the Lot  does pass QC and is rejected. The majority of the assays are performed by accredited third-party diagnostic labs.  It takes about 6 to 8  weeks for all testing to be completed and reported to QC in order that the C of A can be prepared, completed, signed and published.

The NorthBio C of A is comprised of the following information:

 A . Lot Identification information :

 Lot Number, Serum Type, Grade, Origin, Catalog Number and Expiration Date

 B.  Four columns of testing information:

                1. Test – Identifies what is being measured           .              

                2. Test Method – Identifies method of testing.

                3. Specification – The acceptable value, range, or condition of the characteristic being measured.

                4. Result – the actual value obtained from testing.  Must comply with specifications or theLotwill fail QC.       

  C. The Statement of Origin –

Signed Statement confirming the Country of Origin of the crude blood, that the blood was aseptically collected in U.S.D.A approved abattoirs, the country where the serum was processed and filtered, and that the Facilities are registered with Medical Devices (U.S.) following the guidelines of the  F.D.A. (US) and H.P.F.B. (Canada).

 Example: Lot SF07012

 A. Lot Identification Information

                 Lot Number :     e.g.    SF07012

                Serum Type:    e.g.    Fetal Bovine Serum

                Serum Grade:  e.g.  Cell Culture Grade

                Origin:              e.g. Canadian – Raw Sera was obtained from Fetal Blood collected in the country ofCanada

                Cat. Number:  e.g. NBSF-702            

                Lot Expiry Date: e.g. May 2015

B. Anayltical Tests Performed and Reported

 Sterility and Endotoxin

Aerobic Bacteria [Sterility assessment]

Anaerobic Bacteria [Sterility assessment]

Fungi [Sterility assessment]

Mycoplasma [Sterility assessment]

NOTE: Mycoplasmas infect many mammalian cell types.  Unlike bacterial contaminations, a    mycoplasma contamination is not obvious.  A suspected mycoplasma contamination must be confirmed either by PCR or long term cell culture involving special staining techniques .   Mycoplasma can alter cell physiology, growth rate, protein expression and cell appearance.

Endotoxin

Endotoxin, or lipopolysaccharide from gram neg bacterial cell wall, is an indicator of prior bacterial growth occurring in raw sera prior to sterile filtration.  Carefully handled and processed sera will have lower inherent Endotoxin levels.  Endotoxin per se is not detrimental to most cell growth, and can induce faster cell division in some cell types.   However, the presence in sera of very low levels of Endotoxin can contribute to unwanted cellular processes.  For example, background cellular response in proliferation assays, reducing the relative increase in proliferation attributable to the factor being studied.  When culturing Endotoxin sensitive cells it is best to use sera with the lowest level of Endotoxin possible.

Viral Safety Testing

Viral Safety Testing is performed in full compliance with the US Federal Code or Regulations (9CFR Section 113.53). Many Bovine diseases are highly transmissible and can have potentially devastating socio-economic effects on animal and human populations irrespective of national borders.  For this reason the production and global transport of animal serum is highly regulated.   Viral tests are performed and reported in order to satisfy National Veterinary Regulatory Authorities around the world, facilitating the global transport of FBS.  Most counties accept the guidelines of the USDA and/or EU blah blah.  However, sera may also be required to meet specific criteria of national Veterinary Health Authorities.   Performing and reporting mandated testing for various Bovine Viruses helps to protect domestic agricultural industries as well as human health from pathogens that might be inadvertently imported by contaminated serum.

BVD

Bovine viral diarrhea (BVD) is a disease of cattle which reduces productivity and increases death loss. BVD is a notifiable disease and eradication programs are administered in many countries worldwide.  Geneally two subtypes: BVD I and BVD II, a more severe hemorrhagic form occurring in theUSandCanada.  BVD in FBS can infect many types of cell cultures and modify cell physiology, confounding cell culture research. 

HA and CPE

9CFR testing requires that FBS is tested for CytoPathic Effects (CPE) and HemeAbsorbing agents (HA).  The tests used are broad range indirect tests that detect for many viruses, including IBR and PI3.

Bovine Parvo

Bovine parvovirus (BPV), also known as Hemadsorbing Enteric Virus, is a member of the parvivirus group, with three significant sub-species: BPV1, 2 and 3.  BPV most commonly causes diarrhoea in neonatal calves and respiratory and reproductive disease in adult cattle. The distribution of the virus is worldwide.

Bovine Adeno 3 and Bovine Adeno 5

Bovine Adeno viruses, of which there are 10 subtypes, cause gastrointestinal, respiratory and ocular disease in Cattle depending on the subtype.  Infection can reduce productivity.  Healthy animals may harbor virus. 

BRSV

Bovine respiratory syncytial virus (BRSV) is an RNA virus classified as a pneumovirus in the paramyxovirus family. This virus was named for its characteristic cytopathic effect—the formation of syncytial cells.  BRSV infections are associated with respiratory disease in cattle.

REO Virus

Reovirus is a dsDNA virus that can affect the Gastrointestinal system and respiratory tract.  Originally though to be somewhat benign, reoviruses are now known to be associated with serious disease (e.g. Bluetonge disease).

Rabies

The rabies virus is a single stranded, enveloped, neurotropic  RNA virus that causes fatal disease in human and animals.

Blue Tongue

Bluetongue disease or catarrhal fever is a very serious, non-contagious, non-zoonotic, insect-borne,  viral disease of ruminants. It is caused by the Bluetongue virus (BTV), a type of reovirus.  Bluetongue disease has warranted more attention by Veterinary authorities as the geographic distribution of the disease is moving into more northern countries, which is thought to be a result of global warming.

BVD Antibody

Bovine Viral Diarrhea (BVD) Serum Neutralization Assay.  Tests for the amount of Antibody to BVD I and BVD II present in the sera.  It is an indicator of prior exposure to the virus. 

Biochemistry          

Total Protein (30 – 45 g/l)

Measured to confirm age of donor animals.  Fetal Bovine Serum contains significantly less protein than Calf or Adult Bovine Serum.  Also an indicator of 100 % Fetal Serum content.

 The following key Biochemical characteristics are measured and reported.

Albumin – Most abundant serum protein. Acts as carrier protein for hydrophobic steroids and fatty acids.

Globulins – Group of serum proteins comprised of of alpha 1, alpha 2, beta and gamma globulins.

Bilirubin – Heme containing breakdown product of Hemoglobin, normally present in blood plasma.

ALT – Alanine transaminase (ALT), also called serum glutamic pyruvate transaminase (SGPT) test.

AST – aspartate aminotransferase (AST), also called called serum glutamic oxaloacetic transaminase (SGOT) test.

LDH – Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate. LDH is often used as a marker of tissue breakdown as LDH is abundant in red blood cells and can function as a marker for hemolysis. A blood sample that has been handled incorrectly can show false-positively high levels of LDH due to erythrocyte damage.

Cholesterol  

Glucose

Urea

Creatinine

Sodium

Potassium

Calcium

Phosphorus

Osmolality (Specification: 280 – 340 mOsm/Kg)

A measure of all the chemical particles present in the serum Lot.  Serum Lots that fall outside of range indicate the serum was diluted or contains added factors.

 Serum Identity

Ouchterlony method performed to confirm serum as Bovine.

IgG – A measure of IgG concentration in the FBS Lot.  FBS contains inherently low IgG concentrations (Specification < 250 mg/ml) compared with Calf or Adult Bovine Serum.  High IgG levels suggests calf or adult sera has been blended with FBS.

Hemoglobin

Hemoglobin in sera comes from Red Blood Cells that lysed during raw sera collection and/or processing.  While not normally detrimental to cell culture, the hemoglobin concentration is an indicator of the care taken during collection, the temperature at which the clotting blood was held, as well as the length of time between collection and separation of the raw sera from the blood clot.  Hemoglobin concentrations above 25 mg/dl suggest inferior collection and handling of the raw sera.  Poor collection and handing will correlate with a high Endotoxin level in the serum, as bacterial growth will occur over prolonged periods of time prior to sterile filtration.

pH

Serum value must fall within the specified range,

Specific Gravity

Measure of density of serum relative to water.  It is another measure the chemical content of serum.  Serum should have a Specific Gravity above 1.010 g/L.

ALP – Alkaline phosphatase (ALP) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids.  Bioactivity assay.

GGT – Gamma-glutamyl Transferase test.  Bioactivity assay.

Chloride

Uric Acid

Triglyceride

 Cell Culture Performance Testing

Plating Efficiency

A relative performance assay measuring attachment and proliferation of an adherent diploid cell line.  Plating Efficiency is an indicator or the robustness of the FBS Lot.  That is to say, are nutrients present in sufficient concentration in the serum to permit a colony to form from a single cell in a single well.  

Population Doubling

A assay to determine the cell population doubling time of a diploid cell culture supported by the FBS Lot.  This is a relative test as each every cell line will have its own inherent doubling rate.

Cell Toxicity

An assay that measures the ability of the FBS Lot to support growth of a diploid cell line when plated at very low concentration in 30 % FBS.  Assures the FBS Lot will not be toxic to cells in culture.

Growth Promotion

A relative performance assay measuring theLot’s ability to support the continuous growth of a diploid cell line thru multiple passages.  Indicates robustness of the FBS Lot.

Appearance

Visual determination of color and clarity.

 C. The Statement of Origin

This serum has been derived from blood aseptically collected in approved abbatoirs.  The serum was aseptically processed and filter sterilized in canada in a facility registered with Medical Devices (U.S.) following guidelines of F.D.A. and H.P.F.B. (Canada). The Country of Origin of the crude blood (Donor cow ) is Canada

 

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