Services Archive - Page 4 of 5

Environmental Monitoring

Even in the most advanced and secured clean room environment, microbial contamination is unavoidable. Environmental monitoring review of microbial controlled environments such as clean rooms and Laminar air flow hoods, is required in order to assure control is being maintained.

Sampling methods rely on growth of microorganisms on media such as Soyabean Casein Digest Agar (TSA) and Sabouraud dextrose agar (SDA) which supports the growth of a wide range of bacteria, yeast, molds and fungi.

Hy Laboratories offer the service of our skilled and certified samplers. Our samplers will come to your facility and perform the entire sampling process. We provide the plates and an Active Air Sampler and all other needed equipment according to the costumers needs.
As well as routine sampling, a PQ validation of clean rooms is also offered including planning and advising, writing of protocol, performing the sampling and issuing a final PQ report.
After the sampling process our sampling team delivers all plates and information to the Laboratory for the incubation step. Incubation time and temperature varies between different growth media. At the end of the incubation period, colony forming units are being counted and reported. Following the incubation and count, an identification of bacterial growth may be provided

We also offer complimentary tests for Environmental: Clean room validation, Bacterial Identification, Ecology & Morphology, Disinfectant Efficacy

Dialysis water

Monitoring dialysis water quality is crucial to avoid excesses of known or suspected harmful elements being carried in the water and transmitted to the patient. Therefore, it is very important for the dialysate to meet the norms and conditions specified by the health ministry. It is recommended that every dialysis system will be checked monthly and after replacement or treatment of any component of the system.

In our lab we perform several tests to assess the quality of the dialysate:

TVC (Total viable count) – Gives an estimate regarding the concentration of all microorganisms such as bacteria, yeast, and molds, Pseudomonas aeruginosa, Fecal coliforms.
BET – test the dialysate for the presence and quantification of endotoxins by performing BET (Bacterial Endotoxin Test) also known as LAL test.
ICP-MS – test the dialysate for harmful heavy metals presence.
*For microbial testing – all dialysis water samples are tested within 6 hours from sampling time.

We also offer a Complementary test for Dialysis Water: Bacterial Identification, Ecology, and Morphology .

Mycoplasma Testing

Mycoplasma is the smallest free-living, self-replicating organism, found mostly on the outer surface of the cell membrane and rely on their hosts for nutrients. Consequently, mycoplasma has adverse effects on the characteristics of a contaminated cell line including changes in growth, morphology, metabolism, synthesis of DNA, RNA and protein, the antigenicity of cell membranes, and more.

Mycoplasma frequently infects cell cultures and cell-derived biological products (e.g., vaccines and recombinant protein), but the contaminated matrix does not display any visible signs of contaminations (such as changes in turbidity, pH or cytology). Moreover, once found, mycoplasma contamination is difficult to eliminate since most routine antibiotics used in cell culture are ineffective against mycoplasma, and common filtrations techniques fail to remove mycoplasma due to their small size.
Most contaminations are caused by 8 species of mycoplasma and it is estimated that 5-35% of cell cultures in current use are infected by mycoplasma.
Therefore, testing for mycoplasma is a necessary quality control requirement to assure the safety of biotechnological products and related materials introduced during the manufacturing process.

As a leading service lab in Israel, we offer both microbial and molecular testing methods: mycoplasma culture method (28 days), nested PCR method (1-3 days) and the latest innovative Real-time PCR (8-24 hours).
While both the microbial and molecular methods have similar sensitivity (as required by the Pharmacopeia), the most essential differences between those methods are that the culture test detects only viable mycoplasma, whereas a PCR-based test detects both live or dead mycoplasma, but with fastest turnaround time.
Our standard operation procedure relies on USP<63> and EP<2.6.7> and GMP approved.

We offer complementary services and products for Mycoplasma Testing:
Cell Culture, Virology as well as Hy-Mycoplasma detection Kit

Disinfectant Efficacy

The purpose of this study is to determine the efficacy of the disinfectant reagents used for the sanitization of surfaces in controlled manufacturing facilities and performed according to USP <1072>.

The disinfectants efficacy is evaluated by the determination of survival rate of selected microorganisms spiked on coupons (such as PVC, stainless steel, glass etc.) or test tubes after being exposed to selected disinfectants for a pre-defined contact time.

The study can be performed with ATCC challenge microorganisms and with Sponsor environmental isolates (Wild Strains).

We also offer complimentary tests for disinfectants efficacy: Bacterial Identification

Cleaning & Disinfection

The manufacturing of reusable medical device must accompany detailed instructions on how to reprocess the device between patient uses in the hospitals. Cleaning of the device is a critical step in reprocessing of any device after it has been used on a patient. Failure to remove foreign material from the device can interfere with the effectiveness of subsequent disinfection and/or sterilization.

Residues of protein and TOC are being tested as part of Cleaning Validation, after performing a shortened cleaning procedure according to TIR30.

Log reduction of chosen microorganisms is being tested as part of Disinfection Validation, after performing a shortened disinfection procedure according to TIR12.

Bioburden

Materials and products that are to be sterilized should be examined to determine the level of bioburden (microbial load/count) in the article, prior to its final sterilization.

Monitoring of in-process bioburden of pharmaceutical components and products is an essential element of the overall contamination-control program for appropriate sterilization process control.

Bioburden monitoring should be designed for the recovery of a broad range of microorganisms that are likely to be present in the material being processed.

Pre-sterilization bioburden analysis should be conducted on samples that are representative of materials produced during routine preparation and processing

We also offer Complementary tests for Bioburden: Bacterial Identification, Ecology, and Morphology.

Sterility & LAL

Sterility test:

We are considered to be the most critical lab in Israel for sterility test product release. That specialty in sterility testing is due to our vast experience in adopting rapid methods for sterility such as BacT/ALERT and HB&L (see below) and by the fact that we test and support the critical companies in Israel from Cell therapy, Biopharma, and medical devices sectors.

Sterility testing is performed in order to establish the presence or absence of viable microorganisms and is carried out in a clean room, under aseptic conditions with highly trained technicians. Sterility test may be carried out using a Membrane Filtration technique or by Direct Inoculation of the culture medium with the product to be examined. Samples are incubated for 14 days in two types of media, which allow the detection of both aerobic and anaerobic microorganisms.

Hy Laboratories provides sterility tests in a Clean Room Class 1000 (ISO 6/Grade B) and under laminar airflow hood Class 100 (ISO 5/GradeA) conditions and serves the healthcare, pharmaceutical, medical device, and cell therapy industries.

We also offer complimentary tests for Sterility: Bacterial Identification, Ecology, Morphology, Clean Room Validation, Disinfectant Efficacy & BET (bacterial endotoxin test)

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BET (bacterial endotoxin test):

The bacterial endotoxin test, which is also known as LAL (Limulus Amebocyte Lysate), is an assay for the detection and quantification of bacterial endotoxins, a component of the cell walls of gram-negative bacteria, in drugs and biological products. Endotoxins are fever-inducing substances that can be harmful or fatal if administered to humans above certain concentrations.

The endotoxin test is derived from the fact that the horseshoe crab (Limulus Polyphemus) blood cells (amebocytes) react with bacterial endotoxins.

We provide services in 3 assays of the Bacterial endotoxin test:

· LAL Gel-Clot assay – a qualitative assay, considered as the “Gold Standard” in pharmacopeial monographs. The test involves mixing the sample and the LAL in a test tube and incubating, a positive result is indicated by the formation of a clot at the bottom of the tube. Suitable for all sample types.

· Kinetic Turbidimetric assay – a quantitative photometric assay, utilizing the clot formation as a parameter. The test involves mixing the sample and the LAL and placing the test tube for incubation inside a 96 tube spectrophotometer, which tracks the change in absorbance as the clot is forming compared to a standard curve. Suitable for drug, medical devices, and water testing.

· Kinetic Chromogenic assay – a quantitative colorimetric assay, quite similar to the turbidimetric assay. Tracks the change in color of the sample-LAL mix compared to a standard curve. suitable for medical devices and water testing.

*Both turbidimetric and chromogenic assays involve the use of a 21 CFR part 11 compliant software.

qPCR testing

Gene expression (GMP certified)

Reverse transcription-quantitative PCR (RT-qPCR) is considered the gold standard for accurate, sensitive, and fast measurement of gene expression. The RT-qPCR method for gene expression determination is based on relative or absolute quantification using TaqMan or SYBR green assays. Relative quantification is performed by normalizing the qPCR values obtained for the tested gene to those of a known, stably expressed, gene and those in a reference setting (e.g. reference cells or animal). Absolute quantification is based on a standard curve, which is prepared from samples of known template concentration.

Residual DNA (GMP certified)

The requirement for quantification of residual DNA impurities in biopharmaceuticals is based on safety concerns.
The procedure for detection of host cell residual DNA in a tested sample by quantitative PCR (qPCR) includes DNA purification from the tested sample, qPCR detection of residual DNA using TaqMan or SYBR green assays, and primers/probe designed for the tested host cell DNA. Parallel spiking of host cell DNA into the tested sample, following by the same procedure, is used as a positive control.

Gene copy number (GMP certified)

Genetic stability testing is used to analyze a production strain’s stability by demonstrating that the expression system has not undergone any mutations or rearrangements that would affect the integrity of the product. Determination of a transgene copy number in the genome is one of the major tests required as part of genetic stability testing. The qPCR method for Gene Copy Number (GCN) determination is based on normalization of the qPCR values obtained for the tested gene to those of a known single copy gene of the relevant organism.

DNA Biodistribution (GMP certified)

Biodistribution analysis is a key component in the evaluation of gene therapy protocols’ efficacy and determines the distribution of gene therapy vectors in distant or intended tissues. DNA biodistribution qPCR analysis in tested tissues is based on the use of targeted DNA (e.g. human Alu primers and probe) vs. the tested animal endogenous control used in order to normalize the amount of template used. DNA amounts are determined compared to a control tissue or a standard curve equation generated for targeted DNA per targeted animal DNA.

Microorganism & Pathogen Identification (GMP certified)

qPCR has been shown to be a great tool for microorganism (including viruses) presence test and quantitation due to its speed, accuracy, and its enormous dynamic range. The microorganism amounts can be quantitated in either a relative or an absolute manner, using a standard curve.

A standard curve is generated using the target microorganism/virus nucleic acids (DNA or RNA) and encompasses the expected range of the microorganism quantity. Then, the tested sample raw value obtained in the qPCR, is used to estimate the microorganism quantity in the sample.

We also offer Complementary tests for QPCR: Sanger sequencing, Next-generation sequencing.

DNA Fragment (STR)

DNA Fragment Analysis is the characterization of DNA fragments based on their size, including specific Gene Fragment. Labeling each fragment with a specific fluorescent dye, multiple fragments can be separated by size using capillary electrophoresis, and analyzed in a single sample. Hylabs offers tailor-made Gene fragment analysis for a variety of applications such as:

Microsatellite analysis (STR) for animal breeding, linkage mapping, and pathogen sub-typing
Cell Authentication
Fingerprint for plants and cell lines
SNP Genotyping for allele discrimination
gDNA fingerprinting for genome typing
CRISPR editing events identification

We also offer Complementary tests for DNA Fragment (STR) and Gene Fragment:
Sanger Sequencingc, Next-generation sequencing

Microbial Identification

Bacterial Identification – BID (GMP certified):

Sequencing of the Ribosomal RNA (rRNA) genes, followed by comparison of the resulting sequences to ribosomal gene databases, has proven to be the most accurate method available for bacterial identification, providing the highest sensitivity and specificity. BID service support customers’ regulatory requirements including environmental monitoring, tracking and trending analysis and sterility testing.

Fungal Identification – FID (GMP certified):

Sequencing of the Ribosomal RNA (rRNA) genes, followed by comparison of the resulting sequences to ribosomal gene databases, has proven to be the most accurate method available for yeast and mold (fungal) identification, providing the highest sensitivity and specificity. FID service support customers’ regulatory requirements including environmental monitoring, tracking and trending analysis and sterility testing.

RAPD:

Randomly amplified polymorphic DNA (RAPD) is a powerful molecular method for diversity typing of bacteria and yeasts. This method consists of using a single arbitrary short primer that generates polymorphic fragments following PCR amplification and gel electrophoresis. These polymorphic fragments, used as fingerprints, allows discrimination between different strains within a species.

MLST:

Multi-locus sequence typing (MLST) is a highly discriminatory method of characterizing bacterial isolates on the basis of the sequences internal fragments of seven housekeeping genes. The data obtained by MLST can be used to address basic questions about the evolutionary and population biology of bacterial species. A major advantage of MLST is the ability to compare the results obtained in different studies via online databases.

MALDI-TOF-MS:

As a leading service lab in Israel we offer both methods – MALDI-TOF-MS and the molecular biology sequence.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) is a powerful analytical mass spectrometry technique that is used in our lab for the identification of cultured bacteria. By identifying the unique chemical signature of each sample, bacteria can be identified down to the species level using the MALDI-TOF-MS. Samples are isolated on growth media and incubated until significant growth occurs (usually between 24-48 hours). Only when growth is observed in the isolated sample, identification procedures can take place. The MALDI-TOF-MS pulls bacterial particles mixed with ions from the matrix into a vacuum and measures each particle’s mass according to its time of flight, giving a unique signature to each sample. The information gathered is then compared to our existing database to identify the specific species of the bacteria. In case the isolate is not found in the MALDI library, it can be identified by the r-RNA sequence and that isolate can be added to the MALDI library. Also, in case the isolate is mold/fungi/yeast – molecular identification is recommended.

Ecology and Morphology:

Morphology – In addition to the identification of microorganisms we offer characterizing of microorganism’s morphology on an agar plate. This method is used to help distinguish between groups of microorganisms.

Ecology – In addition to the identification of microorganisms we offer ecology description of the identified microorganism based on known databases.

Bacterial subculture:

A subculture is a new microbiological culture made by transferring a colony of microorganism from a previous culture to fresh growth medium. A subculture is needed in order to isolate a specific microorganism from a mixture or prior to identification process.

Gram Stain test:

Although Gram stain is a very old method, it is still being used as a rapid method to release cellular products before transplantation. We are the only lab worldwide that has been approved by the FDA for that unique method. Gram stain is a differential staining technique, used for differentiation of bacteria into two groups: gram-negative and gram-positive. The differentiation is made on the basis of their cell wall structure, which serves to give the organism its size and shape as well as to prevent osmotic lysis. The material in the bacterial cell wall which confers rigidity is peptidoglycan.

Gram-positive cell walls have a thick peptidoglycan layer beyond the plasma membrane – Gram-positive cell walls stain blue/purple with the Gram stain.
Gram-negative cell walls are more complex, they have a thin peptidoglycan layer and an outer membrane beyond the plasma membrane – Gram-negative cells will stain pink with the Gram stain.

In our lab, gram staining is used for the detecting and differentiation of bacteria from biological products produced in cell substrates, specimens and bacterial cultures.

Storing of Microbial Stock:

Creating a frozen bacterial culture for future analysis. Following Environmental monitoring, Bioburden, MLT or BID tests there is an option to prepare a stock culture of the contaminant for an analysis at a later date, stored at -80C.