Archives: Services

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. 

 

 

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.

Using all the knowledge and experience we accumulated at Hy Laboratories, at our GMP laboratory, we can provide an array of microbiological and molecular biology services. We can tailor our services and methods to match most challenges and proof of concepts. From a proof of concept for a new product to preparing for a submission to any regulatory agent.

Using the knowledge of the manufacturing department at Hy Laboratories and the experience of the laboratory staff we provide small scale aseptic manufacturing, mainly for R&D level or animal experiments. These small scale manufacturing is in a GMP environment

Expression and purification (E. coli)

E. coli expression is one of the most widely used systems for recombinant proteins because of its many advantages that include low setup and running costs, short timeline from cloning to protein recovery, high recombinant protein production levels and scalability from small to very large culture volumes.

We offer:

• Codon optimization and gene synthesis
• Pilot expression optimization
• Purification of tagged or tag-free protein
• Tag removal
• QC by SDS-PAGE (native or denatured conditions)

Protein Quantification

Many biochemical applications require the quantification of total protein amount per sample. Hylabs can perform total protein quantification assays for samples such as tissue, cultured cells, bacterial culture, and more. We can perform protein quantification and standard curve for data interpretation using fluorescent or colorimetric detection methods, while employing the reagents relevant for each method such as Bradford and BCA

Host Cell Protein Analysis (HCP)

A biopharmaceutical analytical test, for the detection and quantification of residual low-level protein impurities related to the process of drug generation within host organisms. HCP analysis is aimed at identifying residual endogenously expressed proteins remaining in a preparation following its purification. HCPs can hamper drug efficacy, and their identification is critical for biopharmaceutical companies according to regulations.

Incubation & Count test:

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 regulatory 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. Samples which arrive at our lab are incubated in controlled and calibrated incubators. Incubation time and temperature varies between different growth media. At the end of the incubation period, colony forming units (CFU) are being counted and reported.

Following the incubation and count, an identification of bacterial growth can be provided

Growth Promotion Test (GPT):

A growth promotion test (GPT) ensures that the medium is capable of supporting or inhibiting the growth of indicator microorganisms, both ATCC strains and customers’ wild strains. Our lab provides GPT for culture solid media and broths using various methods such as: Direct Inoculation, Pour Plate, Spreading on Surface and Membrane Filtration. In case of broths media, the medium is inoculated with a small number of microorganisms and the result is qualitative, either “growth” or “no growth”. For GPT on solid media, the test is quantitative and the recovery of the spiked microorganisms should be 50-200 % from spiked control plates.

Media fill:

Media fill is designed to evaluate and validate aseptic performance either by qualified personnel or of assembly lines producing sterile products. Using a sterile microbiological growth medium in place of the actual articles, the test assess whether the aseptic procedures are adequate to prevent contamination during real time production.

Microbial Limit Test (MLT):

This test is intended for quantitative enumeration of bacteria and fungi that may grow under aerobic conditions of non-sterile products/raw materials (according to USP <61>) and to test for the absence of specified microorganism (according to USP <62>).

The products/raw materials to be tested are diverse: solutions, creams, powders, tablets, inhalers, medical cannabis and more.

This test is intended for samples that are classified as non-sterile.

Before routine testing on a product, a validation is  recommend to assure the suitability of the method to the product.

Recovery of Biological Indicators (BIs):

An important aspect of the sterilization validation program involves the use of biological indicators when appropriate. A biological indicator (BI) is a well-characterized preparation of a specific microorganism with a known resistance to a specific sterilization process. Sterilization alternatives: steam, dry heat, gas, radiation, vapor, chemical, or filtration. After completion of the sterilization procedure, the BIs is removed and transferred to the lab within a noted time (NMT 4 h), then added to a suitable medium and incubated for 7 days. Sterilization indicators respond to sterilization process parameters in a non-quantitative fashion; i.e., they show passing or failing results.

Verification of spore population:

The aim of the test is to evaluate the total count of viable spores in biological indicators (standardized inoculated carriers) pre-sterilization.

The correct use of biological indicators (BIs) in the development, validation, and control of sterilization processes requires that their population and resistance be accurately known.

When evaluating vendor-supplied BIs, the viable spore count shall be between 50% and 300% of the manufacturer’s stated value.

Antibiotic Potency:

The activity (potency) of antibiotics can be demonstrated either by chemical methods or by microbial methods. A reduction in antimicrobial activity may not be adequately demonstrated by chemical methods thus the microbiological assay is the standard analytical method.

The cylinder-plate assay depends on diffusion of the antibiotic from a vertical cylinder through a solidified agar layer in a Petri dish. The growth of the specific microorganisms inoculated into the agar is prevented in a circular area or “zone” around the cylinder containing the solution of the antibiotic. The activity of antibiotics is calculated by measuring the inhibition zone in comparison to a standard curve. Antibiotic Potency is tested according to USP <81>

Autoclave cycle:

Autoclave Cycle – We can offer you autoclave sterilization cycle according to your needs. Starting from small scale (dentist autoclave) to a larger industrial scale autoclaves. Those cycles can be based on our validations if that’s suitable, or perform your specific validation cycles.

Sterilization validation:

Sterilization validation is a crucial part of the development of medical device in order to assure the SAL (sterility assurance level) needed for a medical device. At Hy Laboratories we perform sterilization validation for products going through Radiation sterilization (Gamma/Beta), Ethylene Oxide sterilization, Autoclave, STERRAD sterilization and Dry Heat. We have vast experience in setting those validations with complexed and combined medical devices including submission to the CE and FDA.

Antimicrobial Preservative effectiveness (AET)

Antimicrobial preservatives are substances added to aqueous pharmaceutical products. Nonsterile dosage forms may have preservatives added to protect them from growth of microorganisms inadvertently introduced during or subsequent to the manufacturing process. In the case of sterile articles packaged in multiple-dose containers, antimicrobial preservatives are added to inhibit the growth of microorganisms that may be introduced from repeatedly withdrawing individual doses. The aim of the test is to demonstrate the effectiveness of added antimicrobial preservatives by inoculating the product with appropriate number of microorganisms. The products are divided into categories, the test procedure and specifications are determined according to the product category and pharmacopoeia. Our standard operation procedure relies on USP<51> & EP<5.1.3>.