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FAQ

Frequently asked questions

Recombinant DNA technology is a robust nucleic acid-based tool that has opened up a new age for a variety of scientific innovations. Using it, a gene or multiple genes can be identified, cut, and inserted into the genome of another organism. Recombinant DNA technology has been effectively used to produce various human proteins in microorganisms such as recombinant human serum albumin. The choice of recombinant products is increasing in the market over the existing products due to plenty of benefits it offers. BSA and HSA have been used for a long time but possesses some serious concerns of viral infectivity, purity, endotoxins, and immunogenicity. Recombinant technology majorly uses three components:

1. Host organism 2. Vector 3. Enzymes (Restriction enzyme, Polymerase, and ligase)

The target gene sequence of the protein and vector (in which the target sequence will be inserted) is cleaved using the specific restriction enzymes to produce complementary ends. The sequence is then ligated with the vector using ligase and amplified later with the polymerase to get multiple copies. The vector containing the target gene sequence is then transformed into the yeast-based expression system (Pichia Pastoris), which is scaled up for production of the protein.

Recombinant human serum albumin (rHSA) can be expressed in various prokaryotic and eukaryotic hosts. However, pre-clinical and clinical trials prove yeast produced rHSA to be safe and efficient. Recombinantly produced human serum albumin can be a superior alternative having high purity and batch-to-batch consistency. It is an endotoxin-free preparation with the least coloration. The yeast produced rHSA may have multiple industrial applications. It can be used as a stabilizer in drug formulations, as an efficient reagent in cell culture and research, and also as a blood volumizer in therapeutics.

The use of rHSA provides added advantages such as

Lack of impurities and serum components
Greater purity
Lack of human pathogens
Batch-to-batch consistency
Assured supply

Plasma-derived HSA can be replaced with highly pure rHSA as a preferable alternative for its clinical, pharmaceutical, and research applications. The advantage of such substitution may significantly improve the performance and quality of therapeutics, maintaining product safety, and consistency.

The yeast-based Pichia Pastoris expression system is the best option for the production of recombinant human serum albumin due to easy scale-up and high purity. Additionally, the post-translational modification property of the expression system, which offers efficient proteolytic processing, protein folding, disulfide bond formation, and adept glycosylation are major reasons for considerations.
The methylotrophic yeast expression system has proven to be highly successful in rHSA production since they produce correctly folded tertiary protein structure which is secreted into the medium directly thus easing the extraction process. The Pichia Pastoris system secretes a very minimal level of endogenous proteins as the culture medium supporting its growth does not contain any added protein. Hence, the secreted heterologous protein will consist majorly of the total protein in the culture medium. There are many benefits of this system such as:

Synthesis of complex tertiary structures
Very high success rate
Efficient and fast
Feasibility in purification
Easy scale-up
Regulatory approval

An antigen is a protein structure present on the surface of bacteria or virus that is known for eliciting immune responses in the host, stimulating the production of complementary antibodies. With core expertise in recombinant technology, we create high-quality antigens for biotechnology and pharmaceutical markets. Lately, PCR based molecular diagnostic methods have been used widely but their complex handling, expensiveness often restricts them as the first choice. Antibody-based diagnostic tests are also common for various diseases, but it faces some challenges such as delay in diagnosis and false-positive results. These challenges raise the need for highly robust and effective diagnostic kits to steadfast the detection of complex viruses. In light of this, antigen-based diagnostic kits are proving to be highly effective. Nowadays, antigens have been widely used in several commercial antigen-based detection immunoassay kits.

When manufacturing antigen-dependent diagnostic kits for viruses-like HIV, HBV, and HCV, it is always better to go for the recombinant version. The biosafety implications involved with the handling of viruses often raise concerns and challenges during the manufacturing process. Therefore, it is better to work with recombinantly produced antigens expressed in a robust system. Multiple expression systems have been explored earlier by researchers for the production of antigens, which include both prokaryotic and eukaryotic systems. However, systems like E.coli were not considered to be robust and efficient as they lack requisite post-translational modification. The E.coli based expression systems lack the machinery, which can help produce proteins (Antigen) with proper conformation. This, in turn, leads to poor specificity and sensitivity when used in the diagnostic kits.

We at Lazuline Biotech use the mammalian expression system (Pichia Pastoris), which overcomes the challenges in other expression systems. The methylotrophic yeast-based expression system is apt to produce recombinant proteins (antigens) as it offers proper glycosylation and folding properties. Thus, recombinant antigens produced using the system closely resemble those found in nature and we are able to design diagnostic kits that offer high sensitivity and specificity, giving robust results.

Enterokinase, also known as enteropeptidase is an enzyme that plays a crucial role in digestion among humans and other animals. It is majorly produced by cells localized in the duodenum. Enterokinase is known to be involved in various underlying biological pathways to support a cascade of events. One such important role is in the conversion of trypsinogen (a zymogen) into trypsin (the active form), which in turn helps in the activation of several digestive enzymes in the pancreas. Considering the important role of enterokinase in the digestive process, its absence can result in severe digestive disorders.
It is a serine protease that is anchored to the intestinal system with the help of a disulfide-linked heavy chain and a light chain consisting of the catalytic subunit. Conventionally, the enterokinase has been isolated from various living organisms which is later purified and concentrated in the requisite ratios. Though proven to be effective, there are some concerns about viral infectivity, endotoxins, and purity of the enzyme. To overcome these challenges researchers are looking forward to a recombinant version of the enzyme which can be produced using an appropriate eukaryotic or prokaryotic based expression system.

Hepatitis C Virus (HCV) antigen is a protein present on the surface of the virus. It is the core antigen present on the virus surface and mainly observed in blood post 2 weeks of infection or invasion of the virus into the host. Considering the simple and less expensive resources involved in the viral loading testing of the virus, some of the researchers suggest it to be of great use in resource-limited settings.
One of the biggest advantages of using HCV core antigen testing is that the same testing platform can be used for anti-HCV testing. This allows reflex HCV core antigen testing in samples that are anti-HCV positive.
Worldwide, more than 200 million people are infected by the Hepatitis C virus (HCV). At least 50 million of these patients are potential targets of liver cirrhosis or cancer. Hepatitis C is mainly transmitted from person to person through blood contact. Therefore, almost every country has made it mandatory to screen blood donors for HCV. In order to produce a diagnostic kit, three to four HCV antigens are used: i) Core antigen, ii) NS3, iii) NS4, and iv) NS5.
HCV antigens are widely used in diagnostic kits, as the cases of HCV have increased among people recently. HCV cases are high among HIV positive cases. The effectiveness, reliability, quick and cost-effective pricing of the HCV antigen testing has enhanced its place in the market as an alternative to Hepatitis C Polymerase Chain Reaction, which is used for long for the diagnosis of acute HCV infection.

With over 40 million infected people, AIDS is one of the worst humanitarian crises the world is facing. With no cure available, the most important step to combat this crisis is through prevention. The primary requirements for prevention are education and identifying the infected.
The concern about HIV started during the early AIDS epidemic when there was a lack of any significant tests to identify the infected patients carrying the virus. However, the efforts of researchers and scientists soon helped in the development of the HIV antibody test late in 1985 which was initially designed for screening of blood products and not for the diagnosis of AIDs. Later the first-generation diagnostic assays were developed, which helped detect HIV with 6-12 weeks of viral exposure. However, one common challenge everyone was facing during that time was of false-positive results. To combat this challenge, two test algorithms were designed which work using a western blot or immunofluorescence test. The second and third-generation HIV tests helped in reducing the window of detection to 3 weeks from 6 weeks, post-infection. To further reduce the window, fourth-generation tests came into picture which was mainly dependent on p24 antigen detection for the screening of the samples. As a result, the window was reduced to 11 to 14 days along with reduced false-negative results. The fifth-generation HIV test overcomes the challenge in the earlier generation of detecting both antigen and antibody yet incapable to differentiate them. The series of inventions and discoveries have helped reduce the test window to up to 2-week post-exposure with reduced number false-positives, highlighting the evolution of HIV antigen-based testing that has fueled the diagnostic industry with quality-driven outcomes.

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