Biochemistry, Molecular Biology, Physiology, Microbiology, Immunology, Pharmacology & Drug Discovery
Preparation Lecture Notes, Multiple Choice Questions, Scientific Discoveries

MCQ on Heme synthesis & Related Inherited Disease (Porphyria)

August 27, 2019

Multiple Choice Question on Heme synthesis & Related Inherited Disease (Porphyria)

1) Heme is a tetrapyrrole structure consisting of Fe+2 ions in the center of the porphyrin ring. Which of the following proteins consist of heme?
a) Myoglobin
b) Hemoglobin 
c) Cytochrome
d) All of the above



2) Besides erythroid precursor cells, which of the following is the site for the synthesis of heme?
a) Kidney
b) Spleen
c) Liver
d) Heart

3) Which of the following is not the precursor for the synthesis of Heme?
a) Glycine
b) Succinyl CoA
c) Both of the above
d) None of the above

4) Which of the following is the rate-limiting enzyme for the synthesis of heme?
a) delta-aminolevulinic acid synthase 1
b) Uroporphyrinogen synthase III
c) Protoporphyrinogen oxidase
d) Ferrochelatase


5) The synthesis of heme involves both cytosolic and mitochondrial cellular compartment. Which of the following enzyme-catalyzed reaction does not occur in the cytosol?
a) delta-aminolevulinic acid synthase 1
b) Uroporphyrinogen synthase III
c) Protoporphyrinogen oxidase
d) Ferrochelatase

6) Lead positioning causes the increases the accumulation and urinary excretion of coproporphyrin III and ALA in the urine. Which of the following enzyme is inhibited by lead metal?
a) ALA synthase and Protoporphyrin oxidase
b) ALA synthase and Ferrochelatase
c) ALA dehydratase and Protoporphyrin oxidase
d) ALA dehydratase and Ferrochelatase

7) The heme, hematin and Cytochrome P450 represses the synthesis of the following enzyme thereby reducing heme synthesis.
a) delta-aminolevulinic acid synthase 1
b) Uroporphyrinogen synthase III
c) Protoporphyrinogen oxidase
d) Ferrochelatase

8) Which of the following intermediate of the heme synthetic pathway is water-insoluble and excreted via feces via biliary tract?
a) delta-aminolevulinic acid
b) Porphobilinogen
c) Uroporphyrinogen
d) Protoporphyrin


9) The porphyrias are a group of metabolic disorders that result from partial deficiencies of the enzyme of the heme biosynthetic pathway. The porphyria may be classified as acute and nonacute. Which of the following porphyria is not acute type?
a) Acute intermittent porphyria
b) Hereditary coproporphyria
c) Variegate porphyria
d) Porphyria cutanea tarda

10) Identify porphyria that does not have accumulation and increased excretion of delta-aminolevulinic acid and porphobilinogen among the following:
a) Acute intermittent porphyria
b) Hereditary coproporphyria
c) Congenital erythropoietic porphyria
d) Variegate porphyria

11) Porphyria Cutanea Tarda (PCT) is the most common porphyria characterized by photosensitivity, skin lesions. Which of the following statement is false regarding PCT
a) PCT is caused by the deficiency of uroporphyrinogen decarboxylase enzyme
b) Accumulation of photo-sensitizers such as uroporphyrinogen and carboxyl-substituted porphyrinogen
c) Accumulation of ALA (delta-aminolevulinic acid) and PBG (porphobilinogen)
d) None of the above

12) Which of the following is the most sensitive method for the measurement of porphobilinogen?
a) Colorimetric Ehrlich's reaction method
d) Ion exchange chromatography based method
c) HPLC coupled MS
d) Spectral analysis of porphobilinogen

13) All of the porphyrias are inherited in autosomal dominant fashion except the following two autosomal recessive conditions
a) ALA synthase deficiency
b) ALA dehydratase deficiency
c) Congenital erythropoietic porphyria
d) Hereditary coproporphyria

14) The enzyme deficient in erythropoietic protoporphyria is
a) delta-aminolevulinic acid synthase 1
b) Uroporphyrinogen synthase III
c) Protoporphyrinogen oxidase
d) Ferrochelatase

15) The intravenous administration of hemin is required for reducing symptoms during acute porphyria attacks. The hemin decreased the  gene synthesis of the following enzyme
a) delta-aminolevulinic acid synthase 1
b) Uroporphyrinogen synthase III
c) Protoporphyrinogen oxidase
d) Ferrochelatase

Answers
1-d) All of the above
2-c) Liver
3-d) None of the above
4-a) delta-aminolevulinic acid synthase
5- b) Uroporphyrinogen synthase III
6-d) ALA dehydratase and Ferrochelatase
7)- a) delta-aminolevulinic acid synthase 1
8)-d) Protoporphyrin
9)-d) Porphyria cutanea tarda
10)- c) Congenital Erythropoietic porphyria
11)-c) Accumulation of ALA (delta-aminolevulinic acid) and PBG (porphobilinogen)
12) -c) HPLC coupled MS
13)- b) ALA dehydratase deficiency & c) Congenital erythropoietic porphyria
14)-d) Ferrochelatase
15-a) delta-aminolevulinic acid synthase 1


Heme Synthesis & Porphyria (Diagram NEMJ)

MCQ on Heme synthesis & Related Inherited Disease (Porphyria) MCQ on Heme synthesis & Related Inherited Disease (Porphyria) Reviewed by Biotechnology on August 27, 2019 Rating: 5

Medical Microbiology: MCQ on Human Immunodeficiency Virus (HIV/ AIDS)

August 27, 2019

Multiple Choice Question on Human Immunodeficiency Virus (HIV/AIDS)


1) There are two types of HIV (Human immunodeficiency virus) viruses- HIV 1 & HIV 2. HIV 1 virus is found worldwide.  HIV 2 (a less pathogenic than HIV 1) is mainly found in which part of the world?
a) Asia
b) West Africa
c) Northern Europe
b) North America

2) Which of the following is an important molecule present in the outer membrane of the HIV that helps to enter the host cell?
a) Polysaccharides
b) Glycoproteins
c) Proteins
d) Lipopoysaccharides

3) All of the following are the examples of the route through which HIV can be transmitted from one person to another, EXCEPT?
a) Unprotected sexual contact with an infected person
b) From infected mother to the fetus
c) From the mosquito bite carrying the virus
d) Exposure to contaminated blood and blood products

4) HIV belongs to which of the following genus member of the virus?
a) Orthomyxovirus
b) Lentivirus
c) Parvovirus
d) Reovirus

5) All of the following statements regarding HIV infection in human is true, EXCEPT?
a) Person once infected will remain infected for life if untreated
b) Monocytes and macrophages are the major reservoirs of the virus
c) The opportunistic infections in AIDS are mainly due to the loss of cell-mediated immunity
d) The center of the virus has the main antigenic properties

6) Which of the following enzyme is required for viral replication and plays a critical role in the pathogenesis of HIV infection?
a) RNA polymerase
b) DNA polymerase
c) RNA polymerase II
d) Reverse transcriptase

7) The envelope protein gp120 (Glycoprotein 120) is required for the attachment of the HIV virus to CD 4 receptors of target host cells. Identify the immune cells that consist of CD 4 receptors?
a) T helper cells
b) Monocytes
c) Macrophages
d) Dendritic cells

8) Identify the chemokine receptor cell present in the host macrophages that helps in the primary attachment of HIV?
a) CxCR 4
b) CCR5
c) Both of the above
d) None of the above

9) Which of the following is an important HIV antigen in determining the early detection of HIV infection?
a) p24
b) gp120
c) Pol gene
d) Gp120

10) In many patients, the chronic HIV infection progresses to AIDS (Acquired Immunodeficiency Syndrome) which is characterized by multiple opportunistic infections. Which of the following common bacterial infections is seen globally in HIV infected patients?
a) Pneumocystis carinii pneumonia
b) Tuberculosis
c) Candidiasis
d) Toxoplasmosis

11) When was the first human AIDS case reported in the USA?
a) 1990
b) 1982
c) 1981
d) 1991

12) Highly active antiretroviral therapy (HAART) is a combination therapy that includes protease inhibitor class drugs like indinavir and saquinavir and other drugs prescribed for HIV infection. What is the main goal of these drugs?
a) Inhibitors of the enzyme protease
b) Inhibits viral replication and viral load
c) Prevents the interaction between the virus and the coreceptor
d) All of the above

13) All of the following are the examples of a biological specimen that can be taken for the laboratory diagnosis of HIV infection, EXCEPT?
a) Blood
b) Saliva with the presence of blood
c) Genital secretions
d) Urine with no presence of blood

14) Which of the following drug can significantly reduce the transmission of HIV infection from mother to baby?
a) Acyclovir
b) Zidovudine
c) Ceftriaxone
d) None of the above

15) All of the following are the current preventive methods of HIV infection, EXCEPT?
a) Safe and protected sex
b) Use of available vaccines
c) Use of sterile injection needles
d) All of the above


Multiple Choice Answers
1)- b) West Africa
2)- b) Glycoprotein
3)- c) From the mosquito bite carrying the virus
4)- b) Lentivirus
5)- d) The center of the virus has the main antigenic properties
6)- d) Reverse transcriptase
7)- a) T helper cells
8)- b) CCR5
9)- a) p24
10)- b) Tuberculosis
11)- c) 1981
12)- b) Inhibits viral replication and viral load
13)- d) Urine with no presence of blood
14)- b) Zidovudine
15)- b) Use of available vaccines 
Medical Microbiology: MCQ on Human Immunodeficiency Virus (HIV/ AIDS) Medical Microbiology: MCQ on Human Immunodeficiency Virus (HIV/ AIDS) Reviewed by Biotechnology on August 27, 2019 Rating: 5

RNAi based therapeutics (Givosiran) for treatment of Acute Hepatic Porphyria

August 20, 2019

RNAi based therapeutics for treatment of Acute Hepatic Porphyria

RNAi based therapeutics of acute hepatic porphyria is recent example of how biochemical understanding of the metabolic pathways pave a path for drug discovery. Porphyria is a group of rare disease caused by a deficiency of the enzymes in the heme synthetic pathway. The heme synthetic pathway consist of nine steps catalyze by different enzymes. Please refer to pathway for detail below.

What is the therapeutic target enzyme of heme synthesis?


Among these nine enzymes, the repression of delta-amino levulinic acid synthase 1 (ALAS1)  is a lead candidate for the therapeutic development for following reason:
a) ALS 1 is a rate-limiting enzyme for the synthesis of heme and the presence of high heme repress the ALS1 synthesis thereby inhibiting the overall pathway.
b) ALS1 catalyzes the synthesis of ALA, an intermediate substrate for the downstream enzymes in the pathway. ALA and PBG have a neurotoxic effect and lead to neuronal damage. Therefore reduction of these intermediates, in theory, reduces the toxic effect.
c) Hematin a heme product has proven to mitigate the toxic effect of toxic metabolites by repressing ALS1. Therefore, direct inhibition of ALS1 should mitigate symptoms of any porphyria.

How does RNAi based therapeutics work?

Anylam Therapeutics developed an RNAi based therapeutic candidate (Givosiran) to selectively repress ALAS1 and reduce the disease symtoms. This ALAS1 siRNA is conjugated with GALNAc that selectively binds to the liver receptor and internalized into the liver tissue. In the liver tissue, it binds to the ALAS1 mRNA, thereby reducing the expression of the ALAS1.
Mechanism of action of RNAi based therapeutics for suppression of ALAS1

What are the clinical outcomes from clinical study ENVISION?

Anylam submitted the new drug application and market authorization application to FDA to FDA and EMA respectively. Both regulatory agencies granted the fast track review and the decision is expected in 2020. The filing was based on the efficacy and safety data from phase 3 ENVISION which is a randomized, double-blind, placebo-control trial with 94 subjects enrolled in the study. The study result showed significant reduction of primary endpoint composite annualized attacks (74%) in the treatment group compared to the placebo. The composite annualized attacks defined as the attack that require hospitalization, urgent healthcare visit or hemin administration of relief of symptoms. 

Snapshot of Efficacy Date: Positive outcome of RNAi based therapy for Acute Hepatic Porphyria
The biochemical endpoints included the estimation of aminolevulinic acid and porphobilinogen in the plasma. The sustained 90% reduction of ALA and PBG in the treatment group was observed a surrogate biomarker for RNAi induced repression of ALAS1 protein. Overall the study shows the significant improvement in the subjects with acute intermittent porphyria.

As with RNAi class drug, mild liver toxicity was observed in the subset of the subjects. The estimated glomerular filtration rate is stable in the study enrolled subject. In porphyrias, liver disease and chronic kidney disease may also exist before the treatment, and treatment has not shown to greatly aggravate these chronic diseases.

Summary 

Based on these clinical data available for public, this drug is expected to obtain approval and would be available for the treatment for acute hepatic porphyria. While successful clinical studies are pivotal for showing clinically safety and efficacy in humans, other factors including CMC, drug manufacturers have a significant impact on the approval process. I am hopeful that this drug will make into the market, would greatly impact the much need Acute Hepatic Porphyria populations.



RNAi based therapeutics (Givosiran) for treatment of Acute Hepatic Porphyria RNAi based therapeutics (Givosiran) for treatment of Acute Hepatic Porphyria Reviewed by Biotechnology on August 20, 2019 Rating: 5

What are the methods to improve drug tolerance in ADA assay?

August 07, 2019

What are the methods to improve drug tolerance


1. Acid Dissociation
2. Solid-phase extraction with acid dissociation (SPEAD)
3. Affinity Capture Elution (ACE)
4. Bead extraction and acid dissociation (BEAD)
5. Precipitation and acid dissociation (PandA)

1. Acid Dissociation methods:


Acid-base treatment is a widely adopted dissociation method for improving the drug tolerance of the assay. The method is based on the principle that acid or base breaks any drug: antibody complex interaction thereby allowing the detection/capture reagents to the ADAs. The method involves incubation of ADA:drug complex with weak acid with pH 2-3 (e.g 300mM acetic acid, 0.1 mM Glycine) followed by neutralization with base (1 mM Tris HCl, pH 9.5).  But when a high concentration of circulating drugs is present, there is a higher probability that ADA re-associates with the drug during the neutralization step. The acid may also negatively affect the assay signal/noise ratio and assay sensitivity. In those cases, an alternative sample extraction technique may be appropriate. The detail protocols may be obtained from the following MSD link

2. Solid-phase extraction with acid dissociation (SPEAD) method:


The solid-phase extraction and acid dissociation sample treatment method are performed in two-steps: a) A biotin-avidin capture technique physically separate ADA and ADA-drug complexes from the sample matrix, which eliminates the excess unbound drug and other serum proteins that can interfere in the assay.  b) The acid dissociation step removes the ADA from the biotin-avidin complex but does not dissociate the biotin-streptavidin bond. The high pH after neutralization encourages the binding of ADA and ADA: drug complex to the surface of a microtiter ELISA plate. The SPEAD treatment method has shown to improve drug tolerance by 10-100 folds than without sample treatment while retaining the sensitivity of the assay. (Smith  et al. 2007)

Overview of method for SPEAD


3. Affinity Capture Elution (ACE) method:

The ACE method is based on the principle where first acid treatment dissociates the ADA-drug complexes followed by neutralization in the solid-phase allowing ADA to be affinity captured. After washing away excess drug, ADA is diluted off with acid and subsequently bound to a fresh surface. Bound ADA are subsequently detected by the addition of biotinylated drug followed by streptavidin-HRP and substrate. (Bourdage et al., 2007). ACE treatment method has shown to improve drug tolerance up to 500 ug/ml while retaining the sensitivity of the assay below 500 ng/ml.

The recently described ACE- bridging assay format combines the affinity and capture step of ACE format followed by dual-labeled drug bridging of the bridging assay. Although bridging assay is highly sensitive but prone to interference by a high concentration of unlabeled drugs which competes with a labeled reagent for ADA binding. The ACE- bridging assay format overcomes the poor drug tolerance of bridging assay by removing the excess drug. The ACE-bridging assay was able to tolerate high drug concentration while retaining the sensitivity of the assay below 100ng/ml. (Chen et al, 2016)

MSDB uses an initial acid dissociation to break up potential drug–ADA complexes (indicated by dashed line). ADA are measured by ECL detection through a bridging ELISA format using biotin and sulfo-tagged drug for capture and detection, respectively. SPEAD uses biotinylated drug for ADA capture followed by acid dissociation (indicated by dashed line) to allow selective ADA transfer to a second plate. Detection occurs by ECL using a sulfo-tagged drug. ACE uses an initial acid dissociation to break up potential drug–ADA complexes (indicated by dashed line) followed by ADA capture on a drug-coated polystyrene plate. A second acid-dissociation step is used to selectively transfer the ADA to a second plate for colormetric detection using biotinylated drug and streptavidin HRP (Future Science 2010).


4. Biotin Extraction and Acid Dissociation (BEAD) method:

This method utilizes dissociation of ADA: drug complex with acid followed by biotin drug in neutralization buffer allowing the ADA to bind to the biotinylated drug. Biotinylated drug: ADA was extracted with streptavidin-coated magnetic beads. Second acid treatment of separated biotinylated drug: ADA( magnetic beads ) allows selective extraction of ADA from any residual drugs. This method was implemented in an accurate measurement of neutralizing antibodies in LC-MS format as well as cell-based NAb format (Jiang et al. 2014, Xu et al. 2015, Niu H et al. 2017).

5. PandA method:

In the PandA method, the ADA: free drug complex formed in the presence of excess free drug is precipitation using PEG. The precipitates are treated with acid and dissociated free ADA is captured onto the large capacity surface and detected using a specific detection agent. The assay with this treatment method was able to tolerate high drug concentration while retaining the sensitivity of the assay below 100 ng/ml. (Zoghbi et al, 2015)

Summary and Conclusion:

The characterization of drug tolerance is a crucial step in assay development. It is the regulatory expectation and best practice to prove the drug tolerance of the assay above maximum drug concentration (Cmax) in the circulation. To use any acid treatment method, the assay method should be characterized using treatment methods including cut point, sensitivity, selectivity etc.

What are the methods to improve drug tolerance in ADA assay? What are the methods to improve drug tolerance in ADA assay? Reviewed by Biotechnology on August 07, 2019 Rating: 5

ELISPOT: Quantification of T cell response

August 06, 2019

ELISPOT Principle 




ELISPOT: Quantification of T cell response ELISPOT: Quantification of T cell response Reviewed by Biotechnology on August 06, 2019 Rating: 5

MALS (Multi Angle Stable Light Scattering)

August 06, 2019

MALS (Multi Angle Stable Light Scattering) 



MALS (Multi Angle Stable Light Scattering) MALS (Multi Angle Stable Light Scattering) Reviewed by Biotechnology on August 06, 2019 Rating: 5

SURFACE PLASMON SPECTROSCOPY (BIACORE)

August 06, 2019

Principle of Surface Plasmon Resonance - Biacore



Biacore Evaluation Software

SURFACE PLASMON SPECTROSCOPY (BIACORE) SURFACE PLASMON SPECTROSCOPY (BIACORE) Reviewed by Biotechnology on August 06, 2019 Rating: 5

Mass Spectrometry

August 06, 2019

Mass Spectrometer Principle & Application

Mass Spectrometry Mass Spectrometry Reviewed by Biotechnology on August 06, 2019 Rating: 5

Technology Review: Gyrolab Platform and its application in Biomarker, Pharmacokinetic and immunogencity testing

August 06, 2019
Introduction to Gyrolab 

-Gyrolab® immunoassay platforms miniaturize, integrate and automate the analysis of large numbers of samples in parallel to minimize user error, significantly increase productivity and cut time to develop and validate assays to obtain study results.

-Gyrolab software, designed for 21 CFR part 11 compliance, makes data collection, evaluation, and migration into your existing LIMS straightforward.



Gyrolab Instrumentation and Principle-I 



Gyrolab Instrumentation and Principle-II






References for PK and Biomarker Assay 

Development and validation of an alpha fetoprotein immunoassay using Gyros technology.
 
Given et al. 
J Pharm Biomed Anal. 2012 May;64-65:8-15.
Abstract
Circulating alpha fetoprotein (AFP) is a diagnostic and prognostic biomarker for hepatocellular carcinoma (HCC) with potential utility as a pharmacodynamic endpoint in rodent tumor models. This application is limited, however, by low sample volumes, highlighting the need for sensitive, sample-sparing biomarker assay methods. In order to improve the utility of AFP as an oncology biomarker, we developed a method for AFP using the Gyrolab™, an automated microimmunoassay platform. Commercially available antibodies were screened to identify optimal combinations that were then used in a multi-factorial design of experiments (DOE) to optimize reaction conditions. Analytical validation included assessments of accuracy and precision (A&P), and dilutional linearity/hook effect, as well as reagent and sample stability. The method is reliable, with total error, a measure of accuracy and precision, less than 30% for all concentrations tested. AFP concentrations were measurable in diseased mice and undetectable in normal mice. Therefore, this novel, low volume AFP immunoassay is suitable for pre-clinical drug development, where its miniaturized format facilitates serial sampling in rodent models of cancer.
Click here to read full text 


Overcoming disease-specific matrix effect in a clinical pharmacokinetic assay using a microfluidic immunoassay technology. 
Williams et al. 
Bioanalysis. 2017 Aug;9(16):1207-1216
Abstract: 
AIM: Etrolizumab, a humanized monoclonal antibody, has demonstrated clinical remission in a Phase II study of ulcerative colitis patients. In the Phase III program, a second indication, Crohn's disease was added. The pharmacokinetic ELISA used in the Phase I/II studies in normal human and ulcerative colitis sera exhibited matrix interference in the Crohn's disease population, necessitating implementation of a new technology. Methodology & results: Optimization of the original ELISA and assay redevelopment using different antibody pairs did not result in substantive improvements, necessitating implementation of an alternative technology for assay development. CONCLUSION: We highlight the challenges encountered with optimization/redevelopment of the original ELISA and discuss results of the new assay on the Gyros platform.

Click here for full text

Bioanalytical platform comparison using a generic human IgG PK assay format. 
Leary BA et al.
Immunol Methods. 2013 Nov 29;397(1-2):28-36
Abstract
A comparison of four different ligand-binding assay technology platforms (ELISA, Meso Scale Discovery®, Gyros® and AlphaLISA®) was conducted using quantitative assays for the measurement of a human IgG₁ monoclonal antibody (MAb) in rat serum. The assays used common reagents for Fc-specific measurement to determine total levels of a human IgG MAb drug analyte and all were fully optimized for use on each platform. Mock MAb study samples were prepared and analyzed using all platforms to assess assay performance. Assay parameters such as sensitivity, dynamic range, minimum required dilution and sample volume as well as other considerations such as per-run cost, technology availability, requisite equipment and necessary reagent modifications were evaluated toward the determination of a default go-to assay platform for monoclonal antibody biotherapeutics in this laboratory. Based primarily on superior assay performance, Meso Scale Discovery and Gyros were selected from the four technologies evaluated as our default platforms for non-regulated (discovery) study support. As an adjunct, immunoaffinity LC-MS/MS was explored as an alternate platform for generic Fc quantitation and was found to perform similarly to the ligand-binding assays.
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Immunogenicity ADA assays

Solution ELISA as a platform of choice for development of robust, drug tolerant immunogenicity assays in support of drug development.
Mikulskis et al. 
J Immunol Methods. 2011 Feb 28;365(1-2):38-49.
Abstract 
Humanized monoclonal antibody therapeutics are in many ways indistinguishable from the anti-therapeutic/anti-drug antibodies generated in humans. Therefore, immunogenicity assessments to such therapeutics pose unique challenges in clinical trials especially when significant drug interference is encountered. There are several technology platforms based on the bridging immunogenicity assay format, which have been successfully used for detection and quantification of anti-drug antibodies (ADA) in serum or plasma samples. Enzyme-Linked Immunosorbent Assay (ELISA) and Electrochemiluminescent (ECL) immunoassay formats are among the most popular technology platforms. Pretreatment of samples with acid can also be used to lower drug interference. While ECL technology platform offered many advantages over traditional solid-phase ELISA methods, reliance on a single (or limited) vendor source became a significant concern within the biopharmaceutical industry especially for immunogenicity assays that need to be implemented over a period of many years in support of a single drug development program. We describe herein a systematic evaluation of solid-phase ELISA, GYROS, AlphaLISA, ECL Immunoassay, and solution ELISA platforms for detection of anti-drug antibodies with the goal of selection and development of a robust technology platform that meets the desired performance characteristics for most immunogenicity assays and can be easily implemented in a typical immunoassay laboratory. As part of this effort the Design of Experiments (DOE) approach was utilized in optimization of sample acid treatment conditions in order to improve drug tolerance in the evaluated assay platforms. After the initial evaluation of various technology platforms, a solution ELISA format was chosen for further development to support clinical trials for a humanized therapeutic antibody. As part of the assay development, flexible use of digoxigenin and 6-(2,4-dinitrophenyl) aminohexanoic acid (DNP) for labeling antibodies was evaluated and is presented in this manuscript. In addition, simple methods for evaluation and qualification of streptavidin-coated plates and overcoming soluble target interference in solution ELISA have also been investigated and highlights of these investigations are discussed. The selection of the solution ELISA format was based on availability of generic reagents, achievement of optimal drug tolerance and robust assay performance on a platform that is readily available in many laboratories. This approach removed the heavy reliance on specialized equipment sourced from a single vendor and assay conditions described here are broadly applicable to other immunogenicity assays across many biologics both during clinical development setting and in the post-marketing arena.

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Improving anti-drug antibody assay performance in Gyrolab for therapeutic recombinant antibody Infliximab
Cecilia Bill(Master thesis)
Abstract
Monoclonal antibodies can be used as targeting therapies for several diseases. One major concern when using these therapies is anti-drug antibodies which may hamper the drugs efficiency. Gyrolab is an automated platform which can be used to develop bridging immunoassays where the anti-drug antibodies affinity towards the monoclonal antibody is utilized. Anti-drug antibody immunoassay development on Gyrolab is limited mainly by three factors which may inappropriately affect signal intensity levels. In this project different variants of bridging immunoassays based on drug Fab fragments have been developed for monoclonal antibody Infliximab, with the purpose to illustrate the effects of these three factors.
Findings indicate that an assay based completely on drug Fab fragments is more sensitive compared to an assay based on intact drug since less affected by unspecific interactions between drug reagents and complex formations. Surprisingly findings also indicate that an assay based completely on drug Fab fragments is affected by human anti-hinge antibodies which decrease assay sensitivity. The most optimal assay variant is based on the combination between intact capture drug and Fab fragment as detection. This variant is insensitive to false positive reactions caused by Rheumatoid factor and human anti-hinge antibodies, less prone to form unspecific interactions between drug reagents and complex formations in the presence of anti-drug antibodies. The optimal assay variant also demonstrates best drug tolerance in combination with acid dissociation.

Technology Review: Gyrolab Platform and its application in Biomarker, Pharmacokinetic and immunogencity testing Technology Review: Gyrolab Platform and its application in Biomarker, Pharmacokinetic and immunogencity testing Reviewed by Biotechnology on August 06, 2019 Rating: 5

Digital Droplet PCR : Investigating Biodistribution for Gene Therapy

August 06, 2019


Digital Droplet PCR Principle (ddPCR)


Digital Droplet PCR : Investigating Biodistribution for Gene Therapy Digital Droplet PCR : Investigating Biodistribution for Gene Therapy Reviewed by Biotechnology on August 06, 2019 Rating: 5

Principle of Chromatography: Ion Exchange, Size Exclusion and Affinity Chromatography

August 06, 2019

Ion exchange chromatography



Affinity Chromatography




Principle of Hydrophobic Interaction Chromatography



Size Exclusion Chromatography





AKTA






Principle of Chromatography: Ion Exchange, Size Exclusion and Affinity Chromatography Principle of Chromatography: Ion Exchange, Size Exclusion and Affinity Chromatography Reviewed by Biotechnology on August 06, 2019 Rating: 5

FDA raise concern over data manipulation for Zogensma (Gene therapy for SMA)

August 06, 2019
 Zolgensma is the first gene therapy product approved for the treatment of children less than two years of age with Spinal Muscular Atrophy (SMA). Norvastis obtained the approval of the drug May 2019 & has been a blockbuster with a price tag of 2 million dollars. In the recent  FDA inspections, FDA observed that the standards were not meet in accordance to GMP guidance. Those include
  • Failure to follow the standard procedures
  • Failure to report complete data sets for the potency testing of the gene therapy product
  • Failure to thoroughly review any unexplained discrepancy whether or not the batch has been already distributed.
  • Laboratory records do not include completed records of any testing and standardization of laboratory standards.

What action FDA is taking?

FDA are carefully assessing the issue of the manipulation of the product testing data used in the production process and are conducting a thorough assessment of the information from a recently completed inspection.
The agency will use its full authorities to take action, if appropriate, which may include civil or criminal penalties.

 What is Zolgensma?

Zolgensma is the first gene therapy product approved for the treatment of children less than two years of age with Spinal Muscular Atrophy (SMA). The product is an adeno-associated virus vector-based gene therapy that targets the cause of SMA. The vector delivers a fully functional copy of human SMN gene into the target motor neuron cells. A one-time intravenous administration of Zolgensma results in expression of the SMN protein in a child’s motor neurons, which improves muscle movement and function, and survival of a child with SMA.

What were the clinical evidences from clinical trial?

The drug was approved on May 24 after careful review of the BLA packets including manufacturing, nonclinical safety data, clinical safety and efficacy data. According to FDA "The safety and effectiveness of Zolgensma is based on an ongoing clinical trial and a completed clinical trial involving a total of 36 pediatric patients with infantile-onset SMA between the ages of approximately 2 weeks and 8 months at study entry. The primary evidence of effectiveness is based on results from the 21 patients treated with Zolgensma in the ongoing clinical trial. In this trial, there are 19 remaining patients, who range in age from 9.4 to 18.5 months; 13 of these 19 patients are at least 14 months of age. Compared to the natural history of patients with infantile-onset SMA, patients treated with Zolgensma also demonstrated significant improvement in their ability to reach developmental motor milestones (e.g., head control and the ability to sit without support)."

 How did agency find out about the discrepancy?

Subsequently, on June 28, following the FDA’s approval of the product, the agency was informed by AveXis Inc., the product’s manufacturer, about a data manipulation issue that impacts the accuracy of certain data from product testing performed in animals submitted in the biologics license application (BLA) and reviewed by the FDA.

FDA raise concern over data manipulation for Zogensma (Gene therapy for SMA) FDA raise concern over data manipulation for Zogensma (Gene therapy for SMA) Reviewed by Biotechnology on August 06, 2019 Rating: 5
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