Glycosylation is a key structural and functional element found on a wide variety of biotherapeutics. As such, alterations in glycan profile can significantly affect the efficacy of a drug through, for example, half life in the bloodstream or biological activity as well as being a potential source of immunogenicity. The glycan profile can be selected and controlled through the choice of cell line as well as control of bioreactor conditions. The use of analytical techniques that provide structural data on this type of post translational modification are vital in the development and characterisation of biologics. Techniques in glycan structural characterisation are discussed in this presentation.
Bioanalytical Method Development and Validation..
Bioanalytical method validation include all the procedure that demonstrate that a particular method used for quantitative measurement of analyte in given biological matrix are reliable and reproducible for intended use.
Bioanalytical Method Development and Validation..
Bioanalytical method validation include all the procedure that demonstrate that a particular method used for quantitative measurement of analyte in given biological matrix are reliable and reproducible for intended use.
Gyros Protein Technologies is one of the leading platform for the Nanoliter-Scale Immunoassays.
Specialties in Immunoassays in the biotherapeutic workflow, peptide synthesis for academic and industrial applications.
FirstSource Laboratory Solutions is one of the authorized suppliers of Gyros Immunoassays.
For more details visit us at https://www.firstsourcels.com/gyrolab
Joe McCarthy, Dell Tech Laboratories Lab Services Manager will introduce you to Corrositex®/OECD 435 and demonstrate:
• How easily you can perform Corrositex®
• What you need to perform Corrositex®
• Time & cost advantages of Corrositex®
• Regulatory acceptance of Corrositex®
For full webinar video see: https://www.youtube.com/watch?v=sDPreBmZnPM&t=94s
This presentation details the definition of cell cytotoxicity and cell viability, the difference between the two term and methods of assessment of cells in culture for presence and absence of cytotoxic chemicals or metabolites.
Target identification, target validation, lead identification and lead
Optimization.
• Economics of drug discovery.
• Target Discovery and validation-Role of Genomics, Proteomics and
Bioinformatics.
• Role of Nucleic acid microarrays, Protein microarrays, Antisense
technologies, siRNAs, antisense oligonucleotides, Zinc finger proteins.
• Role of transgenic animals in target validation.
In this slide contains types of HPLC Columns, Plate theory and Van Deemter Equation.
Presented by : Malarvannan.M (Department of pharmaceutical analysis).
RIPER,anantpur.
In silico drug designing is the drug design which can be carried out in silicon chip,i.e., within computers. The slides are helpful to know a brief description about in silico drug designing.
Nowadays, Cell and Molecule Analysis are critical steps for studying cellular mechanisms in any Life Sciences lab, as it offers additional information about Intracellular Molecular Targets in a real Biological environment.
Canvax™ offers a wide range high quality of Cell-based tools and Molecule Detection Kits designed for greater accuracy and increased Throughput for most common measurements like Cell Viability, Proliferation, Cytotoxicity, Reporter Gene or Oxidative Stress Assays.
BioTech Journey through the Phases of Commercializing a ProductCBG Benefits
This presentation was delivered by Breck Arnzen of the ArnzenGroup and Susan Nemetz of The NemetzGroup at the HR BioTech Connect Group meeting in January 2013.
They shared a variety of guidance to help companies navigate the process of commercializing a product in the BioTech industry.
Gyros Protein Technologies is one of the leading platform for the Nanoliter-Scale Immunoassays.
Specialties in Immunoassays in the biotherapeutic workflow, peptide synthesis for academic and industrial applications.
FirstSource Laboratory Solutions is one of the authorized suppliers of Gyros Immunoassays.
For more details visit us at https://www.firstsourcels.com/gyrolab
Joe McCarthy, Dell Tech Laboratories Lab Services Manager will introduce you to Corrositex®/OECD 435 and demonstrate:
• How easily you can perform Corrositex®
• What you need to perform Corrositex®
• Time & cost advantages of Corrositex®
• Regulatory acceptance of Corrositex®
For full webinar video see: https://www.youtube.com/watch?v=sDPreBmZnPM&t=94s
This presentation details the definition of cell cytotoxicity and cell viability, the difference between the two term and methods of assessment of cells in culture for presence and absence of cytotoxic chemicals or metabolites.
Target identification, target validation, lead identification and lead
Optimization.
• Economics of drug discovery.
• Target Discovery and validation-Role of Genomics, Proteomics and
Bioinformatics.
• Role of Nucleic acid microarrays, Protein microarrays, Antisense
technologies, siRNAs, antisense oligonucleotides, Zinc finger proteins.
• Role of transgenic animals in target validation.
In this slide contains types of HPLC Columns, Plate theory and Van Deemter Equation.
Presented by : Malarvannan.M (Department of pharmaceutical analysis).
RIPER,anantpur.
In silico drug designing is the drug design which can be carried out in silicon chip,i.e., within computers. The slides are helpful to know a brief description about in silico drug designing.
Nowadays, Cell and Molecule Analysis are critical steps for studying cellular mechanisms in any Life Sciences lab, as it offers additional information about Intracellular Molecular Targets in a real Biological environment.
Canvax™ offers a wide range high quality of Cell-based tools and Molecule Detection Kits designed for greater accuracy and increased Throughput for most common measurements like Cell Viability, Proliferation, Cytotoxicity, Reporter Gene or Oxidative Stress Assays.
BioTech Journey through the Phases of Commercializing a ProductCBG Benefits
This presentation was delivered by Breck Arnzen of the ArnzenGroup and Susan Nemetz of The NemetzGroup at the HR BioTech Connect Group meeting in January 2013.
They shared a variety of guidance to help companies navigate the process of commercializing a product in the BioTech industry.
PEGS the essential protein and antibody engineering summit Nicole Proulx
PEGS - The Essential Protein Engineering Summit is the premier event for antibody and protein science research and the biologics industry, with more than 1,800 participants in attendance from over 30 countries. Join us in Boston this May 4-8, 2015 to share insights and best practices with colleagues, connect and form new collaborations during copious networking opportunities, learn from world-renowned thought leaders, discover industry trends and find solutions to current challenges. PEGS is knowledge-sharing at its best.
PepTalk: The Protein Science Week is an annual gathering where industry experts from around the world convene to share case studies, unpublished data, breakthroughs and solutions that support and enhance research, and to gain new perspectives on the evolution of biologics.
Increasing knowledge of genetics and cell processes leads to potential new biologic (and drug) targets at each step in the protein-production process. This leads to new therapies, which in turn lead to new understanding of diseases. Here is an update on relatively new drugs called biologics...
Journey in the Development of Biologics Through End of Phase 3
Our Goals
To better understand the FDA’s CMC requirements and expectations for biologic manufacturing and product testing
To better visualize a cost-effective, risk-managed approach to manage these manufacturing processes and products through clinical development into market approval
To better appreciate the challenges involved with controlling safety, potency, and impurity profiles for these products
Advances in LC/MS methodologies for the characterisation of complex glycoprot...Quality Assistance s.a.
Dr. Arnaud Delobel from Quality Assistance spoke on "Advances in LC/MS methodologies for the characterisation of complex glycoproteins (case study: Etanercept)" at the "Challenges and Opportunities in Protein Analytics" day in Brussels.
For more information on our expertise and services, visit: www.quality-assistance.com
Follow us on social media:
LinkedIn: https://www.linkedin.com/company/quality-assistance
Twitter: https://twitter.com/QA_Belgium
Facebook: https://www.facebook.com/QualityAssistanceBelgium
Google +: https://plus.google.com/103676189647965359292
Quality Assistance S.A. is a leading European Contract Research Organisation providing the pharmaceutical industry with all the analytical services required by EMA and FDA regulations for the development and marketing of innovative human medicinal products.
We assist our clients from candidate selection, through non-clinical and clinical studies, to marketing authorisation, using our state-of-the-art, product-dedicated expertise in analytical sciences.
For each customer and each project, we design customised solutions, define analytical protocols, develop and validate specific new analytical methods and perform characterisation, stability, pharmacokinetic, biomarker and immunogenicity studies as well as batch release testing, in order to evaluate the Quality, Safety and Efficacy of the given drugs.
Group presentation that contains:
- outlining the basics of translation
- experimental evidence that shows proteins from synonymous mRNA sequences differ
- hypothesis for how synonymous codons effect the resulting protein structure
- the methodology I use to test for the conservation of codon choice within related proteins
Los días 20 y 21 de octubre de 2016, la Fundacion Ramón Areces organizó un simposio internacional para analizar las 'Enfermedades raras de la piel: de la clínica al gen y viceversa'. El doctor Fernando Larcher Laguzzi, del CIEMAT-Universidad Carlos III de Madrid-IIS Fundación Jiménez Díaz, ejerció de coordinador.
A reading report for <A Secreted Slit2 Fragment Regulates Adipose Tissue Ther...星云 王
A reading report for <A Secreted Slit2 Fragment Regulates Adipose Tissue Thermogenesis and Metabolic Function
>, only for private study use, please do not use it for profit or public.
Biosimilar Development Regulatory, Analytical, and Clinical Considerations SGS
The development pathway of a biosimilar is unlike that of a novel biotherapeutic. While there is an increased requirement for analytics throughout a biosimilars development project, and a Phase II clinical trial is generally omitted, careful consideration must be given to the planning of the other phases of development. Many regulatory authorities reference a “step-by-step” approach to establishing biosimilarity. This presentation will provide a look at the multi-stage development for a biosimilar, including a review of the regulatory landscape, structural characterization techniques for biosimilarity assessment, and early phase clinical research challenges
Creative Biolabs' dedicated team of neuroscience CRO scientists want the same thing as you do: to find a cure for the devastating diseases of the central nervous system.
https://neuros.creative-biolabs.com/
Best possible natural ligands which were enlisted on NPACT website were screened ( aid of major drug likeness parameters - pkCSM) and docked with the 2OJG(Target protein) using autodock.
El lunes y martes 20 y 21 de noviembre coordinamos un simposio internacional en la Fundación Ramón Areces, sobre los defectos del transporte de aminoácidos.
SGS First Quarter 2024 Sales Update Presentation EN.pdfSGS
Get the financial highlights and an overview of our performance per business. You can view our financial reports here: https://www.sgs.com/en/investors/results
Get the financial highlights and an overview of our performance per business. You can view our financial reports here: www.sgs.com/en/our-company/investor-relations/reports-and-presentations
Get the financial highlights and an overview of our performance per business. You can view our financial reports here: www.sgs.com/en/our-company/investor-relations/reports-and-presentations
SGS 2022 Full Year Results Alternative Performance Measures ReportSGS
This document presents and defines the Group’s alternative performance measures (APMs), not defined by IFRS which are used
to evaluate financial and operational performance. Where relevant, a reconciliation to the information included in our IFRS consolidated
financial statements is presented. Management deems these performance measures as a useful source of information when taking
strategic decisions and managing the operations. These APMs are disclosed in the annual report, the half year report and other external
communications to investors, as well as available under: www.sgs.com/en/our-company/investor-relations/reports-and-presentations
Get the financial highlights and an overview of our performance per business. You can view our financial reports here: www.sgs.com/en/our-company/investor-relations/reports-and-presentations
Discover our Corporate Sustainability report to get an overview of how we contribute to sustainability, demonstrated by case studies from our operations and services, and a summary of performance.
We've delivered a strong financial performance in 2021, making significant progress on our new strategic plan.
#SGS #SGSGroup #WeAreSGS #FinancialResults
SGS 2021 Full Year Results Alternative Performance MeasuresSGS
We've delivered a strong financial performance in 2021, making significant progress on our new strategic plan.
#SGS #SGSGroup #WeAreSGS #FinancialResults
Learn about our collaboration on a range of innovative circular projects with Dutch Government.
Discussion with three experts on safe construction.
First recycled content declarations for manufacturers.
Get the financial highlights and an overview of our performance per business. You can view our financial reports here: www.sgs.com/en/our-company/investor-relations/reports-and-presentations
Antibiotic Stewardship by Anushri Srivastava.pptxAnushriSrivastav
Stewardship is the act of taking good care of something.
Antimicrobial stewardship is a coordinated program that promotes the appropriate use of antimicrobials (including antibiotics), improves patient outcomes, reduces microbial resistance, and decreases the spread of infections caused by multidrug-resistant organisms.
WHO launched the Global Antimicrobial Resistance and Use Surveillance System (GLASS) in 2015 to fill knowledge gaps and inform strategies at all levels.
ACCORDING TO apic.org,
Antimicrobial stewardship is a coordinated program that promotes the appropriate use of antimicrobials (including antibiotics), improves patient outcomes, reduces microbial resistance, and decreases the spread of infections caused by multidrug-resistant organisms.
ACCORDING TO pewtrusts.org,
Antibiotic stewardship refers to efforts in doctors’ offices, hospitals, long term care facilities, and other health care settings to ensure that antibiotics are used only when necessary and appropriate
According to WHO,
Antimicrobial stewardship is a systematic approach to educate and support health care professionals to follow evidence-based guidelines for prescribing and administering antimicrobials
In 1996, John McGowan and Dale Gerding first applied the term antimicrobial stewardship, where they suggested a causal association between antimicrobial agent use and resistance. They also focused on the urgency of large-scale controlled trials of antimicrobial-use regulation employing sophisticated epidemiologic methods, molecular typing, and precise resistance mechanism analysis.
Antimicrobial Stewardship(AMS) refers to the optimal selection, dosing, and duration of antimicrobial treatment resulting in the best clinical outcome with minimal side effects to the patients and minimal impact on subsequent resistance.
According to the 2019 report, in the US, more than 2.8 million antibiotic-resistant infections occur each year, and more than 35000 people die. In addition to this, it also mentioned that 223,900 cases of Clostridoides difficile occurred in 2017, of which 12800 people died. The report did not include viruses or parasites
VISION
Being proactive
Supporting optimal animal and human health
Exploring ways to reduce overall use of antimicrobials
Using the drugs that prevent and treat disease by killing microscopic organisms in a responsible way
GOAL
to prevent the generation and spread of antimicrobial resistance (AMR). Doing so will preserve the effectiveness of these drugs in animals and humans for years to come.
being to preserve human and animal health and the effectiveness of antimicrobial medications.
to implement a multidisciplinary approach in assembling a stewardship team to include an infectious disease physician, a clinical pharmacist with infectious diseases training, infection preventionist, and a close collaboration with the staff in the clinical microbiology laboratory
to prevent antimicrobial overuse, misuse and abuse.
to minimize the developme
Leading the Way in Nephrology: Dr. David Greene's Work with Stem Cells for Ki...Dr. David Greene Arizona
As we watch Dr. Greene's continued efforts and research in Arizona, it's clear that stem cell therapy holds a promising key to unlocking new doors in the treatment of kidney disease. With each study and trial, we step closer to a world where kidney disease is no longer a life sentence but a treatable condition, thanks to pioneers like Dr. David Greene.
Explore our infographic on 'Essential Metrics for Palliative Care Management' which highlights key performance indicators crucial for enhancing the quality and efficiency of palliative care services.
This visual guide breaks down important metrics across four categories: Patient-Centered Metrics, Care Efficiency Metrics, Quality of Life Metrics, and Staff Metrics. Each section is designed to help healthcare professionals monitor and improve care delivery for patients facing serious illnesses. Understand how to implement these metrics in your palliative care practices for better outcomes and higher satisfaction levels.
Global launch of the Healthy Ageing and Prevention Index 2nd wave – alongside...ILC- UK
The Healthy Ageing and Prevention Index is an online tool created by ILC that ranks countries on six metrics including, life span, health span, work span, income, environmental performance, and happiness. The Index helps us understand how well countries have adapted to longevity and inform decision makers on what must be done to maximise the economic benefits that comes with living well for longer.
Alongside the 77th World Health Assembly in Geneva on 28 May 2024, we launched the second version of our Index, allowing us to track progress and give new insights into what needs to be done to keep populations healthier for longer.
The speakers included:
Professor Orazio Schillaci, Minister of Health, Italy
Dr Hans Groth, Chairman of the Board, World Demographic & Ageing Forum
Professor Ilona Kickbusch, Founder and Chair, Global Health Centre, Geneva Graduate Institute and co-chair, World Health Summit Council
Dr Natasha Azzopardi Muscat, Director, Country Health Policies and Systems Division, World Health Organisation EURO
Dr Marta Lomazzi, Executive Manager, World Federation of Public Health Associations
Dr Shyam Bishen, Head, Centre for Health and Healthcare and Member of the Executive Committee, World Economic Forum
Dr Karin Tegmark Wisell, Director General, Public Health Agency of Sweden
CHAPTER 1 SEMESTER V - ROLE OF PEADIATRIC NURSE.pdfSachin Sharma
Pediatric nurses play a vital role in the health and well-being of children. Their responsibilities are wide-ranging, and their objectives can be categorized into several key areas:
1. Direct Patient Care:
Objective: Provide comprehensive and compassionate care to infants, children, and adolescents in various healthcare settings (hospitals, clinics, etc.).
This includes tasks like:
Monitoring vital signs and physical condition.
Administering medications and treatments.
Performing procedures as directed by doctors.
Assisting with daily living activities (bathing, feeding).
Providing emotional support and pain management.
2. Health Promotion and Education:
Objective: Promote healthy behaviors and educate children, families, and communities about preventive healthcare.
This includes tasks like:
Administering vaccinations.
Providing education on nutrition, hygiene, and development.
Offering breastfeeding and childbirth support.
Counseling families on safety and injury prevention.
3. Collaboration and Advocacy:
Objective: Collaborate effectively with doctors, social workers, therapists, and other healthcare professionals to ensure coordinated care for children.
Objective: Advocate for the rights and best interests of their patients, especially when children cannot speak for themselves.
This includes tasks like:
Communicating effectively with healthcare teams.
Identifying and addressing potential risks to child welfare.
Educating families about their child's condition and treatment options.
4. Professional Development and Research:
Objective: Stay up-to-date on the latest advancements in pediatric healthcare through continuing education and research.
Objective: Contribute to improving the quality of care for children by participating in research initiatives.
This includes tasks like:
Attending workshops and conferences on pediatric nursing.
Participating in clinical trials related to child health.
Implementing evidence-based practices into their daily routines.
By fulfilling these objectives, pediatric nurses play a crucial role in ensuring the optimal health and well-being of children throughout all stages of their development.
Defecation
Normal defecation begins with movement in the left colon, moving stool toward the anus. When stool reaches the rectum, the distention causes relaxation of the internal sphincter and an awareness of the need to defecate. At the time of defecation, the external sphincter relaxes, and abdominal muscles contract, increasing intrarectal pressure and forcing the stool out
The Valsalva maneuver exerts pressure to expel faeces through a voluntary contraction of the abdominal muscles while maintaining forced expiration against a closed airway. Patients with cardiovascular disease, glaucoma, increased intracranial pressure, or a new surgical wound are at greater risk for cardiac dysrhythmias and elevated blood pressure with the Valsalva maneuver and need to avoid straining to pass the stool.
Normal defecation is painless, resulting in passage of soft, formed stool
CONSTIPATION
Constipation is a symptom, not a disease. Improper diet, reduced fluid intake, lack of exercise, and certain medications can cause constipation. For example, patients receiving opiates for pain after surgery often require a stool softener or laxative to prevent constipation. The signs of constipation include infrequent bowel movements (less than every 3 days), difficulty passing stools, excessive straining, inability to defecate at will, and hard feaces
IMPACTION
Fecal impaction results from unrelieved constipation. It is a collection of hardened feces wedged in the rectum that a person cannot expel. In cases of severe impaction the mass extends up into the sigmoid colon.
DIARRHEA
Diarrhea is an increase in the number of stools and the passage of liquid, unformed feces. It is associated with disorders affecting digestion, absorption, and secretion in the GI tract. Intestinal contents pass through the small and large intestine too quickly to allow for the usual absorption of fluid and nutrients. Irritation within the colon results in increased mucus secretion. As a result, feces become watery, and the patient is unable to control the urge to defecate. Normally an anal bag is safe and effective in long-term treatment of patients with fecal incontinence at home, in hospice, or in the hospital. Fecal incontinence is expensive and a potentially dangerous condition in terms of contamination and risk of skin ulceration
HEMORRHOIDS
Hemorrhoids are dilated, engorged veins in the lining of the rectum. They are either external or internal.
FLATULENCE
As gas accumulates in the lumen of the intestines, the bowel wall stretches and distends (flatulence). It is a common cause of abdominal fullness, pain, and cramping. Normally intestinal gas escapes through the mouth (belching) or the anus (passing of flatus)
FECAL INCONTINENCE
Fecal incontinence is the inability to control passage of feces and gas from the anus. Incontinence harms a patient’s body image
PREPARATION AND GIVING OF LAXATIVESACCORDING TO POTTER AND PERRY,
An enema is the instillation of a solution into the rectum and sig
ICH Guidelines for Pharmacovigilance.pdfNEHA GUPTA
The "ICH Guidelines for Pharmacovigilance" PDF provides a comprehensive overview of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines related to pharmacovigilance. These guidelines aim to ensure that drugs are safe and effective for patients by monitoring and assessing adverse effects, ensuring proper reporting systems, and improving risk management practices. The document is essential for professionals in the pharmaceutical industry, regulatory authorities, and healthcare providers, offering detailed procedures and standards for pharmacovigilance activities to enhance drug safety and protect public health.
The Importance of Community Nursing Care.pdfAD Healthcare
NDIS and Community 24/7 Nursing Care is a specific type of support that may be provided under the NDIS for individuals with complex medical needs who require ongoing nursing care in a community setting, such as their home or a supported accommodation facility.
India Clinical Trials Market: Industry Size and Growth Trends [2030] Analyzed...Kumar Satyam
According to TechSci Research report, "India Clinical Trials Market- By Region, Competition, Forecast & Opportunities, 2030F," the India Clinical Trials Market was valued at USD 2.05 billion in 2024 and is projected to grow at a compound annual growth rate (CAGR) of 8.64% through 2030. The market is driven by a variety of factors, making India an attractive destination for pharmaceutical companies and researchers. India's vast and diverse patient population, cost-effective operational environment, and a large pool of skilled medical professionals contribute significantly to the market's growth. Additionally, increasing government support in streamlining regulations and the growing prevalence of lifestyle diseases further propel the clinical trials market.
Growing Prevalence of Lifestyle Diseases
The rising incidence of lifestyle diseases such as diabetes, cardiovascular diseases, and cancer is a major trend driving the clinical trials market in India. These conditions necessitate the development and testing of new treatment methods, creating a robust demand for clinical trials. The increasing burden of these diseases highlights the need for innovative therapies and underscores the importance of India as a key player in global clinical research.
India Clinical Trials Market: Industry Size and Growth Trends [2030] Analyzed...
Glycan Structural Analysis Throughout Biotherapeutic Development
1. GLYCAN STRUCTURAL ANALYSIS THROUGHOUT
BIOTHERAPEUTIC DEVELOPMENT
Dr Richard Easton
Team Leader Carbohydrate AnalysisTeam Leader, Carbohydrate Analysis
SGS Life Science Services
Wokingham, UK
2. What is glycosylation? What is glycosylation?
What aspects of glycosylation are key? What aspects of glycosylation are key?
H th b i ti t d? How can these be investigated?
2
3. N GLYCOSYLATIONN-GLYCOSYLATION
Attached to Asn in the consensus Attached to Asn in the consensus
sequence …Asn-X-Ser/Thr…where X is
any AA except Pro
Initiated in ER by en bloc transfer of a pre-
formed lipid-anchored conserved glycan
Mature glycans formed by trimming (in ER
and Golgi) and elongation (Golgi)
Found on mammalian, insect, fungal,
bacterial and plant cell proteins
3
4. O GLYCOSYLATIONO-GLYCOSYLATION
Occurs on Ser and ThrOccurs on Ser and Thr
No consensus sequence but some “rules” eg nearby proline,
tandem repeats of Ser/Thr
Initiated in Golgi by addition of a single sugar - usually GalNAc in
mammals
Additi l dd d ti ll t f th Additional sugars are added sequentially to form the core
sequence and then the antennae
Cores 1 & 2 are very common in many glycoproteinsy y g y p
Other monosaccharides may act as linkers depending on the
protein structure eg O-linked Fucose in EGF domains
Cores 1 & 2 are very common in many glycoproteins
Found on mammalian, insect, fungal, bacterial and plant cells
4
9. N-GLYCOSYLATION IN FISH AND BIRDSN-GLYCOSYLATION IN FISH AND BIRDS
some fish glycans are the most
decorated of all known glycans
9
decorated of all known glycans
10. SELECTED MAMMALIAN N GLYCAN STRUCTURESSELECTED MAMMALIAN N-GLYCAN STRUCTURES
(i)
(iii)
(ii)
Erythropoietin
Glycodelin S
Gl d li A
(iv)
(v)
+/-
(vi)
Glycodelin A
+/-
Tamm-Horsfall glycoprotein
M kid
Neutrophil
10
Mouse kidney
21. MONOSACCHARIDE ANALYSIS – BOVINE FETUINO OS CC S S O U
Is my molecule glycosylated?
• Is the glycosylation N linked and if so what type (complex high mannose)?M-Scan Ltd.
Sample: Fetuin standard
Job: 12849 Sample No:Acquired on: 29-Sep-2000 at 13:37:38 by:
100
%
FETST299 Scan EI+
298
6.30e4
21.83
N-Acetyl
neuraminic acid
• Is the glycosylation N-linked and if so what type (complex, high mannose)?
• Is there evidence of O-glycosylation?
Is there evidence of carbohydrate contamination coming through the process?
• High levels of glucose may indicate breakdown of, for example, gel filtration beads
100
0
%
FETST299 Scan EI+
173
1.56e5
16.98
N-Acetyl
g g y , p , g
100
0
%
16.38 21.83
17.13
FETST299 Scan EI+
20411.32 Galactose
N-Acetyl
galactosamine
y
glucosamine
0
100
%
3.69e512.08
12.65 13.12
13.48 16.98 21.83
FETST299 Scan EI+
Mannose
Galactose
Glucose
100
%
FETST299 Scan EI+
TIC
3.99e6
10.28
7.72
7.00
21.83
11.32
12.08
11.47
16.98
13.12
12.65
13.50
Arabitol (IS)
21
8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00
Time0
7.72
23. PERMETHYLATIONPERMETHYLATION
Population analysis by MALDI MS Population analysis by MALDI-MS
Identification of glycan compositions and an initial
assessment of the types of structures presentassessment of the types of structures present
Electrospray analysis
A determination of glycan antennae present in the
sample through fragmentation of the glycans.
Identification of unwanted epitopes eg GalαGalp p g
GC-MS
Identification of monosaccharide linkages in the
population
23
24. MALDI MS ANALYSIS OF N GLYCANSMALDI-MS ANALYSIS OF N-GLYCANS
Secretory glycoprotein
Antibody
24
25. FRAGMENTATION IN ELECTROSPRAY IONISATIONFRAGMENTATION IN ELECTROSPRAY IONISATION
O
OO O
CH2OR
O
CH2OR
+
OO O
OR OR OR OR
H
H NA
A-type oxonium ion cleavage
HexNAc
or
Sialic Acid
O
CH2OR
+
O
OR OR
25
OR OR
27. MS/MS OF THE SIGNAL AT M/Z 895 5MS/MS OF THE SIGNAL AT M/Z 895.5
298.2
CH2OMe
CH2OMeMeOCH2
CH
CHNMeAc
CHOMe
CHOMe
OHC
CC
MeOC
HH
H
C O
H
OMeOC
MeOCH
H
COOMe
AcNMeC
C
H
O
MeOCH
CHOMe
CH2OMe
OMeOC
H
H
H
C
H
C
OMe
AcNMeC
620.3
520.3
376 2/398 2376.2/398.2
NeuAcHexHexNAcitol + Na+
27
28. MS/MS OF THE SIGNAL AT M/Z 895 5MS/MS OF THE SIGNAL AT M/Z 895.5
298.2
298.2
CH2OMe
CH2OMeMeOCH2
CH
CHNMeAc
CHOMe
CHOMe
OHC
CC
MeOC
HH
H
C O
H
OMeOC
MeOCH
H
COOMe
AcNMeC
C
H
O
MeOCH
CHOMe
CH2OMe
OMeOC
H
H
H
C
H
C
OMe
AcNMeC
NeuAcHexHexNAcitol + Na+
28
29. MS/MS OF THE SIGNAL AT M/Z 1256.6
CH2OMe
CH
CHNMeAc
O
C O
H
CH2OMe
M OCH
MeOCH
MeOCH2
620.3
CH
CHOMe
CHOMe
CH
OHC
CC
MeOC
HH
H
H
OMeOC
MeOCH
H
H
H
COOMe
C
H
C
AcNMeC
C
H
O
CH2H
OMe
MeOCH
MeOCH2
881.5
659.3376.2/398.2
OC
MeOCH
H
H
H
COOMe
C
H
C
AcNMeC
C
H
O
284.2
H
OMe 506.3
376.2/398.2
NeuAcHexHexNAcitol + Na+
29
30. MS/MS OF THE SIGNAL AT M/Z 1256.6
CH2OMe
CH
CHNMeAc
O
C O
H
CH2OMe
M OCH
MeOCH
MeOCH2
CH
CHOMe
CHOMe
CH
OHC
CC
MeOC
HH
H
H
OMeOC
MeOCH
H
H
H
COOMe
C
H
C
AcNMeC
C
H
O
CH2H
OMe
MeOCH
MeOCH2
659.3
OC
MeOCH
H
H
H
COOMe
C
H
C
AcNMeC
C
H
O
659.3 H
OMe
659.3
NeuAcHexHexNAcitol + Na+
30
31. LINKAGE ANALYSIS
45
GC/EI-MS analysis – Spectrum of 1,2-linked Mannose
LINKAGE ANALYSIS
OAc
OAc
CH3O
CH2OCH3
H
45
H OAc
CH2OCH3
117
161
306
129OAc
OCH3 OAc
D
H OCH3
H OCH3
161
205
190
234129
130
129
130
CHDOAc
H OAc 277
74
146
161 74
190
161
190
31
32. LINKAGE ANALYSIS
73
GC/EI-MS analysis – spectrum of 1,3,6-linked Mannose
LINKAGE ANALYSIS
OA
CH2OAc
73
H OAc
CH2OAc
145
189
306
129
OAc
OAc
CH3O
H
D H OAc
H OCH3
189
261
190
234
118 129
OAc OCH3
CHDOAc
H OCH3
305
74
118
129
74
234
189
32
33. LINKAGE ANALYSIS
45
GC/EI-MS analysis – spectrum of 1,4-linked N-Acetylglucosamine
LINKAGE ANALYSIS
OAc
OAc
AcO
CH2OCH3
H
45
H OAc
CH2OCH3
117
189
347
OAc
OCH3 NCH3Ac
D
OCH3H
H OAc
189
233
203
275117
CHDOAc
H NCH3Ac305
74
159117
74
159
233
33
36. 2 AB STACKED CHROMATOGRAMS2-AB STACKED CHROMATOGRAMS
Five separate Mab N glycan preparationsFive separate Mab N-glycan preparations
G0F G1F G2F
Run 1 0.59 0.32 0.09
Run 2 0.58 0.32 0.09
Run 3 0.59 0.32 0.09
Run 4 0.58 0.33 0.09
Run 5 0 58 0 32 0 09Run 5 0.58 0.32 0.09
Average 0.584 0.322 0.09
SD 0.0055 0.0045 0.00
%CV 0.94 1.40 0.00
36
37. 2 AB STACKED CHROMATOGRAMS2-AB STACKED CHROMATOGRAMS
Diff i t f M b tiDiffering amounts of Mab preparations
100μg
10μg
50μg
37
38. TAKE HOME MESSAGESTAKE HOME MESSAGES
Mass spectrometric analysis provides detailed structural information on
composition, antennal structures and linkage.
Chromatography provides a unique glycan profile due to the precise
structure and associated interactions of the glycans with the column
t i Thi fil b d f i t hi h thmatrix. This profile can be used as a reference against which other
batches can be compared. Useful for characterisation, stability, forced
degradation studies.
The use of LC/ES-MS of proteolytic digests allows sites of glycosylation
to be isolated and identified. These sites can then be analysed using the
b t h i t d t i th t f th l t h itabove techniques to determine the nature of the glycans at each site.
38