SFKKIF002U Biopharmaceuticals: Protein Production and Analysis
This course aims to provide students with the required practical laboratory competencies for preparatory and analytical research with proteins during pharmaceutical development. During the course the students will gain competency with the key in-vitro techniques for working with proteins in a laboratory. Specifically, the course covers recombinant protein expression, protein purification and analysis of proteins and in particularly how these aspects are closely interconnected for quality control/analysis of protein biopharmaceuticals in the phamacopoiea. Topics will be dealt with from a practical perspective relevant to both academic and biotech/pharmaceutical drug discovery/development environments.
The most relevant course topics are:
- Physicochemical properties of peptides and proteins and challenges to in-vitro handling and laboratory work with proteins (protein stability, pH, temperature, concentration, aggregation, unfolding)
- Introduction to protein sequence databases (Uniprot, Protein Data Bank)
- Recombinant protein expression in host cells (E.coli, yeast, mammalian cells)
- Principles of protein separation and chromatographic purification (affinity, ion-exchange, size-exclusion, HIC, HPLC)
- Basic methods for analysis of primary protein structure (SDS-PAGE, intact mass analysis by mass spectrometry (MS))
- Advanced methods for analysis of primary structure and covalent modifications (amino acid analysis, LC-MS, IEF, peptide mapping analysis by enzymatic digestion and MS)
- Methods for analysis of the higher-order structure of protein biopharmaceuticals during development (fluorescence, CD, NMR, SEC, DLS, HDX-MS etc.)
- Quality control of protein biopharmaceuticals in the pharmacopoiea
- Methods for quantitation of proteins in pure samples (spectroscopy, biochemical assays) and in complex biological samples (host cell protein analysis, pharmacokinetics, drug metabolism
This course is centered around a practical component that involves a series of open structured laboratory sessions where students in groups are provided an unknown protein sample and access to lab stations with equipment for purifying and analyzing proteins. The laboratory work in this course should therefore be viewed as a mini-research project rather than a predefined series of laboratory exercises. By applying theory learned during the lectures, the students will plan their experimental work in an independent manner and subsequently carryout the purifcation and analysis/quality control of a protein of interest in the unknown sample.
As part of this laboratory-based course, the students will write a group report that describes a detailed description and interpretation of results obtained during their laboratory project. Furthermore, each student must prepare an appendix to the group report on a individually chosen study topic of interest in preparatory or analytical biopharmaceutical science.
Finally, students of the course will get the opportunity to participate in a one-day excursion to Novo Nordisk A/S where two in-house scientists will give lectures on how protein production and analysis is performed at a large biopharmaceutical company like Novo Nordisk. This excursion will also include a tour of facilities at Novo Nordisk for large scale protein expression and purification.
Formål / Objective
The objective of this course is to provide the students with the knowledge and practical competencies neccesary for experimental work with proteins in a laboratory setting. The specific aim is for students to acquire abilities and skills with the handling, purification and analysis of proteins and/or be able to communicate proficiently with researchers in a biotech drug discovery/development environment.
Målbeskrivelse / Course outcome
Theoretical knowledge: At the end of the course the students should be able to:
- Describe the primary structure, covalent modifications and higher-order structure of proteins and how these determine protein function.
- Understand the basic chemical and physical properties of proteins in solution (charge, solubility, hydrophobicity, affinity).
- Explain the principles of recombinant protein expression and the strengths/weaknesses of different host cell systems.
- Describe the common methods for protein purification and their underlying physical principles for protein separation
- Understand the common methods for analysis of protein primary structure and common covalent modifications of proteins
- Understand the common methods for analysis of higher order protein structure and physical size/oligomerization.
- Identify the strengths and weaknesses of different analytical methods to analyze the primary or higher-order structure of protein biopharmaceuticals during development.
- Understand methods for quality control of protein biopharmaceuticals in the pharmacopoiea.
- Discuss and critically evaluate the use of protein purification and protein analysis in the scientific literature and during biopharmaceutical development.
Practical competencies: At the end of the course the students will have acquired sufficient skills to be able to:
- perform affinity chromatography to purify a protein from crude cell extract
- apply gel electrophoresis (SDS-PAGE) to identify protein components in a complex mixture
- perform size-exclusion chromatography for fine-grade purification of a protein from other protein components
- determine the concentration of protein in a sample by UV absorbance spectroscopy
- employ analytical HPLC to determine protein purity and purification yield
- prepare and execute ESI and MALDI mass spectrometry to determine the molecular mass and quality control of a purified protein
- perform enzymatic digestion and MALDI mass spectrometry to identify and localize stress-induced covalent modifications to the primary structure of a purified protein
- apply fluorescence spectroscopy to assess the higher-order structure of a protein
- to search bioinformatics databases for the aminoacid sequence, molecular mass and chemical properties of a target protein
- to search bioinformatics databases for structural information for a target protein and visaluze higher-order protein structure using molecular graphics software.
Lecture notes and research/review papers on purification and analysis of proteins available on the course homepage
- Laboratory protocols and course appendix
- Chapters from the book: "Protein Analysis and Purification - Benchtop techniques". 2nd edition, Ian M. Rosenberg. Birkhauser.2005.
- Chapters from the book: "Methods for Structural Analysis of Protein Pharmaceuticals". Edited by Wim Joskoot and Dan Crommelin. AAPS, 2005.
• Laboratory: 56 hrs
• Project work (individual study report): 20 hrs
- Practical exercises
- 7,5 ECTS
- Type of assessment
- Written examination, 1 hour under invigilationThe multiple choice-test is made up of a number (typically 25) of statements to which the student has to decide whether they are true or false.
- Exam registration requirements
- 1 ) Participation in all laboratory exercises.
2 ) In order to be able to take the exam, the laboratory group report and oral presentation of the group report must be approved.
3 ) In order to be able to take the exam, the student‘s report and oral presentation of the "Independent Study Report" must be approved.
No written aids.
Apart from the standard programs and IT tools listed under The Faculty of Health and Medical Sciences at http://pc-eksamen.ku.dk/pc_exam students will at this exam also have access to MathType
- Marking scale
- passed/not passed
- Censorship form
- No external censorship
- Exam period
- block 3
Criteria for exam assesment
In order to pass the exam the student should obtain points corresponding to at least 45% of the maximal number of points.
- A correct answer results in: +1 point
- A wrong answer results in:- 1 point
- A blank answer results in: 0 points
Students requiring a grade for the exam, should contact the course responsible.