SFAB21005U Biopharmaceuticals: Protein Production and Analysis

Volume 2021/2022
Education

BSc Programme in Pharmacy (elective)

BSc Programme in Medicinal Chemistry (elective)

BSc Programme in Chemistry (elective)

Content

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 industry and 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, 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 laboratory project that involves a series of open structured laboratory sessions where students in groups are provided the means for expressing, purifying and analyzing an unknown protein through access to state-of-the-art equipment at various lab stations. The laboratory work in this course should therefore be viewed as an independent 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 carry out the expression, purifcation, analysis, quality control and identification of their unknown protein.

As part of this lab-based course, the students will write a group report that describes a detailed description and interpretation of results obtained during their laboratory project. Each student group will present their report to the class. Furthermore, student groups (MSc students only) will perform an individual study on a individually chosen study topic of interest in preparatory or analytical biopharmaceutical science which will be presented to the class.

Finally, students of the course will get the opportunity to participate in a one-day excursion to Novo Nordisk A/S where 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 and opportunities for networking and Q/A sessions.

Learning Outcome

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 expression, handling, purification and analysis of proteins and be able to communicate proficiently with researchers in a biotech drug discovery/development environment.

At the end of the course, students are expected to be able to:

Knowledge

  • 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 chemical/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.
  • understand methods for quality control of protein biopharmaceuticals in the pharmacopoeia.
  • understand methods for quantitative analysis of protein biopharmaceuticals.

 

Skills

  • perform recombinant expression of a protein using E.coli host cells
  • perform cell lysis and centrifugation to extract proteins from E.coli cells
  • perform affinity chromatography to purify a protein from crude cell extract
  • 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
  • perform reversed-phase chromatography with UV of a protein sample
  • perform enzymatic digestion and reversed-phase chromatography with UV detection to characterize the primary structure of a purified protein
  • perform enzymatic digestion and reversed-phase chromatography with MS detection to characterize the primary structure of a purified protein
  • perform fluorescence spectroscopy and CD spectroscopy analysis of a protein sample
  • search bioinformatics databases for the aminoacid sequence, molecular mass and chemical properties of a target protein.
  • search bioinformatics databases for structural information for a target protein and visualize higher-order protein structure using molecular graphics software.

 

Competences

  • discuss and critically evaluate current approaches for recombinant expression, purification and analysis of protein biopharmaceuticals.
  • evaluate the growth and protein expression yields of a host cell culture
  • evaluate purity and identity of protein components in a complex mixture by gel electrophoresis (SDS-PAGE) analysis
  • evaluate protein purity by reversed-phase HPLC analysis
  • determine the molecular mass of a protein by reversed-phase HPLC combined with mass spectrometry
  • determine the primary structure of protein by peptide mapping analysis
  • evaluate the higher-order structure of a protein by fluorescence spectroscopy and CD spectroscopy
  • evaluate the strengths and weaknesses of different host cells for recombinant expression of protein pharmaceuticals.
  • evaluate the strengths and weaknesses of different methods for protein purification.
  • evaluate the strengths and weaknesses of different analytical methods to analyze the primary or higher-order structure of protein biopharmaceuticals.
  • Lecture slides and research/review papers available on the course homepage

  • Lab station manuals available on the course homepage

  • OPTIONAL: Chapters from the book: "Protein Analysis and Purification - Benchtop techniques". 2nd edition, Ian M. Rosenberg. Birkhauser. 2005.

  • OPTIONAL:Chapters from the book: "Methods for Structural Analysis of Protein Pharmaceuticals". Edited by Wim Joskoot and Dan Crommelin. AAPS, 2005.
If you are applying for the course as a credit transfer student, you must have passed SFAB20015U Biopharmaceuticals -bioorganisk kemi, SFAB20017U Farmaceutisk fysisk kemi II - kinetik og transportfænomener and SFAB20035U Farmaceutisk analytisk kemi or have acquired similar competencies within protein structure, bioorganic chemistry, microbiology, thermodynamics (physical chemistry), and analytical chemistry in another course. Documentation for corresponding competencies in the form of a course description and an exam result must be attached to your application.
• Lectures: 20 hrs
• Laboratory: 56 hrs
• Project work (individual study report): 20 hrs
  • Category
  • Hours
  • Lectures
  • 20
  • Preparation
  • 128
  • Practical exercises
  • 56
  • Exam
  • 2
  • Total
  • 206
Continuous feedback during the course of the semester
Credit
2,5 ECTS
Type of assessment
Course participation
In order to obtain a course certificate the students should:
•Participate to a satisfactory level in all laboratory exercises.
• Submit and gain approval of a laboratory group report
• Take part in group and/or individuel presentation of the laboratory group report
Marking scale
passed/not passed
Censorship form
No external censorship
Criteria for exam assesment

To pass the course the student must be able to:

Knowledge

  • 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 chemical/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.
  • understand methods for quality control of protein biopharmaceuticals in the pharmacopoeia.
  • understand methods for quantitative analysis of protein biopharmaceuticals.

 

Skills

  • perform recombinant expression of a protein using E.coli host cells
  • perform cell lysis and centrifugation to extract proteins from E.coli cells
  • perform affinity chromatography to purify a protein from crude cell extract
  • 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
  • perform reversed-phase chromatography with UV of a protein sample
  • perform enzymatic digestion and reversed-phase chromatography with UV detection to characterize the primary structure of a purified protein
  • perform enzymatic digestion and reversed-phase chromatography with MS detection to characterize the primary structure of a purified protein
  • perform fluorescence spectroscopy and CD spectroscopy analysis of a protein sample
  • search bioinformatics databases for the aminoacid sequence, molecular mass and chemical properties of a target protein.
  • search bioinformatics databases for structural information for a target protein and visualize higher-order protein structure using molecular graphics software.

 

Competences

  • evaluate the growth and protein expression yields of a host cell culture
  • evaluate purity and identity of protein components in a complex mixture by gel electrophoresis (SDS-PAGE) analysis
  • evaluate protein purity by reversed-phase HPLC analysis
  • determine the molecular mass of a protein by reversed-phase HPLC combined with mass spectrometry
  • determine the primary structure of protein by peptide mapping analysis
  • evaluate the higher-order structure of a protein by fluorescence spectroscopy and CD spectroscopy
  • evaluate the strengths and weaknesses of different host cells for recombinant expression of protein pharmaceuticals.
  • evaluate the strengths and weaknesses of different methods for protein purification.
  • evaluate the strengths and weaknesses of different analytical methods to analyze the primary or higher-order structure of protein biopharmaceuticals.
Credit
5 ECTS
Type of assessment
Written examination, 1 hour under invigilation
Multiple Choice
The 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.
Aid
Without aids

There is access to the following at the exam on Peter Bangs Vej:

  • R – Statistical programme
  • MathType - formel programme
  • Maple

 

usb-stick is not allowed

Marking scale
passed/not passed
Censorship form
No external censorship
Criteria for exam assesment

To pass the course the student must be able to:

Knowledge

  • 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 chemical/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.
  • understand methods for quality control of protein biopharmaceuticals in the pharmacopoeia.
  • understand methods for quantitative analysis of protein biopharmaceuticals.

 

Skills

  • perform recombinant expression of a protein using E.coli host cells
  • perform cell lysis and centrifugation to extract proteins from E.coli cells
  • perform affinity chromatography to purify a protein from crude cell extract
  • 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
  • perform reversed-phase chromatography with UV of a protein sample
  • perform enzymatic digestion and reversed-phase chromatography with UV detection to characterize the primary structure of a purified protein
  • perform enzymatic digestion and reversed-phase chromatography with MS detection to characterize the primary structure of a purified protein
  • perform fluorescence spectroscopy and CD spectroscopy analysis of a protein sample
  • search bioinformatics databases for the aminoacid sequence, molecular mass and chemical properties of a target protein.
  • search bioinformatics databases for structural information for a target protein and visualize higher-order protein structure using molecular graphics software.

 

Competences

  • evaluate the growth and protein expression yields of a host cell culture
  • evaluate purity and identity of protein components in a complex mixture by gel electrophoresis (SDS-PAGE) analysis
  • evaluate protein purity by reversed-phase HPLC analysis
  • determine the molecular mass of a protein by reversed-phase HPLC combined with mass spectrometry
  • determine the primary structure of protein by peptide mapping analysis
  • evaluate the higher-order structure of a protein by fluorescence spectroscopy and CD spectroscopy
  • evaluate the strengths and weaknesses of different host cells for recombinant expression of protein pharmaceuticals.
  • evaluate the strengths and weaknesses of different methods for protein purification.
  • evaluate the strengths and weaknesses of different analytical methods to analyze the primary or higher-order structure of protein biopharmaceuticals.

 

Students requiring a grade for the exam, should contact the course responsible.