Biopharmaceuticals: Protein Production and Analysis

Course 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 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.

Education

MSc Programme in Pharmacy or Pharmaceutical Sciences (Danish programmes cand.pharm and cand.scient.pharm) - elective

MSc Programme in Medicinal Chemistry - elective

MSc Programme in Pharmaceutical Sciences (English programme) - elective

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 handling, purification and analysis of proteins and/or 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 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 pharmacopoiea.
  • 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 visaluze higher-order protein structure using molecular graphics software.

 

Skills

  • identify the strengths and weaknesses of different analytical methods to analyze the primary or higher-order structure of protein biopharmaceuticals during development.
  • 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 enzymatic digestion and MALDI mass spectrometry to identify and localize stress-induced covalent modifications to the primary structure of a purified protein

 

Competences

  • discuss and critically evaluate the use of protein purification and protein analysis in the scientific literature and during biopharmaceutical development.
  • apply gel electrophoresis (SDS-PAGE) to identify protein components in a complex mixture
  • 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
  • apply fluorescence spectroscopy to assess the higher-order structure of a protein

• Lectures: 20 hrs
• Laboratory: 56 hrs
• Project work (individual study report): 20 hrs

  • 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.

The main academic prerequisites are a basic understanding of protein structure, bioorganic chemistry, microbiology, thermodynamics (physical chemistry), and analytical chemistry.

ECTS
7,5 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:

  • Office (Word, Excel, Onenote and Powerpoint)
  • IO2 – the digital pen
  • Panoramic Viewer
  • Paint
  • Calculator – Windows' own
  • R – Statistical programme
  • ITX MC – multiple choice programme
  • Adobe reader

 

usb-stick is not allowed

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

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 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 pharmacopoiea.
  • 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 visaluze higher-order protein structure using molecular graphics software.

 

Skills

  • identify the strengths and weaknesses of different analytical methods to analyze the primary or higher-order structure of protein biopharmaceuticals during development.
  • 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 enzymatic digestion and MALDI mass spectrometry to identify and localize stress-induced covalent modifications to the primary structure of a purified protein

 

Competences

  • discuss and critically evaluate the use of protein purification and protein analysis in the scientific literature and during biopharmaceutical development.
  • apply gel electrophoresis (SDS-PAGE) to identify protein components in a complex mixture
  • 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
  • apply fluorescence spectroscopy to assess the higher-order structure of a protein

 

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

Single subject courses (day)

  • Category
  • Hours
  • Lectures
  • 20
  • Preparation
  • 40
  • Practical exercises
  • 56
  • Preparation
  • 24
  • Exam
  • 2
  • Preparation
  • 70
  • English
  • 212