Quality control and analysis for biopharmaceuticals

A.Y. 2021/2022
Overall hours
BIO/14 CHIM/06 CHIM/08
Learning objectives
This course will provide the theoretical basis of the main spectroscopic (IR, NMR, CD), mass spectrometric analysis techniques such as IE, CI and ESI, DSC and XRPD. It will give to the student essential tools to determine the structure of organic and bioorganic compounds. Particular importance is given to the analysis of the data and the strategy for their interpretation, also through problems solving strategies.
Basic notions will be given concerning the most popular analytical techniques applied for the characterization of biopharmaceuticals, including chromatography, capillary and gel electrophoresis, mass spectrometry as well as emission and absorption UV-Vis spectroscopy. Moreover, various procedures for potency (biological activity) testing will be shown, including ligand and receptor binding assays, cell culture-based assays, biochemical assays (such as enzymatic assays) and transgenic animal-based assays.
Expected learning outcomes
At the end of the course the student is expected 1) to prove a basic knowledge of the principles of the most popular analytical techniques for the characterization of biopharmaceuticals 2) to be able to plan analytical strategies for the characterization of biopharmaceuticals; 3) to be able to understand the results obtained from instrumental analyses of biopharmaceuticals; 4) to prove a basic knowledge of main methods employed for measuring biological activity of biopharmaceuticals; 5) to learn how to handle the spectroscopic data and to use them in an integrated and comparative way with the aim to extract all the information about the structure of organic and bioorganic molecules; 6) to be able to communicate clearly and with an appropriate language the knowledge and the acquired interpretative ability of the various spectra and 7) to develop a good autonomy for the identification of the structures of organic molecules.
Course syllabus and organization

Single session

Lesson period
First semester
Concerning the modes of delivery of teaching activities during the academic year 2021/2022, more information will be given in the next months, based on the evolution of the public health emergency.
Prerequisites for admission
Knowledge of the fundamentals of physics, chemistry, organic chemistry and biochemistry.
Assessment methods and Criteria
The exam consists of a two-hours written test concerning the program of the module "Spectroscopic methods in bioorganic chemistry" and an oral test concerning the program of the module "Analytical and pharmacodynamic characterization of biopharmaceuticals".
The written test consists of a theory question and an exercise concerning the interpretation of spectral data of an organic molecule.
The oral test is aimed at verifying the knowledge of the fundamentals of biopharmaceutical analysis and biological activity measurement.
Pectroscopic methods in bioorganic chemistry
Course syllabus
GENERAL PART: electromagnetic spectrum and electromagnetic radiation, characterization of electromagnetic radiation, electromagnetic radiation and interactions with matter, spectroscopic techniques.
INFRARED SPECTROSCOPY: Basic principles: frequency of infrared absorption and chemical structure.
The IR spectrum. Characteristic absorption: hydrocarbons (alkanes, alkenes, alkynes, aromatics), alcohols, ethers, halides, carbonyl compounds, amines. IR spectrometers: classic, Fourier transform IR spectrophotometer (FT-IR). Reflection techniques: ATR, DRIFT, RAIRS. Applications of IR spectroscopy.
NUCLEAR MAGNETIC RESONANCE, NMR (Nuclear Magnetic Resonance): general principles, properties of nuclei, magnetic field and electromagnetic radiation (radiofrequency), the resonance phenomenon
Proton magnetic resonance: Chemical shift, Spin-spin coupling, Spin decoupling. Overhauser effect.
13C-NMR spectroscopy: Introduction. Carbon spectrum recording techniques. Chemical displacements. Spin 13C-1H coupling. DEPT and APT spectra. NMR correlation spectroscopy: 1H-1H COSY, 1H-13C HETCOR-HSQC-HMBC. NMR spectroscopy of other important nuclei 15N, 19F, 31P. Applications
MASS SPECTROMETRY. Introduction. Instrumentation, introduction system, sources, analyzers. Low and high resolution molecular ion. Molecular ion recognition. Fragmentations and rearrangements.
Mass spectra of some chemical classes. CIRCULAR DICROISM: Introduction. Principles. Applications
Teaching methods
Theoretical lessons and spectral interpretation exercises
Teaching Resources
R.M. Silverstein, F.X. Webster Identificazione spettrometrica di composti organici Terza edizione-Casa Editrice Ambrosiana. Structure determination of organic compounds, practical exercises E. Rossi, D. Nava, G. Abbiati, G. Celentano, S. Pandini. Casa Editrice Edises
A. Randazzo. Guida pratica all'interpretazione di spettri NMR. Casa Editrice Loghia
Analytical and pharmacodynamic characterization of biopharmaceuticals
Course syllabus
Biopharmaceuticals and in particular therapeutic proteins produced using recombinant DNA technology are complex, heterogeneous molecules, and subject to a variety of enzymatic and / or chemical modifications that can occur during expression, purification and long-term storage.
The course consist of two parts. The first part is concerning the structural characterization of biopharmaceuticals, whereas the second part is concerning their pharmacodynamic characterization.

The structural characterization of latest generation biopharmaceuticals will be taught using as educational example the characterization of antibody-drug conjugates (ADCs) as a comprehensive educational path to teach the most popular analytical techniques and methodological approaches. The program will include the fundamentals of:
1) Chromatography (high performance liquid chromatography)
2) Electrophoresis (gel and capillary electrophoresis)
3) UV-Vis spectroscopy (absorption and emission techniques)
4) Mass spectrometry (ESI and MALDI ionization)
The fundamentals of hyphenation of such analytical techniques and the principles of sample preparation and purification necessary for sample analysis will be discussed as well.

Concerning the pharmacodynamic characterization of biopharmaceuticals the determination of the efficacy of such products refers to the quantitative measurement of the biological activity of a given product. Biological activity is a critical quality attribute; therefore, efficacy assays are an essential component of quality control. During the course, different procedures for tests will be illustrated, including:
1) Assays based on the use of cell cultures
2) Biochemical tests (e.g. enzymatic tests)
3) Assays based on the use of experimental animals (animal, transgenic models, etc.)

Furthermore, essays referring to:
4) Microbiological contamination of cell cultures
5) Genetic drift of cell cultures
Teaching methods
The course is delivered by classroom lectures, in which the topics are illustrated both with by slides and by exercises on the blackboard. To promote student's engagement some topics are delivered through flipped classrooms, breakout rooms, as well as interactive live quizzes and Q&A sessions.
Teaching Resources
Slides of the room lessons and other material available online at the Ariel website of the course.

Antibody-Drug Conjugates, Methods and Protocols, edited by L.NathanTumey (2020), ebook available at the university online library service, ISBN: 1-4939-9929-X; 1-4939-9928-1.

Analytical Characterization of Biotherapeutics (capitoli 1,2 e 7), edited by Jennie R. Lill and Wendy Sandoval (2017), ebook available at the university online library service, ISBN: 1-119-38442-7; 1-119-38443-5; 1-119-05310-2.
Analytical and pharmacodynamic characterization of biopharmaceuticals
Lessons: 48 hours
Pectroscopic methods in bioorganic chemistry
CHIM/06 - ORGANIC CHEMISTRY - University credits: 3
Lessons: 24 hours
By appointment
Via Golgi 19. Entrance C, IV floor
on demand, to be scheduled via email
Via L. Mangiagalli 25, first floor, room 1066