Analytical strategy lecture “Biotherapeutics” – CSL Behring

December 2019

AGENDA

Who we are All

Analytical strategy problem solving Sandro Manni

Experience report Sandro Manni

Early career talents @ CSL Lisa Furrer

Q & A Recruiting All

Who we are

Daria Schüpbach

WHO WE ARE

Sandro Manni Lisa Furrer

Senior Scientist Method Development R&D HR Specialist

Analytical strategy –

Problem solving

Students

6

BIOTHERAPEUTICS CASE

Tasks:

a) Describe the mechanism of action of serine proteases.

b) Which methods could be applied to monitor both the presence and

proteolytic activity of coagulation factor contaminants in therapeutic

immunoglobulins or other blood plasma products such as albumin.

c) How could serine protease contaminants, which are present in trace

amounts, contribute to proteolytic activity, and are as-yet-

uncharacterized, be isolated and identified? Describe a possible

preparative experimental setup and feasible analytical method for

identification.

Literature:

1) Etscheid M, Breitner-Ruddock S, Gross S, Hunfeld A, Seitz R, Dodt

J, Vox Sang. 2012 Jan; 102(1):40-6

2) Germishuizen WA, Gyure DC, Stubbings D, Burnouf T, Biologicals.

2014 Sep;42(5):260-70.

Case:

Therapeutic intravenous immunoglobulins (IVIG) produced from blood plasma

of healthy donors are used to treat various autoimmune, infectious and

idiopathic diseases such as primary immunodeficiency disease, chronic

inflammatory demyelinating polyneuropathy, chronic lymphocytic lymphoma

or Kawasaki syndrome. IVIG products must contain the full range of natural

human polyclonal immunoglobulins in order to exert their protective and

modulatory effects.

The occurrence of Thromboembolic events (TEE) including pulmonary

embolism, deep vein thrombosis, myocardial infarction or stroke are rare

adverse events associated with IVIG administration that may depend on

patients precondition, the route of administration (rapid infusion) or protease

contamination by proteins with proteolytic activity (such as coagulation

factors). In the case of the latter, such contaminating proteins originate from

the source plasma, are imperfectly removed during the manufacturing

process, and eventuate in the final product.

In the past, competitor IVIG products have been shown to contain plasma

kallikrein and activated coagulation Factor XI (FXIa). Trace amounts of FXI

were primarily responsible for TEE (Etscheid et al, 2012). Several coagulation

factors (including FXI or Factor X) are serine-type proteases, which act by

cleaving and further activating downstream proteins to engage the

coagulation cascade.

7

SAMPLE SOLUTION - TASK A)

Active Site (catalytic triad):1) Serine: Serine protease active sites contain serine or in some

cases cystein – substrate binding2) Histidine: proton acceptor, donator3) Aspartate: protons acceptor

(A)(B) Serine/Cystein acts as a nucleophile during catalysis by donating an electron to the carbonyl carbon of the peptide bond to be hydrolyzed

(B)(C) Acyl serine is formed and a proton donated to the departing amyl group by the Histidine residue

(C)(D) The acyl enzyme is then hydrolyzed, the carboxylic acid product is released, and the active site is regenerated

8

SAMPLE SOLUTION - TASK B)

SAMPLE SOLUTION - TASK B)

SAMPLE SOLUTION - TASK B)

SAMPLE SOLUTION - TASK B)

Inhibitor Profiling

• C1 inhibitor (85%): complement cascade

• ATIII +/- Heparine: coagulation cascade

• PPACK II (80%): Kallikrein specific

• Aprotinin: non specific, serine proteases

• PPACK: non specific, serine proteases

Inhibitor Profiling

• C1 inhibitor (85%): complement cascade

• ATIII +/- Heparine: coagulation cascade

• PPACK II (80%): Kallikrein specific

• Aprotinin: non specific, serine proteases

• PPACK: non specific, serine proteases

Sandwich-ELISA

SAMPLE SOLUTION - TASK B)

TASK C) – ANALYTICAL STRATEGY

Analyte Aspect Method SOP / Kit No. Assay type Group

Complement Factor Bb Content ELISA BASB

Complement Factor C2 Content ELISA BASB

Complement Factor C4 Content ELISA BASB

Complement Factor C5a Content ELISA BASB

Complement Factor D Content ELISA BASB

Factor II (Thrombin)Content ELISA BASB

Activity Chromogenic substrate BASC

Factor IXContent ELISA BASB

Activity (FIXa-like) Chromogenic substrate BASC

Factor X Activity (FXa-like) Chromogenic substrate BASC

Factor XI

Content ELISA BASB

Activity (FXIa-like)Chromogenic substrate BASC

Thrombin clot generation

Factor XIIContent ELISA BASB

Activity (FXIIa-like) Chromogenic substrate BASC

High-molecular weight

KininogenContent ELISA BASB

Kallikrein

Content (Total) ELISA BASB

Content (Pre-kallikrein) Chromogenic substrate BASC

Activity (KLK-like) Chromogenic substrate BASC

Non-kallikrein proteases Activity Chromogenic substrate MD

Pre-kallikrein Activator Activity Chromogenic substrate BASC

PlasminogenContent ELISA BASB

Activity (Plasmin) Chromogenic substrate BASC

Activity (Plasminogen) Chromogenic substrate BASC

Pre-kallikrein Activator Activity Chromogenic substrate BASC

α-FXIIa Inhibitor Content Chromogenic substrate BASC

β-FXIIa Inhibitor Content Chromogenic substrate BASC

TASK C) – ANALYTICAL STRATEGY

TASK C - ANALYTICAL STRATEGY

16

SAMPLE SOLUTION - TASK C)

Activity-based protein profiling

(ABPP) is a direct method of

quantifying enzyme activity using

active site-targeted small-molecule

covalent probes that selectively label

active but not inhibitor-bound

enzymes.

• HRM-MS™ detection

~7000 proteins per

sample

• Acquisition of peptides

in parallel mode (DIA)

• Deconvolution using

proprietary algorithms

• quantification of ~150

pre-defined proteins

• Very sensitive

TASK C) – MS ANALYSISDiscovery Proteomics

Targeted Proteomics

18

TASK C) - ABPP

TASK C) – HYDROLASE PROBE SELECTION

pulldown SN

TASK C) – WORKFLOW AND ANALYSIS

pulldown SN

TASK C) – WORKFLOW AND ANALYSIS

TASK C) – CHALLENGES

• pKa of Imidazol (His 57): 6.4

• pH > 6.4 favors deprotonated form, reactivity withbasic amino acids side chains

• pH < 6.4 favors protonated form=> conjugate acid disables inhibitor to react with His

TASK C) – CHALLENGES

• pKa of Imidazol (His 57): 6.4

• pH > 6.4 favors deprotonated form

• pH < 6.4 favors protonated form=> conjugate acid disables inhibitor to react with His

• Acidic pH will favor

TASK C) – MODIFIED ABPP APPROACH ENGINEERING OF INHIBITORS

Profiling with engineered inhibitors Pulldown experiments

Specific Inhibition of unknownproteolytic activity

Experience report

Sandro Manni

26

MY WAY TO CSL

BSc in Biochemistry

& Molecular Biology

Medicinal Chemistry

MSc in Molecular Life Science

Neuroimmunology, Inflamation

Research Internship

Molecular Structure

Research

PhD in Biochemistry

Tumor Biology,

Vascular Biology

Postdoc in Tumor Biology

Jr. Scientific Affairs

Manager

Postdoc in Tumor Immunology

Immunotherapy, Checkpoint

inhibitors, CAR-T, TCR engineering

80 % Scientist MD R&D (1 year)

80 % Senior Scientist MD R&D

(2.5 years)

20 % Project Manager

Clinical Diagnostics

ORGANISATIONAL LOCATION

R&D

Plasma Recombinant

ResearchProduct

Development

BioanalyticalSciences

ProcessDevelopment

TechOps & Impurity

Medical Devices

Pathogen Safety

CMCBreakthrough

Technology

MethodDevelopment

BAS Chemistry BAS Biology

Bern

Projects & Systems

GROUP TASKS

1. To develop fit-for-purpose bioanalytical methods for

R&D

2. To perform bioanalytical studies and research projects

3. Ad-hoc troubleshooting

4. Adhere strongly to scientific principles

5. Bring a mindset of flexibility

6. Build meaningful collaborations inside & outside

7. Exhibit innovative spirit

MAIN COLLABORATORS

MD

PD

TechOps & IDM

QC & Quality

BAS groups

Research

Commercial & Clinical

• Impurity Analysis• Investigations

• Authority questions

• Failed-assay investigations• Method transfer• Measurements for research projects

• Biological understanding

• Specific expertise

• Market-knowledge

• Process development• Collaborations for experimental designs

SOME GROUP CAPABILITIES

Target Analysis

Main Product

2° structure analysis

3° structure analysis

MW variants (including aggregation)

Hydrophobicity variants

Charge variants

Potency assays

PTMs (e.g. oxidation & deamidation)

Process-related

impurities

Residual protein identification, quantification

Residual protein activity

General Sub-visible particles

CURRENT IN-GROUP TECHNOLOGIES

FTIR Fluorometer

HPLC /

UHPLC

Cap.

Electrophoresi

s (CE)

Multi-mode platereader

Multi-angle light

scattering (MALS)

Fluorescence-infrared imager

Dynamic Light Scattering

Instrument (DLS)

UV/VIS

MSAnalytical

Ultracentrifugation

Phage

Display

FOURIER TRANSFORM INFRARED SPECTROSCOPY

1) Quantitative analysis of protein structure

2) Structure based classification (PCA)

3) Structure-stability relationship

FLUORESCENCE SPECTROSCOPY

1) Protein tertiary structure 2) Structure-aggregation relationship 1) Protein stability

DEVELOPMENT OF STATISTICAL APPROACHES

Currently:

– Design of Experiments (DoE)

• Method optimisation

• Method robustness analysis

– Principle Component Analysis

– Trend & out-of-trend analysis

• Bioanalytical method failure

– Standard curve optimisation

– Longitudinal (Panel data, e.g. time- or wavelength-resolved) data

analysis

35

SCIENTIFIC CHALLENGES

Early career talents @ CSL

Lisa Furrer

GROW WITH US!

CSL-Trainees in Bern

2 YEARS TRAINEE-PROGRAMM

We

lco

me

Day / O

rien

tatio

n

ON-THE-JOB

Assignment 1 Assignment 2 Assignment 3 Assignment 4

SOCIAL LEARNING

Buddy Program

Mentoring Program

FORMAL LEARNING

Insights

(1/2d)

Presentation

Skills (1d)

Project

Management (3d)

Problemsolving

(3d)

Networking Events

Communication

skills (1/2d)

2 Production Trainees

Start in Oct, 2020• Pilot Plant

• Support & Hygiene

• Manufacturing Assistant

• Validierung (Departement Quality

Management)

2 Quality Management Trainees

Start in Oct, 2020• Sterility Assurance

• Compliance/ Quality Assurance

• Lab Automation

• Data Integrity

HOW TO APPLY: WWW.CSLBEHRING.CH/CAREERS

JOB NEWS ALERT

GRADUATES / EARLY CAREER TALENTS AT CSL

Profiles

• Graduates Bachelor, Master or PhD (depends on department needs)

• Relevant fields Life science (e.g. Chemistry, Biology, Microbiology), Engineering, IT

• First working experience Internships or projects with companies (Pharma preferable)

• Mindset and soft skills Strong self-starter, good team-, social- and problem-solving skills, service orientation,

communication skills

• Language Fluent in German and/or English

Q&A

Sandro Manni & Lisa Furrer

Q&A

LET’S STAY CONNECTED

These are exciting times at CSL. We are always looking to add more

great talents to our teams

• Let’s connect:

• Website:

• http://www.cslbehring.com/careers

• http://www.cslbehring.ch/trainees

• E-Mail:

• growwithus@cslbehring.com

Source icons:https://thenounproject.com/

Sandro Manni Lisa Furrer Daria Schüpbach

Have a nice day!

THANK YOU FOR YOUR ATTENTION