clx202 topic 1 (1 slide per page)

Bioprocess Applications

CLx202

CLx202 Course Module

Introduction to Biopharmaceutical Manufacturing Process Sterilisation and its application in the

pharmaceutical industry Sterile and aseptic processing Sterile Filtration Pharmaceutical Cleanrooms Pharmaceutical Utilities

Last edited: Sep 2015 NYP - SCL - BPA - Topic 1 - © NYP 2

WHAT’S SO SPECIAL ABOUT BIOLOGICS? Size does matters


Small Molecule Drugs Molecular weight < 900 Da (daltons) Initially extracted from natural products Often chemically modified Often by chemical synthesis

Aspirin: C9H8O4 (M=118) Penicillin G: C16H18N2O4S (M=334)


Biopharmaceuticals Large molecule weight

Protein Nucleic acid (DNA/RNA)

By genetically engineered organisms

Insulin: C257H379N65O75S6 (M=5808) Avastin: C6638H10160N1720O2108S44

(M≈149000)


Small Molecule Drugs Vs Biopharmaceuticals (in terms of size and complexity)

Small Molecule Small Protein Large Protein

Aspirin 21 atoms 180 Da

Growth Hormone e.g Protropin ~3000 atoms

~22 kDa

Business Jet 15000 kg

Antibody (IgG) e.g. Avastin

~ 25000 atoms ~ 149 kDa

Bicycle 10 kg Car 2000 kg

Abilify 57 atoms 448 Da


Proteins

Amino acid chain

Primary Secondary Quaternary Tertiary

α-helix

β-sheet


Proteins Macromolecules composed of amino acids

as basic component o Mix & match out of 20 amino acids

Consists of C, H, O, N, & S atoms Function as

o Enzymes o Structural skeleton of cells (e.g. actin) o Nutrition o Hormones (e.g. insulin) o Immune system (e.g. antibodies)


Amino Acids

An α-carbon atom with R group

Amino group (-NH2)

Carboxyl group (-COOH)

Exist as zwitterions


20 Amino Acids


Peptide / Amide Bonds

Formed via condensation of 2 or more amino acids

Linked amino acids chains via amide linkages are known as peptides

Peptide bonds form the primary structure of proteins


Posttranslational Modifications Covalent modification of proteins

(generally enzymes-aided) during or after protein biosynthesis


But why need modification? To modify polypeptides into useful and

meaningful proteins To convert immature proteins to fully

functional proteins


Posttranslational Modifications

Biopharmaceuticals/Vaccines


Insulin Hormone (glucose) Erythropoietin Hormone (blood) Lucentis Age-related vision loss Avastin Angiogenesis inhibitor Gardasil/Cervarix HPV vaccine Herceptin Cancer drug Humira Rheumatoid Arthritis Remicade Rheumatoid Arthritis Factor VIII, Factor IX Haemophilia

Trends in Pharmaceuticals

Drugs Biologics


Bio Manufacturing: Production Process

Stock Culture

Shake Flask Seed fermentor Production

fermentor

Supernatant

Product Extraction

Product Purification

Fill & Finish Packaging

Medium Sterilization

Medium Formulation

Medium Raw Material

Culture fluid

Bio mass

Cell separation

2. Sterilisation and its application in the pharmaceutical industry

3. Sterile and aseptic processing 4. Sterilizing Filtration 5. Pharmaceutical clean rooms 6. Pharmaceutical utility systems


UPSTREAM PROCESS Making the product


BACKGROUND Cells multiply and need more space


Fermentation Harvest

Lysis Primary recovery

Clarification Purification

Polishing Buffer exchange

Formulation

mechanical

chemical

thermal

sieve

Bag filters

Precipitation & Depth filtration

centrifugation

UF

IEX

Chrom

UF

SEC

UF


Cell Culture - Upstream


2. Sterilisation and its application in the pharmaceutical industry 3. Sterile and aseptic processing 4. Sterilizing Filtration 5. Pharmaceutical clean rooms 6. Pharmaceutical utility systems

Purification - Downstream



Fill and Finish



FERMENTATION


Fermentation

Utilizes o Microbial, animal and plant cells o Components of cells such as enzymes to

manufacture new products and destroy harmful wastes

Generally reactions where a raw organic feed is converted into product by action of microbes or enzymes.


2 Types of Fermentation


Enzymes promoted − enzyme catalyzed reactions

Micro-organisms/cells promoted o Micro-organisms o Animal cells o Plant cells

Common Fermentation

Baker’s yeast Beer Cheese Penicillin Vaccines Monoclonal antibodies Tamiflu production process (partial)


CELL GROWTH And you thought rabbits multiply fast…


During Fermentation … … Cell growth and production are measured as a function of the culture environment/conditions. Condition examples are:

Temperature pH O2 level (in solution, dissolved O2) Nutrient concentrations Stirrer speed


Batch Culture Kinetics Phases 1. Lag phase

Slow cell growth as cells are adapting to new environment 2. Log / Exponential phase

Exponential cell growth Reaches maximum growth rate

3. Post-log phase Cell growth continues at lower rate than the maximum

4. Stationary phase Cell growth ≈ Cell death ∴ viable cell number maintain constant Cells metabolism still ongoing

5. Death phase Cell growth < Cell death or growth stopped ∴ viable cell number ↓ Cell death rate exceeds cell growth Death becomes obvious


Batch Growth Curve


VCD

t

Lag phase

Log / Exponential phase

Stationary phase

Death phase

Specific Growth Rate, μ Lag phase: μ = 0 Log phase: μ = μmax

Stationary phase: μ = 0 Death phase: μ = negative


Why Exponential Growth?

XdtdXX

dtdX µ=∴∝


Doubling time, td?


Fermentation

Some derived/calculated parameters use to describe fermentation process performance are Specific Growth Rate, μ (unit of per time, e.g. h-1, d-1) Specific Production Rate, QP (unit of mass per cell

per time, e.g. 𝐩𝐩𝐩𝐩𝐜𝐜𝐜𝐜𝐜𝐜𝐜𝐜∙𝐝𝐝𝐝𝐝𝐝𝐝

)

Product Yield or Titer Cell Density or Concentration, CD or X (unit of cells

per vol, e.g. cells/mL, cells/L) Viable Cell Density, VCD (same unit as CD)


Unit Prefix Refresher Prefix Symbol Factor Mass Length Volume peta P 1015 tera T 1012 giga G 109 mega M 106 t kilo k 103 kg km

100 g m m3 deci d 10-1 dm centi c 10-2 cm milli m 10-3 mg mm L, dm3

micro μ 10-6 μg μm mL, cm3 nano n 10-9 ng nm μL pico p 10-12 pg pm femto f 10-15 fm


Product Formation

Basic Assumption o Cells produce all the time

o All cells produce at same rate (QP) How much product does each cell produce per time?


How can we increase production?

Specific Production Rate, QP Unit of pg

cell∙day

Base on previous assumption,


Higher QP ≅ Higher Product Yield

QP of 1 pgcell∙day

means that it takes 1012

cells to produce 1 g of product in a day.

How long would 1011 cells take to produce 1 g?

Man-hour, Cell-day… Man-hour is the amount of work performed by an average worker per hour.

∴ If we want more work done, we can – hire more workers or – have workers work longer hours

Likewise, to get more production in a fermenter, increase amount of cells or extend the production duration


VCD

t

Total Number of Cell-days


IVCD: Time integral of viable cell density

∫=ω

α

dtXIVCD t

Total Number of Cell-days

IVCD: Time Integral of Viable Cell Density

In practical:

∑

∑

⋅+

≈

∆⋅+

≈

−

−

daylast

dayfirst

ii

ii

dVCDVCDIVCD

tVCDVCDIVCD

_

_

1

1

12

2

𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼 = � 𝐼𝐼𝐼𝐼𝐼𝐼𝑡𝑡 𝑑𝑑𝑑𝑑𝑡𝑡

𝑡𝑡0


IVCD Example

Fermenter size = 10 L, Run duration = 4 days

Day 0: X = 3 × 108 cells/L

Day 1: X = 3 × 108 cells/L

Day 2: X = 6 × 108 cells/L

Day 3: X = 1 × 109 cells/L

Day 4: X = 1 × 109 cells/L


Determine QP Fermenter size = 10 L, Run duration = 4 days

IVCDtotal = 2.55 × 109 cell∙day/L The fermentation produced a titer of 510 mg/L.


How much product does each cell produce everyday?

CELL HARVESTING / BIOMASS REMOVAL Clarification or Concentration


Where is the Product?


Cell Harvesting / Biomass Removal

Centrifugation / Separation

•Use centrifugal forces to separate substances of differing denseness

Tangential / Cross Flow Filtration (TFF)

•Use membrane to separate substances of differing size, charge etc.


Cell Harvesting / Biomass Removal

Cell Culture

Supernatant / Permeate

Biomass / Retentate

1: Feed, 2: Feed for liquid water or concentrate recycle, 3: Centripetal pump for clarified liquid, 4: Disc stack, 5: Inlet chamber for product, 6: Concentrate chamber, 7: Nozzles,

8: Concentrate discharge, 9: Clarified liquid discharge Last edited: Sep 2015 NYP - SCL - BPA - Topic 1 - © NYP 48

Cross or Tangential Flow Filtration


DOWNSTREAM PROCESS Removing everything except the product


Downstream Processing

Typically starts after cell separation Mostly Protein Purification steps

o Filtration o Chromatography

Include measures to reduce viral load Last edited: Sep 2015 NYP - SCL - BPA - Topic 1 - © NYP 52

Types of Chromatography

Liquid chromatography o Size exclusion o Hydrophobic interaction o Ion exchange o Affinity


25.5 t • Fermentation

1.43 t

• Primary recovery (cell harvesting, cell disruption, inclusion body recovery)

• Step yield 90%

62.5 t • Chemical conversion (IB solubilisation, DF, IEX, refolding, DF, HIC) • Step yield 60%

2.94 t • Enzymatic conversion • Step yield 90%

15 kg • Final purification (DF, IEX, DF, RP, DF, SEC, UF, crystallisation, filtration) • Step yield 60%


Yield Losses

nLY

−⋅=

%1001%100

Y: Yield L: percent yield loss for one step n: number of steps

%9.59%100

%51%10010

=

−⋅=

Y

Y


Example: o 5% loss per step o 10 steps

Total Yield

Calculate the overall yield of the insulin process!

4321 YYYYYYield itotal ⋅⋅⋅=∏=

=totalYield

Σ – capital Sigma for sums

Π – capital Pi for product

itotal YYield ∏=


FILL & FINISH Sterile or Finished Dosage Form


Common Finished Dosage Forms

Pre-filled syringes Liquid vials Lyophilized vials Cartridges



FILL & FINISH Sterile or Finished Dosage Form

Industrial Lyophiliser

clx202 topic 1 (1 slide per page)

Documents

nyp scl

bpa topic

amino acids chains

immature proteins

proteins macromolecules

meaningful proteins

functional proteins

actin o nutrition o