Introduction to BioLC Columns · Column housing PEEK for 4.0 mm I.D. columns SST for 7.8 mm and 2.1 mm I.D. columns Separation range for globular proteins 10,000 - 1,000,000 Exclusion

The world leader in serving science

Introduction to Thermo Fisher Scientific BioLC Columns

2

Overview

• What are Biomolecules? • Thermo Scientific BioLC Column Portfolio • Columns for Proteins

• Affinity • Size Exclusion •  Ion Exchange • Reversed-phase

• Concluding remarks

3

What are biomolecules?

http://www.sciencemuseum.org.uk/

Biomolecules are organic molecules, especially a macromolecules (large molecules, such as a protein or oligonucleotides) in living organisms*. •  Transcription: gene information stored in

DNA is transferred to RNA and then to proteins.

•  Translation: The process of RNA information being transferred to protein

*Merriam Webster Definition

4

Category Columns

Proteins are made of amino acids

Amino Acids

Thermo ScientificTM Hypercarb / AminoPac

Amino acids together form a peptide

Peptides Thermo ScientificTM AcclaimTM 300 Thermo ScientificTM PepMapTM Thermo ScientificTM PepSwiftTM

Many peptides form a protein.

Proteins Thermo ScientificTM ProPacTM Thermo ScientificTM MAbPacTM BioBasic Thermo ScientificTM AccucoreTM 150 Thermo ScientificTM ProSwiftTM

What is a protein, peptide and amino acid?

5

Why do we need different tools for small molecule vs large molecules?

Small Molecule Pharmaceutical Production

Mix Chemicals Together

Wait for Reaction

Product of 180 MW

https://lppt.epfl.ch/page-51483-en.html

• Immunization of mouse and isolation of their splenocytes

Mouse

• Preparation of myeloma cells to fuse with spleen cells, followed by fusion

Fusion • Clone Screening and selection

Clone Selection

• Characterization

Functional Characterization • Scale up clones

producing desired antibodies in bioreactors

Scale up

An Example Flow Path: Large Molecule Production

6

Thermo Scientific BioLC Column Portfolio

7

Thermo Scientific Columns

LC Columns

Accucore

Acclaim

Hypersil GOLD

Syncronis

Hypercarb

Legacy

Bio Columns

MAbPac

ProPac

GlycanPac

DNAPac

ProSwift

DNASwift

BioBasic

• BioLC Columns are optimized for the needed separation

• Separation need

•  High resolution •  High Throughput

• Biocompatibility • Biomolecule Size

•  Proteins •  Peptides •  Amino Acids

8

Size Exclusion

MAbPac SEC

BioBasic SEC

Ion Exchange

ProPac WCX, SCX, WAX, SAX

MAbPac SCX

BioBasic SAX

ProSwift WCX, SCX, WAX,

SAX

Reversed-phase

BioBasic C18, C8, C4

MAbPac RP

Accucore 150-C18, 150-C4

ProSwift RP

Hydrophobic Interaction

ProPac HIC

MAbPac HIC-10, HIC-20,

HIC-Butyl

Affinity

MAbPac Protein A

ProPac IMAC

ProPac ConA-1S

Glycans

Accucore Amide-HILIC

GlycanPac AXR-1, AXH-1

Thermo Scientific BioLC Columns Portfolio by Technique

9

Analysis Description Columns and Buffers Detection Titer mAb capture, titer & screening

Thermo Scientific™ MAbPac™ Protein A UV

Aggregate Routine screening for aggregates and fragments

Thermo Scientific™ MAbPac™ SEC-1 UV & light scattering

Charge Heterogeneity Routine variant profiling including; lysine truncation, deamidation and acylation

Thermo Scientific™ MAbPac™ SCX-10 Thermo Scientific™ MAbPac™ SCX-10 RS Thermo Scientific™ ProPac™ WCX-10 Thermo Scientific™ CX-1 pH Gradient Buffer Kit

UV

Methionine & Tryptophan Oxidation

Targeted analysis of methionine and tryptophan oxidation

Thermo Scientific™ MAbPac™ HIC-20 Thermo Scientific™ MAbPac™ HIC-10 Thermo Scientific™ ProPac™ HIC-10

UV

Antibody Drug Conjugate (ADC)

Drug to Antibody ratios Thermo Scientific™ MAbPac™ HIC-10 Butyl Thermo Scientific™ MAbPac™ HIC-20 Thermo Scientific™ MAbPac™ HIC-10 Thermo Scientific™ MAbPac™ RP

UV

Antibody Drug Conjugate (ADC) using MS

Drug to Antibody ratios and intact mass

Thermo Scientific™ MAbPac™ SEC-1 Thermo Scientific™ MAbPac™ RP Thermo Scientific™ Acclaim™ SEC-300

Intact or Fragment Mass Intact, light (LC), heavy chain (HC) and fragment (Fab & Fc) analysis

Thermo Scientific™ MAbPac™ RP UV and MS

Native Mass Intact native mass analysis Thermo Scientific™ MAbPac™ SEC-1 Thermo Scientific™ Acclaim™ SEC-300

UV and MS

Bio-Column Selection Guide

10

Selecting the Correct Column for Intact Protein Separation

Cells are grown

Harvest monoclonal antibodies

Affinity

11

Affinity Chromatography Columns

12

Affinity Columns

Affinity

MAbPac Protein A

ProPac IMAC

ProPac ConA-1S

Product Base material Ligand Purpose

Thermo Scientific MAbPac Protein A

Hydrophilic nonporous resin, 12 µm

Protein A Determination of monoclonal antibody (MAb) concentration (titer) in the harvest cell culture

Thermo Scientific ProPac IMAC (Immobilized metal affinity)

IDA grafted nonporous polymer,10 µm

Iminodiacetate Separates proteins based on their affinity for metals.

Thermo Scientific ProPac ConA-1S

Polymeth-acrylate

Concanavalin A (Con A)

Enrichment and purification of Con A binding glycans, glycopeptides, and glycoproteins

13

Protein A Captures MAb

Wash out other proteins

Load Harvested Cell Culture Fluid onto Column

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 -200

1,000

2,000

3,000

4,000

5,200

mAu

Minutes

IgG

Unbound

Elute MAb

MAbPac Protein A Affinity Column: Load, Wash, Elute

14


Cells are grown


Affinity

SEC

15

Size Exclusion Chromatography Columns

16

What is an aggregate and fragment?

Monomer Aggregates

17

Limits on diffusion control the separation • Pore size – controls the relative separation of proteins • Pore volume – controls the overall retention time of each species

Size Exclusion Chromatography Particle pore size

18

Physical Data on a MAbPac SEC column

Bonding Chemistry Diol Silica Substrate Spherical, high-purity porous silica

Particle size 5 µm

Pore size 300 Å

Column housing PEEK for 4.0 mm I.D. columns SST for 7.8 mm and 2.1 mm I.D. columns

Separation range for globular proteins 10,000 - 1,000,000

Exclusion limit for globular proteins >1,000,000

The Hydrophilic boundary layer and the Pore Size are important factors

19

mAb Aggregates by LC/UV

Highest Resolution

Best for LC/MS

Best for Low solvent use

20

Top Four mAb samples with the MAbPac SEC – Global Applicability

Infliximab

Bevacizumab

Rituximab

Trastuzumab

Monomer Aggregate

Fragments

21

Overlay of three chromatography runs of Bevacizumab

22

•  Repeat injections of Bevacizumab on MAbPac SEC-1, 4 x 300 mm, 5µm

•  Excellent column performance up to Injection #1953

Column Lifetime Study

23


Cells are grown


Affinity

SEC Ion Exchange

24

Ion Exchange Chromatography Columns

25

Particle Design of Ion Exchange Columns

Nonporous Polymeric Beads

Products Base material Features ProPac WCX-10, SCX-10, SAX-10 WAX-10

Nonporous Polymeric Bead, 10µm

•  Weak and Strong Cation Exchangers

•  Ultra-high resolution •  Low capacity

MAbPac SCX-10 Nonporous Polymeric Bead, 3, 5 &10µm

•  Strong Cation Exchange •  Ultra-high resolution, •  Low capacity

26

Lysine Variants

Y Y K K

Y Y K

Y Y

Acidic Variants Basic Variants

ProPac WCX-10, 4x250 mm

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 0.0 5.0

10.0 15.0 20.0 mAU

min 2

4x150 mm MAbPac SCX-10 15 min. Total Analysis Time

Improve resolution or sample throughput through chemistry

Acidic Variants

Basic Variants

Y Y K K

Y Y K

Y Y

MAbPac SCX-10, 4x250 mm

Lysine Variants

4x250 mm MAbPac SCX-10 60 min. Total Analysis Time

MAbPac SCX-10 IEC for mAb Analysis

27

Blue trace: MAbPac™ SCX-10 Red trace: ProPac® WCX-10 Tris buffer (pH 8.0)

Comparison of MAbPac SCX-10 and ProPac WCX-10 Columns for mAb Analysis

15 20 25 30 32.5

mAU

0

175

MAb3: pI 8.87

Minutes

mAU

250

MAb1: pI 9.2

4 8 12 18 0

6 10 14 16

mAU

500

MAb2: pI 8.9 min7 8 9 10 11 12 13 14 15 16

mAU

0

2

4

6

8

8 10 12 15 0

9 11 13 14 Minutes

27567

28

-5.0

0.0

5.0

10.0

15.0

20.0

25.0

30.0 1 - SCX_3.2u_0613-307po613#2_4.0x50mm_0.5mL_PDL_1mg_mL_Ult_MES #10 15.0 SR_3u_SCX_22_37_5min_0_5mL_37B_2min_17T_LPump_MESpH5_6_PDLmAU

1

WVL:280 nm

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0-5.0

0.0

10.0

20.0

30.0

35.0 2 - Ag_BioMAb_1.7u_4.6x50mm_A341284USDJA01060_PDL_1mg_mL_Ult_MES #305 10.0 AgBioMAb_WCX_1_7um_0_5mL_30_50_5min_18T_LPump_MESPDLmAU

min

2

WVL:280 nm

MabPac SCX_3 um, 4x50 mm;

Flow: 0.5 mL/min; Gradient: 30-50%B in 5 min;

Agilient BioMab 1_7 um, 4.6x50 mm;

Flow: 0.5 mL/min; Gradient: 22-37%B in 5 min;

A.  20 mM MES, pH 5.6 + 60 mM NaCI B.  20 mM MES, pH 5.6 + 300 mM NaCl

A)  20 mM MES, pH 5.6 + 60 mM NaCI B)  20 mM MES, pH 5.6 + 300 mM NaCl

Comparison of Agilent Bio-MAb 1.7 µm & MAbPac SCX 3 µm

29

ProPac WCX-10 vs. Sepax Antibodix NP-10

PDL-MAb: MES based eluents, pH 5.6

1 - MAB_ANALYSIS_4_16_2008 #581 MAb_4x250_ProPac_WCX_07_027_014FG_2_Ref(MES/Heat Tr) MAb_Analysis_MES_300mM_B_25_46_44B_50min_T73min

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 58.0 -6.0 -5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0

10.0 2 - MAB_ANALYSIS_4_16_2008 #583 MAb_SEPAX_Antibodix_4x250_WCX_SST_05080917665 MAb_Analysis_MES_300mM_B_25_46_44B_50min_T73min 3 - MAB_ANALYSIS_4_16_2008 #585 MAb_SEPAX_Antibodix_4x250_WCX_SST_05080917665 MAb_Analysis_MES_300mM_B_25_46_44B_50min_T73min mAU

min

3

2

1

WVL:280 nm

ProPac WCX-10, 4x 250 mm

SEPAX 7µm, 4x 250 mm

SEPAX 7µm, 4x 250 mm

30

ProPac WCX 10-30% B in 18.75 min 1.5 mL/min

TSK_CM-STAT 10-30% B in 7.5 min 2 mL/min

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 -10.0 0.0

10.0 20.0 30.0 40.0 50.0 60.0 70.0

1

WVL:280 nm

10.0

0.0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 11.3 12.5 13.8 15.0 16.3 17.5 18.8 20.0 21.3 22.5 23.8 25.0 -5.0 0.0

10.0

20.0

30.0

40.0

2

WVL:280 nm

10.0

El. A: 20 mM MES pH 6.0

El. B: 0.5M NaCl in Elunet A

MAb1: MES Eluents

Tosoh recommended conditions

4.6 x 100 mm

4.0 x 250 mm

mAU

Minutes

ProPac WCX-10 vs. TSK gel CM-STAT

31

Protein and MAb Separation on IEX Columns

Salt Gradient pH Gradient

•  Most widely used method

•  Relatively simple to make the buffer

•  Takes longer to optimize the separation condition (pH, salt concentration)

•  Can predict elution profile with pI value

•  Lower salt concentration in collected fractions

•  In many cases, improved resolution was observed

•  Difficult to generate a linear

pH gradient

32

Advantages •  Platform method ð single method

for wide range of mAbs and other proteins

•  Reduced times for method development and method transfer

• Outperforms any other charge variant technology

•  Less variability from effects of column

•  Transferability of method from development to QC

Charge Variant Analysis using pH Gradient

33

pH Gradient Buffers – How Do They Work?

34

Salt vs pH Gradient IEC of MAb Sample

30 min gradient, Thermo Scientific™ MabPac™ SCX-10, 10 µm, 4 × 250 mm column

0.0 5.0 10.0 15.0 20.0 25.0 30.0 0.0

5.0

10.0

15.0

min %B: 10.0

30.0

Salt gradient

0.0 5.0 10.0 15.0 20.0 25.0 30.0 0.0

5.0

10.0

15.0

min %B: 25.0

50.0

25.0 pH gradient

35

Top-selling mAbs analyzed by pH gradient method

Platform method for mAb charge variant analysis

15.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 35.0-10.0

0.0

12.5

25.0

37.5

50.0

70.0

Retention Time (min)

Rituxan

1

2

3

15.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 35.0

-10.0

0.0

12.5

25.0

37.5

50.0

70.0


Herceptin

15.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 35.0

-2.0

5.0

10.0

15.0

18.0


Humira

15.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 35.0

-2.0

5.0

10.0

15.0

20.0


Avastin

36


Cells are grown


Affinity

Denatured / fragmented protein

SEC Ion Exchange

Reversed- Phase Top-down

Middle-down

Bottom up

MAbPac RP

37

Reversed-Phase Chromatography Columns

38

Several Base Materials

Chemistries

BioBasic: Silica Columns with many surface chemistries (use for smaller protein analysis). Can scale up to preparative format

MAbPac RP: Supermacroporous wide pore chemistry (1500A) use for large proteins and mAb analysis. Available 1mm ID for use with MS

Accucore 150: Core enhanced technology Fast separation- Solid Core Alternative to the BioBasic line

Monolith: Fast Mass transfer High loading capacity

Reversed-phase

BioBasic C18, C8,

C4

MAbPac RP

Accucore 150-C18, 150-C4

ProSwift RP

39

General Properties: MAbPac RP

•  Superior resolution power for monoclonal antibodies and related substances

•  High efficiency with low carry-over

•  Excellent MS compatibility

•  Wide operating pH range: 0 – 14

•  High temperature stability: up to 110 oC

•  High throughput

40

Minimized Carryover using Polymeric MAbPac RP

41

Excellent Reproducibility

Column: MAbPac RP, 4 µm Format: 2.1 ×50 mm Mobile phase A: H2O/TFA (99.9 : 0.1 v/v) Mobile phase B: MeCN/ H2O/TFA (90: 9.9 :0.1 v/

v/v/) Gradient:

Time (min) %A %B -1 80 20 0.0 80 20 2.5 50 50 2.7 50 50 2.8 80 20 3.0 80 20

Temperature: 80 ºC Flow rate: 0.6 mL/min Inj. volume: 1 µL Detection: UV (280 nm) Sample: 1. Ribonuclease A (0.5 mg/mL)

2. Cytochrome C (0.5 mg/mL) 3. Lysozyme (0.5 mg/mL) 4. mAb (1 mg/mL)

0.00 0.50 1.00 1.50 2.00 2.50 3.00

-80

-60

-40

-20

0

20

40

60

80

100

120

#1101

#901 #801 #701 #651 #501 #401 #301 #201 #101


1

2

3

4

1100 injections with < 1% RSD

42

Loadability: Three Orders of Magnitude

-0.50

1.25

2.50

-2.0

10.0

18.0

-20

140

1.00 1.50 2.00 2.50 3.00 3.50 4.00

-100

500

900


(a) 0.02 µg

(d) 20 µg

(c) 2 µg

(b) 0.2 µg

y = 2.3101x + 0.0491R² = 1

0

5

10

15

20

25

30

35

40

45

50

0 10 20 30

Area

mAb (µg)


v/v/) Gradient: Time (min) %A %B

-1 85 15 0.0 85 15 2.5 50 50 2.7 50 50 2.8 85 15 4.0 85 15

Temperature: 80 ºC Flow rate: 0.6 mL/min Inj. volume: 1 µL Detection: UV (280 nm) Sample: mAb

Linearity 20 ng to 20 µg

43

Chemical Stability: pH range 0-14


v/v/) Gradient:

Time (min) %A %B -1.0 85 15 0.0 85 15 2.5 50 50 2.7 50 50 2.8 85 15 4.0 85 15

Temperature: 80 ºC Flow rate: 0.6 mL/min Inj. volume: 1 µL Detection: UV (280 nm) Sample: 1. Ribonuclease A (0.5 mg/mL)

2. Cytochrome C (0.5 mg/mL) 3. Lysozyme (0.5 mg/mL) 4. mAb (1 mg/mL)

0.50 1.00 1.50 2.00 2.50 3.00 3.50 -20

0

13

25

38

50

63

75

88

100

113

125

140

After 6 hrs of 0.8 M NaOH wash at 80 °C

Before NaOH wash

1

2

3

4


44

Application: LC/MS Analysis of the Intact Protein Standard Mix

Column: MAbPac RP, 4 µm Format: 1.0 × 150 mm Mobile phase A: H2O/FA (99.9 : 0.1 v/v) Mobile phase B: MeCN/FA (99.0: 0.1 v/v) Gradient:

Time (min) %A %B 0.0 90 10 1.0 80 20 15.0 55 45 16.0 10 90 20.0 10 90 20.1 90 10 25.0 90 10

Temperature: 60 ºC Flow rate: 0.1 mL/min Inj. volume: 1 µL MS Detection: positive-ion mode Mass Spec: Q Exactive™ HF Sample: Pierce Intact Protein Standard Mix,

500 ng/µL (p/n A35526) 1. Protein G (21 kDa) 2. Protein AG (51 kDa) 3. IGF-I LR3 (9 kDa) 4. Thioredoxin (12 kDa) 5. Carbonic Anhydrase II (29kDa) 6. Exo Klenow (68 kDa)

6 8 10 12 14 16 18 Time (min)

0 5

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

100

Rel

ativ

e A

bund

ance

5

2

1

3 6

4

45

BioUHPLC Reversed-phase Column Competition

Vendor Product Type Chemistry Base Material

dp (µm)

Pore Size Å

Column Hardware Back Pressure (bar)

Thermo Hypersil GOLD RP C18/C8/aQ/… Si 1.9 175 SST 1035 bar / 15000 psi

Thermo MAbPac RP Phenyl PS-DVB 4 1500 SST 275 bar/ 4000psi

Thermo Accucore RP C18 / C4 /Amide HILIC Si 2.6 150 SST 1000 bar

Waters BioResolve mAb RP RP Polyphenol Si (BEH) 2.7 450 SST 690 bar / 10,000 psi

Waters BEH300 RP C18 / C4 Si (BEH) 1.7 300 SST 1035 bar / 15000 psi

Agilent RRHD RP Diphenyl Si (Zorbax) 1.8 80/95/300 SST 1200 bar

Phenomenex

BioZen/ Aeris

peptide Aeris

Widepore

RP

XB-C18 / (XB-C8/C4) , XB:

protective isobutyl

sidechains on C18

Si Core/Shell µm Peptide (P): 1.25/0.22

2.6/0.5 Widepore (W):

3.2/0.2

P: 1.7, 3.6 µm W: 200 3.6 µm

P: 100 W: 200 SST

Aeris 1.7 µm: 1,000 bar (15,000 psi).

Aeris 3.6 µm: 600 bar (8,700 psi).

Lon

46

Summary

•  Large molecule production varies greatly from small molecule production, and the correct tools are needed

•  Large molecules include: proteins, therapeutic proteins, monoclonal antibodies, ADCs, and biosimilars

•  We have a wide portfolio of columns for bio separations, regardless of characterization technique •  Affinity •  Size Exclusion Chromatography (SEC) •  Charge Variant Analysis (IEX) •  Hydrophobic Interaction Chromatography (HIC) •  Reversed Phase Analysis (RP)

•  Our columns offer a robust, rugged platform for your separations

•  Our portfolio includes innovative products, such as industry leading products: •  ProPac and MabPac columns •  pH gradient buffers

47

Analysis Description Columns and Buffers Detection Titer mAb capture, titer & screening

Thermo Scientific™ MAbPac™ Protein A UV

Aggregate Routine screening for aggregates and fragments

Thermo Scientific™ MAbPac™ SEC-1 UV & light scattering

Charge Heterogeneity Routine variant profiling including; lysine truncation, deamidation and acylation

Thermo Scientific™ MAbPac™ SCX-10 Thermo Scientific™ MAbPac™ SCX-10 RS Thermo Scientific™ ProPac™ WCX-10 Thermo Scientific™ CX-1 pH Gradient Buffer Kit

UV

Methionine & Tryptophan Oxidation

Targeted analysis of methionine and tryptophan oxidation

Thermo Scientific™ MAbPac™ HIC-20 Thermo Scientific™ MAbPac™ HIC-10 Thermo Scientific™ ProPac™ HIC-10

UV

Antibody Drug Conjugate (ADC)

Drug to Antibody ratios Thermo Scientific™ MAbPac™ HIC-10 Butyl Thermo Scientific™ MAbPac™ HIC-20 Thermo Scientific™ MAbPac™ HIC-10 Thermo Scientific™ MAbPac™ RP

UV

Antibody Drug Conjugate (ADC) using MS

Drug to Antibody ratios and intact mass

Thermo Scientific™ MAbPac™ SEC-1 Thermo Scientific™ MAbPac™ RP Thermo Scientific™ Acclaim™ SEC-300

Intact or Fragment Mass Intact, light (LC), heavy chain (HC) and fragment (Fab & Fc) analysis

Thermo Scientific™ MAbPac™ RP UV and MS

Native Mass Intact native mass analysis Thermo Scientific™ MAbPac™ SEC-1 Thermo Scientific™ Acclaim™ SEC-300

UV and MS

Bio-Column Selection Guide

48

Chromatography Resource Center

http://www.separatedbyexperience.com/chromexpert/

The world leader in serving science

Thank you! © 2018 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representatives for details. PP21861-EN 0718S

Documents

Introduction to BioLC Columns · Column housing PEEK for 4.0 mm I.D. columns SST for 7.8 mm and 2.1 mm I.D. columns Separation range for globular proteins 10,000 - 1,000,000 Exclusion